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Sample records for antibody-antigen docking compensates

  1. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

  2. The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production.

    Science.gov (United States)

    Ushijima, Miho; Uruno, Takehito; Nishikimi, Akihiko; Sanematsu, Fumiyuki; Kamikaseda, Yasuhisa; Kunimura, Kazufumi; Sakata, Daiji; Okada, Takaharu; Fukui, Yoshinori

    2018-01-01

    A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab') 2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.

  3. The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production

    Directory of Open Access Journals (Sweden)

    Miho Ushijima

    2018-02-01

    Full Text Available A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR, which triggers activation of B cells and differentiation into plasma cells (PCs. Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab′2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2–Rac axis in PC differentiation and IgG antibody responses.

  4. Antigenicity of envelop and non-structural proteins of dengue serotypes and their potentiality to elicit specifi antibody

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    Ramesh Venkatachalam

    2015-06-01

    Full Text Available Objective: To find out the antigenic nature of envelop (E and non-structural (NS proteins and their ability to induce specific antibodies, and to investigate specific antibody produced by specific dengue virus (DENV serotypes. Methods: Amino acid sequences of E and NS proteins of dengue serotypes were analysed by using VaxiJen antigen predicition server. The transmembrane of topology analyses were conducted by using transmembrane prediction using hidden markov models. The Hex dock server was used for docking. Results: The antigenicity score and exomembrane potentiality of E and NS proteins were calculated. All those proteins were antigenic; these antigens were made to interact with antibodies such as immunoglobulin A, immunoglobulin G and immunoglobulin M. Higher energy values of immunoglobulin M were found in DENV-1 and DENV-2, and more energy values were found in immunoglobulin G of DENV-3, DENV-4, NS-1, NS-3 and NS-5. Conclusions: In the present study, DENV-1 and DENV-2 are positive to immunoglobulin M and involved in the primary infection. DENV 3, DENV 4 and all the NS proteins (NS-1, NS-3, NS-5 which elicit immunoglobulin G are involved in the secondary infection.

  5. Prediction of site-specific interactions in antibody-antigen complexes: the proABC method and server.

    KAUST Repository

    Olimpieri, Pier Paolo

    2013-06-26

    MOTIVATION: Antibodies or immunoglobulins are proteins of paramount importance in the immune system. They are extremely relevant as diagnostic, biotechnological and therapeutic tools. Their modular structure makes it easy to re-engineer them for specific purposes. Short of undergoing a trial and error process, these experiments, as well as others, need to rely on an understanding of the specific determinants of the antibody binding mode. RESULTS: In this article, we present a method to identify, on the basis of the antibody sequence alone, which residues of an antibody directly interact with its cognate antigen. The method, based on the random forest automatic learning techniques, reaches a recall and specificity as high as 80% and is implemented as a free and easy-to-use server, named prediction of Antibody Contacts. We believe that it can be of great help in re-design experiments as well as a guide for molecular docking experiments. The results that we obtained also allowed us to dissect which features of the antibody sequence contribute most to the involvement of specific residues in binding to the antigen. AVAILABILITY: http://www.biocomputing.it/proABC. CONTACT: anna.tramontano@uniroma1.it or paolo.marcatili@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  6. The interfacial character of antibody paratopes: analysis of antibody-antigen structures.

    Science.gov (United States)

    Nguyen, Minh N; Pradhan, Mohan R; Verma, Chandra; Zhong, Pingyu

    2017-10-01

    In this study, computational methods are applied to investigate the general properties of antigen engaging residues of a paratope from a non-redundant dataset of 403 antibody-antigen complexes to dissect the contribution of hydrogen bonds, hydrophobic, van der Waals contacts and ionic interactions, as well as role of water molecules in the antigen-antibody interface. Consistent with previous reports using smaller datasets, we found that Tyr, Trp, Ser, Asn, Asp, Thr, Arg, Gly, His contribute substantially to the interactions between antibody and antigen. Furthermore, antibody-antigen interactions can be mediated by interfacial waters. However, there is no reported comprehensive analysis for a large number of structured waters that engage in higher ordered structures at the antibody-antigen interface. From our dataset, we have found the presence of interfacial waters in 242 complexes. We present evidence that suggests a compelling role of these interfacial waters in interactions of antibodies with a range of antigens differing in shape complementarity. Finally, we carry out 296 835 pairwise 3D structure comparisons of 771 structures of contact residues of antibodies with their interfacial water molecules from our dataset using CLICK method. A heuristic clustering algorithm is used to obtain unique structural similarities, and found to separate into 368 different clusters. These clusters are used to identify structural motifs of contact residues of antibodies for epitope binding. This clustering database of contact residues is freely accessible at http://mspc.bii.a-star.edu.sg/minhn/pclick.html. minhn@bii.a-star.edu.sg, chandra@bii.a-star.edu.sg or zhong_pingyu@immunol.a-star.edu.sg. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  7. Generation of monoclonal antibodies against highly conserved antigens.

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    Hongzhe Zhou

    Full Text Available BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1 and one mouse self-antigen (TNF-alpha as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system. CONCLUSIONS/SIGNIFICANCE: We developed an efficient and universal method for generating surrogate or therapeutic antibodies against "difficult antigens" to facilitate the development of therapeutic antibodies.

  8. Microradioimmunoassay for antibodies to tumor-associated antigens

    International Nuclear Information System (INIS)

    Huang, J.C.C.; Berczi, I.; Froese, G.; Tsay, H.M.; Sehon, A.H.

    1975-01-01

    A versatile microradioimmunoassay for the detection of antibodies to tumor-associated and other tissue antigens was described. The method involved: the preparation of solid-phase antigen with cultured (already adhered) or noncultured cells (sedimented by centrifugation) fixed to Micro-Test plates with neutral buffered formaldehyde or absolute methanol; the incubation of the antigen with test or control sera; and the incubation of the antigen with radioiodinated antiglobulin antibody. The nonspecific background of radioactivity was reduced to an acceptable level by the fixed cells being precoated in the wells with 0.5 percent bovine serum albumin in phosphate-buffered saline which was also used for the dilution of sera and labeled antiglobulin antibody. Tumor cells in primary cultures gave a high background, as compared to long-term cultures, which was due to the presence of immunoglobulins (most likely tumor-specific antibody). The specific antibody response to a syngeneic mouse tumor was demonstrated by this technique. (auth)

  9. An indirect antibody assay using haptenated antigen and 125I-labelled anti-hapten antibody

    International Nuclear Information System (INIS)

    Aalberse, R.C.; Amsterdam Univ.

    1978-01-01

    Hapten (trinitrophenyl) was coupled to antigen (ovalbumin). The haptenated antigen was bound by anti-ovalbumin antibody and binding was quantitated with 125 I-labelled anti-hapten antibodies. Thus, with a single radioactive reagent, antibodies against a variety of antigens can be detected while the problems inherent in a labelled antiglobulin binding test are avoided. In the ovalbumin system, the haptenated antigen binding test proved to be approximately 20 times as sensitive as the iodinated ovalbumin binding test

  10. Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

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    Edith S Málaga-Machaca

    2017-11-01

    Full Text Available Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

  11. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

    Science.gov (United States)

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Human antibody and antigen response to IncA antibody of Chlamydia trachomatis.

    Science.gov (United States)

    Tsai, P Y; Hsu, M C; Huang, C T; Li, S Y

    2007-01-01

    The high prevalence of C. trachomatis worldwide has underscored the importance of identifying specific immunogenic antigens in facilitating diagnosis as well as vaccine development. The aim of this study is to evaluate IncA antibody and antigen production in natural human infections. Our temporal expression study showed that IncA transcription and protein expression could be detected as early as 4 hours after the start of infection. Antibody responses could be detected in urine and genital swab samples from C. trachomatis-positive patients. It is especially interesting to note that the IncA antigen could be detected in urine. In conclusion, we have identified IncA as an important antigen in human. The potential applicability of the IncA antibody or antigen in the diagnosis as well as to vaccine development for C. trachomatis is also discussed.

  13. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

  14. IFACEwat: the interfacial water-implemented re-ranking algorithm to improve the discrimination of near native structures for protein rigid docking.

    Science.gov (United States)

    Su, Chinh; Nguyen, Thuy-Diem; Zheng, Jie; Kwoh, Chee-Keong

    2014-01-01

    Protein-protein docking is an in silico method to predict the formation of protein complexes. Due to limited computational resources, the protein-protein docking approach has been developed under the assumption of rigid docking, in which one of the two protein partners remains rigid during the protein associations and water contribution is ignored or implicitly presented. Despite obtaining a number of acceptable complex predictions, it seems to-date that most initial rigid docking algorithms still find it difficult or even fail to discriminate successfully the correct predictions from the other incorrect or false positive ones. To improve the rigid docking results, re-ranking is one of the effective methods that help re-locate the correct predictions in top high ranks, discriminating them from the other incorrect ones. Our results showed that the IFACEwat increased both the numbers of the near-native structures and improved their ranks as compared to the initial rigid docking ZDOCK3.0.2. In fact, the IFACEwat achieved a success rate of 83.8% for Antigen/Antibody complexes, which is 10% better than ZDOCK3.0.2. As compared to another re-ranking technique ZRANK, the IFACEwat obtains success rates of 92.3% (8% better) and 90% (5% better) respectively for medium and difficult cases. When comparing with the latest published re-ranking method F2Dock, the IFACEwat performed equivalently well or even better for several Antigen/Antibody complexes. With the inclusion of interfacial water, the IFACEwat improves mostly results of the initial rigid docking, especially for Antigen/Antibody complexes. The improvement is achieved by explicitly taking into account the contribution of water during the protein interactions, which was ignored or not fully presented by the initial rigid docking and other re-ranking techniques. In addition, the IFACEwat maintains sufficient computational efficiency of the initial docking algorithm, yet improves the ranks as well as the number of the near

  15. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    Science.gov (United States)

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  16. Antigen-targeting strategies using single-domain antibody fragments

    NARCIS (Netherlands)

    Duarte, Joao Nuno Silva

    2017-01-01

    Antibodies display high selectivity and affinity and have been the preferred platform for antigen targeting. Despite the development of antigen-delivery systems that enable T cell activation, targeting approaches that enhance antibody responses need improvement. This need specially applies to poorly

  17. Molecular aspects of antibody-antigen interactions : size reduction of a herpes simplex virus neutralizing antibody and its antigen

    NARCIS (Netherlands)

    Schellekens, Gerardus Antonius

    1996-01-01

    Antibody molecules, produced as a response against foreign substances, interact with their antigen in a very specific manner. Antibodies with a predetermined specificity (monoclonal antibodies) can be produced and are widely used in medicine and science as indicator molecules. Genetic engineering of

  18. A radioimmunoassay for human antibody specific for microbial antigens

    International Nuclear Information System (INIS)

    Tew, J.G.; Burmeister, J.; Greene, E.J.; Pflaumer, S.K.; Goldstein, J.

    1977-01-01

    A simple and sensitive method for detecting and quantitating antibody specific or microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and -log 2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens

  19. Neuronal surface antigen antibodies in limbic encephalitis

    Science.gov (United States)

    Graus, F; Saiz, A; Lai, M; Bruna, J; López, F; Sabater, L; Blanco, Y; Rey, M J.; Ribalta, T; Dalmau, J

    2008-01-01

    Objective: To report the frequency and type of antibodies against neuronal surface antigens (NSA-ab) in limbic encephalitis (LE). Methods: Analysis of clinical features, neuropathologic findings, and detection of NSA-ab using immunochemistry on rat tissue and neuronal cultures in a series of 45 patients with paraneoplastic (23) or idiopathic (22) LE. Results: NSA-ab were identified in 29 patients (64%; 12 paraneoplastic, 17 idiopathic). Thirteen patients had voltage-gated potassium channels (VGKC)-ab, 11 novel NSA (nNSA)-ab, and 5 NMDA receptor (NMDAR)-ab. nNSA-ab did not identify a common antigen and were more frequent in paraneoplastic than idiopathic LE (39% vs 9%; p = 0.03). When compared with VGKC-ab or NMDAR-ab, the nNSA associated more frequently with intraneuronal antibodies (11% vs 73%; p = 0.001). Of 12 patients (9 nNSA-ab, 2 VGKC-ab, 1 NMDAR-ab) with paraneoplastic LE and NSA-ab, concomitant intraneuronal antibodies occurred in 9 (75%). None of these 12 patients improved with immunotherapy. The autopsy of three of them showed neuronal loss, microgliosis, and cytotoxic T cell infiltrates in the hippocampus and amygdala. These findings were compatible with a T-cell mediated neuronal damage. In contrast, 13 of 17 (76%) patients with idiopathic LE and NSA-ab (8 VGKC-ab, 4 NMDAR-ab, 1 nNSA-ab) and 1 of 5 (20%) without antibodies had clinical improvement (p = 0.04). Conclusions: In paraneoplastic limbic encephalitis (LE), novel antibodies against neuronal surface antigens (nNSA-ab) occur frequently, coexist with antibodies against intracellular antigens, and these cases are refractory to immunotherapy. In idiopathic LE, the likelihood of improvement is significantly higher in patients with NSA-ab than in those without antibodies. GLOSSARY GAD = glutamic acid decarboxylase; LE = limbic encephalitis; NMDAR = N-methyl-D-aspartate receptor; NSA = neuronal surface antigens; nNSA = novel NSA; SCLC = small-cell lung cancer; VGKC = voltage-gated potassium channels

  20. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

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    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  1. Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

    Science.gov (United States)

    Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

    2016-02-01

    We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, pantibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. [Limbic encephalitis with antibodies against intracellular antigens].

    Science.gov (United States)

    Morita, Akihiko; Kamei, Satoshi

    2010-04-01

    Limbic encephalitis is a paraneoplastic syndrome that is often associated with small cell lung cancer (SCLC), breast cancer, testicular tumors, teratoma, Hodgkin's lymphoma and thymoma. The common clinical manifestations of limbic encephalitis are subacute onset, cognitive dysfunction, seizures and psychiatric symptoms. Paraneoplastic neurological disorders are considered to occur because of cytotoxic T cell responses and antibodies against target neuronal proteins that are usually expressed by an underlying tumor. The main intracellular antigens related to limbic encephalitis are Hu, Ma2, and less frequently CV2/CRMP5 and amphiphysin. The anti-Hu antibody, which is involved in cerebellar degeneration and extensive or multifocal encephalomyelitis such as limbic encephalitis is closely associated with a history of smoking and SCLC. The anti-Ma2 antibody is associated with encephalitis of the limbic system, hypothalamus and brain-stem. For this reason, some patients with limbic encephalitis have sleep disorders (including REM sleep abnormalities), severe hypokinesis and gaze palsy in addition to limbic dysfunction. In men aged less than 50 years, anti-Ma2 antibody encephalitis is almost always associated with testicular germ-cell tumors that are occasionally difficult to detect. In older men and women, the most common tumors are non-SCLC and breast cancer. Limbic encephalitis associated with cell-surface antigens (e.g., voltage-gated potassium channels, NMDA receptors) is mediated by antibodies and often improves after a reduction in the antibody titer and after tumor resection. Patients with antibodies against intracellular antigens, except for those with anti-Ma2 antibodies and testicular tumors, are less responsive. Early diagnosis and treatment with immunotherapy, tumor resection or both are important for improving or stabilizing the condition of limbic encephalitis.

  3. Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

    Science.gov (United States)

    Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

    2018-05-01

    The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

  4. Proposed docking interface between peptidoglycan and the target recognition domain of zoocin A

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yinghua [Department of Chemistry, University of Alabama, Tuscaloosa, AL 35487 (United States); Simmonds, Robin S. [Department of Microbiology and Immunology, University of Otago, Dunedin (New Zealand); Timkovich, Russell, E-mail: rtimkovi@bama.ua.edu [Department of Chemistry, University of Alabama, Tuscaloosa, AL 35487 (United States)

    2013-11-15

    Highlights: •Peptidoglycan added to zoocin rTRD perturbs NMR resonances around W115. •Simulations predict docking to a shallow surface groove near W115. •The docking interface is similar to mammalian antibody–antigen sites. •EDTA binds to a distinct surface site. -- Abstract: A docking model is proposed for the target recognition domain of the lytic exoenzyme zoocin A with the peptidoglycan on the outer cell surface of sensitive bacterial strains. Solubilized fragments from such peptidoglycans perturb specific backbone and side chain amide resonances in the recombinant form of the domain designated rTRD as detected in two-dimensional {sup 1}H–{sup 15}N correlation NMR spectra. The affected residues comprise a shallow surface cleft on the protein surface near W115, N53, N117, and Q105 among others, which interacts with the peptide portion of the peptidoglycan. Calculations with AutoDock Vina provide models of the docking interface. There is approximate homology between the rTDR-peptidoglycan docking site and the antigen binding site of Fab antibodies with the immunoglobin fold. EDTA was also found to bind to rTRD, but at a site distinct from the proposed peptidoglycan docking site.

  5. Antigen-antibody reactions of UV-irradiated phage DNA

    International Nuclear Information System (INIS)

    Fink, A.

    1976-01-01

    The observation of others could be confirmed that UV-irradiated DNA is a better immunogen than unirradiated DNA. The author's immune sera contained a high amount of antibodies with a specific action against photoproducts in the DNA. The thymine dimer was identified as relevant photoproduct and thus as antigenic determinant. In comparison, the amount of unspecific antibodies reacting with denaturated DNA was low and varied between sera. Thymin-dimer antibodies showed a high specificity without cross-reaction with other pyrimidine dimers such as anti CC and anti CT; they belong to the class of IgG molecules. UV-irradiated dinucleotide dTpT is sufficient to induce the formation of antibodies reacting with the cis-syn thymine dimers in UV-irradiated DNA. Antibody binding is proportional to the UV doses applied to the DNA. When using completely denaturated DNA, there is a linear increase changing into a plateau at higher doses. The extent of antigen-antibody binding is strongly dependent on the degree of denaturation of the DNA. With increasing denaturation, the antibody binding of the DNA increases. The antigen-antibody reaction can thus be used to estimate the degree of denaturation of the DNA. There were no signs of an influence of the degree of denaturation of the DNA on the quantum yield of thymine dimers. The different amounts of antibodies is therefore due to the masking of thymine dimers in native DNA. When irradiating intact phage particles, there was no sign of an influence of the phages' protein covers on the antibody binding capacity of DNA compared with DNA irradiated in vitro. (orig.) [de

  6. Development of recombinant antigen array for simultaneous detection of viral antibodies.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV, Rubella virus (RV core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs. The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

  7. Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen

    Directory of Open Access Journals (Sweden)

    Anita Verma

    2018-02-01

    Full Text Available Toxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA, the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.

  8. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

    Directory of Open Access Journals (Sweden)

    Martin Kreutz

    Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

  9. Potential radioimmunoassay system for detection of Hanganutziu-Deicher type heterophile antigen(s) and antibodies in tissues and fluids

    Energy Technology Data Exchange (ETDEWEB)

    Mukuria, J C; Naiki, Masaharu; Hashimoto, Masato; Nishiura, Katsumi; Okabe, Masahiro; Kato, Shiro

    1985-06-12

    A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The SVI-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. Different H-D antigen-active molecules were compared for heterophile H-D antigen potency. Eight different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attmept to correlate expression of H-D antigen on tissues with elevation of H-D antibodies.

  10. Mother-Newborn Pairs in Malawi Have Similar Antibody Repertoires to Diverse Malaria Antigens.

    Science.gov (United States)

    Boudová, Sarah; Walldorf, Jenny A; Bailey, Jason A; Divala, Titus; Mungwira, Randy; Mawindo, Patricia; Pablo, Jozelyn; Jasinskas, Algis; Nakajima, Rie; Ouattara, Amed; Adams, Matthew; Felgner, Philip L; Plowe, Christopher V; Travassos, Mark A; Laufer, Miriam K

    2017-10-01

    Maternal antibodies may play a role in protecting newborns against malaria disease. Plasmodium falciparum parasite surface antigens are diverse, and protection from infection requires allele-specific immunity. Although malaria-specific antibodies have been shown to cross the placenta, the extent to which antibodies that respond to the full repertoire of diverse antigens are transferred from the mother to the infant has not been explored. Understanding the breadth of maternal antibody responses and to what extent these antibodies are transferred to the child can inform vaccine design and evaluation. We probed plasma from cord blood and serum from mothers at delivery using a customized protein microarray that included variants of malaria vaccine target antigens to assess the intensity and breadth of seroreactivity to three malaria vaccine candidate antigens in mother-newborn pairs in Malawi. Among the 33 paired specimens that were assessed, mothers and newborns had similar intensity and repertoire of seroreactivity. Maternal antibody levels against vaccine candidate antigens were the strongest predictors of infant antibody levels. Placental malaria did not significantly impair transplacental antibody transfer. However, mothers with placental malaria had significantly higher antibody levels against these blood-stage antigens than mothers without placental malaria. The repertoire and levels of infant antibodies against a wide range of malaria vaccine candidate antigen variants closely mirror maternal levels in breadth and magnitude regardless of evidence of placental malaria. Vaccinating mothers with an effective malaria vaccine during pregnancy may induce high and potentially protective antibody repertoires in newborns. Copyright © 2017 American Society for Microbiology.

  11. Rapid production of antigen-specific monoclonal antibodies from a variety of animals

    Directory of Open Access Journals (Sweden)

    Kurosawa Nobuyuki

    2012-09-01

    Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

  12. I-125 input into antibodies molecules specific to australian antigen

    International Nuclear Information System (INIS)

    Abdukayumov, A. M.; Chistyakov, P.G.; Garajshina, G. R.

    1999-01-01

    There are experimental data on I-125 input into antibodies molecules specific to superficial antigen of hepatitis B virus (australian antigen). Three ways of input are submitted: with the help of T chloramine usage, Bolton-Hunter Reagent and with the help of iodogen. There are also comparative characteristics of iodized products obtained: molar radioactivity, radiochemical frequency, immuno - reactivity. The report also discusses advantages and disadvantages of the used methods for inputting I-125 into antibodies to australian antigen in order to study the possibility of creating radio immunological test system for detecting superficial antigen of B hepatitis

  13. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  14. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    International Nuclear Information System (INIS)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-01-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae

  15. Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.

    Science.gov (United States)

    Tsaltas, G; Ford, C H

    1993-02-01

    Methods following the process of binding and internalization of antibodies to cell surface antigens have often employed low pH isoosmolar buffers in order to dissociate surface antigen-antibody complexes. One of the most widely used buffers is a 0.05 M glycine-HCL buffer pH 2.8. Since the efficacy of action of this buffer was critical to a series of internalization experiments employing monoclonal antibodies (Mabs) to carcinoembryonic antigen (CEA) expressing cancer cell lines in this laboratory, we tested its performance in a number of different assays. Our results indicate that this buffer only partially dissociates antigen-antibody bonds and therefore can introduce major inaccuracies in internalization experiments.

  16. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  17. Monoclonal antibodies to carcino-embryonic antigen

    International Nuclear Information System (INIS)

    Teh, Jinghee; McKenzie, I.F.C.

    1990-01-01

    With the aim of producing new MoAb to colorectal carcinoma, immunization with cell suspensions of a fresh colonic tumour was performed and MoAb 17C4 was obtained. To produce other MoAb to colon cancer, an immunization protocol using fresh tumour, colonic cell lines and sera from patients with colonic tumours was employed and resulted in MoAb JGT-13, LK-4 and XPX-13. MoAb I-1 and O-1 were raised against sera from patients with colon cancer to produce MoAb directed against circulating tumour associated antigens. The six antibodies gave a range of reactions with normal and malignant tissues, indicating that they most likely reacted with different epitopes. Thus, apart from the reactions of 17C4, LK-4 and XPX-13 with fresh and formalin-fixed granulocytes, none of the antibodies reacted with formalin-fixed normal tissues. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to carcino-embryonic antigen polyclonal antibodies, that is the MoAb gave no obvious advantage. 9 refs., 1 tab., 3 figs

  18. Abnormal antigens in breast cancer tissues and production of monoclonal antibodies against one of these antigens

    Energy Technology Data Exchange (ETDEWEB)

    Mohammed, M E. A. [University of Khartoum, Khartoum (Sudan)

    2010-02-15

    Breast cancer is associated with up regulation, down regulation of normal antigens or abnormal antigens. These antigens are very useful candidates as targets for the different breast cancer therapies and for vaccination trials. This study was done to characterize abnormal antigens, extract one of them and to produce monoclonal antibodies against the extracted antigen. One hundred and twenty Sudanese female patients were included in this study after informed consent. The mean age was 47. 2 years (16-80). Two tissue samples were obtained from each patient and they were confirmed as normal and cancerous breast tissues microscopically. 2D PAGE was used to analyze the protein content of samples. LC/MS and nr. fast a database search were used for separation and indentification of the abnormal proteins. Three different patterns of 2D Page results were obtained, the first pattern involved detection of four abnormal proteins in 26.7% of the patient cancerous tissues while they were undetected in the normal tissues of the same patients. In the second 2D PAGE result pattern the cancerous and the normal tissues of 67.5% patients were identical and they did not contain the four abnormal proteins while the third 2D PAGE pattern involved the presence of two abnormal antigens (from the four) in the cancerous tissues of 5.8% of the patients and they were absent from the normal tissues of the same patients. The four abnormal proteins were identified as, human Thioredoxin (D60nmutant), x-ray crystal structure of human galectin-1, retrocopy of tropomyosin 3(rc TPM3) and beta-tropomyosin (isoform 2). The primary and the secondary structures were obtained from the SWISSPROT and the PDB databases. Beta tropomyosin spot was extracted and used as antigen for monoclonal antibody production. Monoclonal antibody against beta- tropomyosin with a concentration of 0.35 mg/ml and a G11 anti beta-tropomyosin hybridoma cell line were produced. The monoclonal antibody was with single bad and

  19. Abnormal antigens in breast cancer tissues and production of monoclonal antibodies against one of these antigens

    International Nuclear Information System (INIS)

    Mohammed, M. E. A.

    2010-02-01

    Breast cancer is associated with up regulation, down regulation of normal antigens or abnormal antigens. These antigens are very useful candidates as targets for the different breast cancer therapies and for vaccination trials. This study was done to characterize abnormal antigens, extract one of them and to produce monoclonal antibodies against the extracted antigen. One hundred and twenty Sudanese female patients were included in this study after informed consent. The mean age was 47. 2 years (16-80). Two tissue samples were obtained from each patient and they were confirmed as normal and cancerous breast tissues microscopically. 2D PAGE was used to analyze the protein content of samples. LC/MS and nr. fast a database search were used for separation and indentification of the abnormal proteins. Three different patterns of 2D Page results were obtained, the first pattern involved detection of four abnormal proteins in 26.7% of the patient cancerous tissues while they were undetected in the normal tissues of the same patients. In the second 2D PAGE result pattern the cancerous and the normal tissues of 67.5% patients were identical and they did not contain the four abnormal proteins while the third 2D PAGE pattern involved the presence of two abnormal antigens (from the four) in the cancerous tissues of 5.8% of the patients and they were absent from the normal tissues of the same patients. The four abnormal proteins were identified as, human Thioredoxin (D60nmutant), x-ray crystal structure of human galectin-1, retrocopy of tropomyosin 3(rc TPM3) and beta-tropomyosin (isoform 2). The primary and the secondary structures were obtained from the SWISSPROT and the PDB databases. Beta tropomyosin spot was extracted and used as antigen for monoclonal antibody production. Monoclonal antibody against beta- tropomyosin with a concentration of 0.35 mg/ml and a G11 anti beta-tropomyosin hybridoma cell line were produced. The monoclonal antibody was with single bad and

  20. Comparative efficacy of antigen and antibody detection tests for human trichinellosis

    International Nuclear Information System (INIS)

    Ivanoska, D.; Cuperlovic, K.; Gamble, H.R.; Murrell, K.D.

    1989-01-01

    Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation

  1. Monoclonal antibody against a serotype antigen of Porphyromonas (Bacteroides) endodontalis and characteristics of the antigen.

    Science.gov (United States)

    Hanazawa, S; Sagiya, T; Amano, S; Nishikawa, H; Kitano, S

    1990-01-01

    Recent studies have demonstrated the presence of three serotypes (O1K1, O1K2, and O1K-) of Porphyromonas (Bacteroides) endodontalis. In the present study, a hybridoma cell line producing monoclonal antibody (BEE11) specific for serotype O1K1 of P. endodontalis was established. The specificity of the antibody was evaluated by enzyme-linked immunosorbent assay and immunoslot blot analysis. BEE11 antibody reacted with strains ATCC 35406, HG 400, and HG 421 of the bacterium. However, it did not react with HG 422 or HG 948. Also, the antibody did not react with any of the black-pigmented Bacteroides strains tested. Although the antibody reacted with total cell envelope and capsule materials, it did not do so with lipopolysaccharide. The antibody reacted with antigen material having a molecular mass of 110 kilodaltons (kDa), as judged from fractionation by Superose 12 prep gel chromatography. When the peak fraction from the Superose 12 column was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, the reactivity was detected as a single band at an apparent molecular mass of about 52 kDa. The antigen material purified partially by high-performance liquid chromatography was sensitive to trypsin, V8 protease, and heating to 80 degrees C but not to neuraminidase. Therefore, the present study shows that BEE11 antibody recognizes a serotype antigen of P. endodontalis which may be a dimer consisting of monomers having molecular masses of approximately 52 kDa and sensitivity to proteases and heat. Images PMID:2370106

  2. A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

    Directory of Open Access Journals (Sweden)

    Yuan-Cheng Cao

    2015-01-01

    Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

  3. Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

    Science.gov (United States)

    Gunasekaran, Manojkumar; Chatterjee, Prodyot K.; Shih, Andrew; Imperato, Gavin H.; Addorisio, Meghan; Kumar, Gopal; Lee, Annette; Graf, John F.; Meyer, Dan; Marino, Michael; Puleo, Christopher; Ashe, Jeffrey; Cox, Maureen A.; Mak, Tak W.; Bouton, Chad; Sherry, Barbara; Diamond, Betty; Andersson, Ulf; Coleman, Thomas R.; Metz, Christine N.; Tracey, Kevin J.; Chavan, Sangeeta S.

    2018-01-01

    The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses. PMID:29755449

  4. Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

    Directory of Open Access Journals (Sweden)

    Manojkumar Gunasekaran

    2018-04-01

    Full Text Available The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR, dorsal root ganglion (DRG sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM into wild-type (WT mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

  5. Antigen spot test (AST): a highly sensitive assay for the detection of antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Herbrink, P; van Bussel, F J; Warnaar, S O [Rijksuniversiteit Leiden (Netherlands)

    1982-02-12

    A method is described for detection of antibodies by means of nitrocellulose or diazobenzyloxymethyl (DBM) paper on which various antigens have been spotted. The sensitivity of this antigen spot test (AST) is comparable with that of RIA and ELISA. The method requires only nanogram amounts of antigen. Since a variety of antigens can be spotted on a single piece of nitrocellulose or DBM paper, this antigen spot test is especially useful for specificity controls on antibodies.

  6. Simultaneous detection of Hepatitis B surface antigen and its antibody by radioimmunoassay

    International Nuclear Information System (INIS)

    Crouzat-Reynes, Gerard; Perigois, Francois; Lecureuil, Michel; Lejeune, Bernard

    1981-01-01

    The authors describe an original radioimmunoassay which allows the simultaneous detection of hepatitis B surface antigen and its antibody in a biological sample. Antigen and antibody are indiscriminately detected in a first step and then distinguished in a second step using the same reagents [fr

  7. A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

    International Nuclear Information System (INIS)

    Goodall, A.H.; Meek, F.L.; Waters, J.A.; Miescher, G.C.; Janossy, G.; Thomas, H.C.

    1982-01-01

    A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available. (Auth.)

  8. Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

    International Nuclear Information System (INIS)

    Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

    1983-01-01

    A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

  9. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

  10. Detection of autoimmune antibodies in localized scleroderma by synthetic oligonucleotide antigens

    DEFF Research Database (Denmark)

    Samuelsen, Simone; Jørgensen, Christian Damsgaard; Mellins, Elizabeth D

    2018-01-01

    In this study, we developed a series of synthetic oligonucleotides that allowed us to investigate the details on the antigen recognition by autoimmune antibodies in localized scleroderma subjects. Besides dramatically improved analytical specificity of the assay, our data suggests a potential...... linking for antibodies to DNA to the biological status of disease state in localized scleroderma. Moreover, introducing chemical modifications into short synthetic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules completely changed the binding titers of corresponding antibodies...... and their clinical relevance. The strongest observed effect was registered for the localized scleroderma skin damage index (LoSDI) on the IgG antibodies to TC dinucleotide-rich double-stranded antigen (p

  11. Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

    OpenAIRE

    Jiménez, T; Díaz, A M; Zlotnik, H

    1990-01-01

    Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Myco...

  12. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    DEFF Research Database (Denmark)

    Samuelsen, Simone V; Solov'yov, Ilia A.; Balboni, Imelda M.

    2016-01-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing...... molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best...... that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies....

  13. Immunoradiometric assay for quantification of serum antibodies to dental plaque antigen in immunized dogs

    International Nuclear Information System (INIS)

    Ahlstedt, S.; Rylander, H.

    1975-01-01

    An immunoradiometric assay (IRMA) was used for quantifying dog serum antibodies to antigens from dental plaque collected from full-grown dogs. The antigens were adsorbed onto the inner surface of plastic tubes and then incubated with dog-anti-plaque serum, 125 I-labelled anti-dog plasma-immunoglobulin was used for quantification of the specific antibodies. Four 10 months old Beagle dogs in excellent gingival health were immunized for 10 weeks with ultrasonicated dog dental plaque. The antibody levels in antisera sampled 6, 8, 10 and 11 weeks after the first antigen injection were 2 to 5 times as high as those recorded before the immunizing period. The variability of the assay as judged from the difference between duplicate samples was found to be 18 percent+-4 (p<0.01) of the mean value and the variability between the same serum ran on different test occasions 13 percent+-7 (p<0.01). The specificity of the antigen-antibody reaction in the immuno assay was tested by inhibition experiments. Preincubation of the antisera with dental plaque antigen significantly inhibited the antigen-antibody reaction in the IRMA, while bovine serum albumin did not. (author)

  14. Immunoradiometric assay for quantification of serum antibodies to dental plaque antigen in immunized dogs

    Energy Technology Data Exchange (ETDEWEB)

    Ahlstedt, S; Rylander, H [Goeteborg Univ. (Sweden)

    1975-01-01

    An immunoradiometric assay (IRMA) was used for quantifying dog serum antibodies to antigens from dental plaque collected from full-grown dogs. The antigens were adsorbed onto the inner surface of plastic tubes and then incubated with dog-anti-plaque serum, /sup 125/I-labelled anti-dog plasma-immunoglobulin was used for quantification of the specific antibodies. Four 10 months old Beagle dogs in excellent gingival health were immunized for 10 weeks with ultrasonicated dog dental plaque. The antibody levels in antisera sampled 6, 8, 10 and 11 weeks after the first antigen injection were 2 to 5 times as high as those recorded before the immunizing period. The variability of the assay as judged from the difference between duplicate samples was found to be 18 percent+-4 (p<0.01) of the mean value and the variability between the same serum ran on different test occasions 13 percent+-7 (p<0.01). The specificity of the antigen-antibody reaction in the immuno assay was tested by inhibition experiments. Preincubation of the antisera with dental plaque antigen significantly inhibited the antigen-antibody reaction in the IRMA, while bovine serum albumin did not.

  15. Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

    Science.gov (United States)

    Liu, Shuangshuang; Li, Shasha; Zhang, Yang; Wang, Ye; Zhu, Yumeng; Wang, Bin; Chen, Zhi-Nan

    2017-01-01

    The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane-spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen-immunoaffinity chromatography purification. The purity of rabbit anti-CD147 polyclonal antibodies was ~99%, and ELISA analysis was able to determine the titer of the rabbit anti-CD147 polyclonal antibodies at 1:512,000. The lowest concentration of the standard CD147 antigen that the sandwich ELISA was able to detect was 31.25 pg/ml. The sandwich ELISA system was composed of anti-hepatoma HAb18 monoclonal antibodies and purified rabbit anti-CD147 polyclonal antibodies. The present study demonstrated that antigen-immunoaffinity chromatography may be a good technique for the purification of polyclonal antibodies, which may be used to detect antigen in sandwich ELISAs. PMID:28487989

  16. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    KAUST Repository

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; D'Aliberti, Deborah; Venza, Mario; Borgogni, Erica; Castellino, Flora; Biondo, Carmelo; D'Andrea, Daniel; Grassi, Luigi; Tramontano, Anna; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2014-01-01

    There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  17. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    Directory of Open Access Journals (Sweden)

    Maria Domina

    Full Text Available There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  18. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    KAUST Repository

    Domina, Maria

    2014-12-04

    There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  19. Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

    NARCIS (Netherlands)

    Dolk, E.; Vliet, C. van; Perez, J.M.J.; Vriend, G.; Darbon, H.; Ferrat, G.; Cambillau, C.; Frenken, L.G.J.; Verrips, T.

    2005-01-01

    In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains

  20. Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability

    Science.gov (United States)

    Schmidt, Michael M.; Thurber, Greg M.

    2010-01-01

    Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids. PMID:18408925

  1. Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies

    International Nuclear Information System (INIS)

    Imai, M.; Nomura, M.; Gotanda, T.; Sano, T.; Tachibana, K.; Miyamoto, H.; Takahashi, K.; Toyama, S.; Miyakawa, Y.; Mayumi, M.

    1982-01-01

    Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants

  2. Preparation and Purification of Polyclonal Antibodies against Mycobacterium Avium Paratuberculosis Antigens in Rabbit

    Directory of Open Access Journals (Sweden)

    Hafezeh Alizadeh

    2012-12-01

    Full Text Available Background and Objective: Johne’s disease is the chronic granulomatous enteritis of ruminants, and a major health hazard worldwide. In recent years, researchers have focused on mycobacterium avium subsp. paratuberculosis (MAP antigens in diagnostic tests. Identification of antibodies against MAP antigens is, therefore, effective for the diagnosis or preparation of vaccine. The aim of this study was to prepare and purify polyclonal antibodies against MAP antigens. Materials and Methods: A New Zealand white rabbit was immunized at a certain time period with MAP antigens and Freund’s adjuvant. After the immunization of the animal, the rabbit was bled to obtain enriched serum. Immunoglobulins were obtained via sedimentation with ammonium sulfate 35% and then IgG was purified by ion exchange (DEAE-cellulose chromatography. Serologic test was used to evaluate the interaction of antigens and antibodies. Results: Ion exchange chromatography of IgG showed one peak, and SDS_PAGE of IgG showed a single band. Serologic test was applied and clear precipitation lines were appeared up to 1:16 dilution, which indicated the high quality of the product. Conclusion: In this study, the humoral immune response was induced well by immunization with MAP antigens in a New Zealand white rabbit and polyclonal antibodies were produced in high titers. Polyclonal antibodies are relatively inexpensive and easy to produce in large quantities and can connect to the more connective sites, resulting in better sensitivity. Identification of polyclonal antibodies via immunological tests can play a significant role in studying MAP disorders.

  3. Peripheral neuropathies associated with antibodies directed to intracellular neural antigens.

    Science.gov (United States)

    Antoine, J-C

    2014-10-01

    Antibodies directed to intracellular neural antigens have been mainly described in paraneoplastic peripheral neuropathies and mostly includes anti-Hu and anti-CV2/CRMP5 antibodies. These antibodies occur with different patterns of neuropathy. With anti-Hu antibody, the most frequent manifestation is sensory neuronopathy with frequent autonomic involvement. With anti-CV2/CRMP5 the neuropathy is more frequently sensory and motor with an axonal or mixed demyelinating and axonal electrophysiological pattern. The clinical pattern of these neuropathies is in keeping with the cellular distribution of HuD and CRMP5 in the peripheral nervous system. Although present in high titer, these antibodies are probably not directly responsible for the neuropathy. Pathological and experimental studies indicate that cytotoxic T-cells are probably the main effectors of the immune response. These disorders contrast with those in which antibodies recognize a cell surface antigen and are probably responsible for the disease. The neuronal cell death and axonal degeneration which result from T-cell mediated immunity explains why treating these disorders remains challenging. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  4. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...... the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice...... ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity...

  5. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    International Nuclear Information System (INIS)

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-01-01

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex

  6. Prevalence of Hepatitis B Antigen/antibody in Patients of Syphilis

    Directory of Open Access Journals (Sweden)

    B N Joshi

    1980-01-01

    Full Text Available In some cases of Hepatitis B antigen positive hepatitis, a history of previous blood transfusion or any parenteral therapy is lacking and evidence for other routes of infections have to be sought. Sexual contact has been suggested as one of the methods of transmission of this infection. To approach the problem from this angle we studied 480 serawhich werepositive for syphilis serology for the presence of HB antigen and antibody by discontinuous counter immune electrophoresis method. It was found to be prevalent to the extentof 5.Z-per centagainst 1.4 per cent found in voluntary blood donors. Our observation agrees with that of other workers that HB antigen/antibody is seen more frequently in patients with positive syphilis serol-ogy.

  7. A radioimmunoassay to screen for antibodies to native conformational antigens and analyse ligand-induced structural states of antigenic proteins

    International Nuclear Information System (INIS)

    Bernotat-Danielowski, S.; Koepsell, H.

    1988-01-01

    A radioimmunoassay is described in which antigenic protein was immobilized by incubating nitrocellulose filters of defined diameter with antigen-containing solutions. Antigenic sites which are sensitive to protein denaturation by drying could be detected with the assay. The assay was also used to screen hybridoma supernatants for antibodies directed against Na + cotransport proteins from renal brush-border membranes. Monoclonal antibodies were selected which showed different binding charactertics depending on whether or not substrates of Na + cotransporters were present. One of the antibodies, which showed different antibody binding after addition of D-glucose or L-lactate, bound to a polypeptide component of the renal N + -D-glucose cotransporter and was able to inhibit Na + gradient-dependent. To investigate the effects of D-glucose and L-lactate on the binding of this antibody concentration dependence was measured. High and low affinity binding sites for D-glucose and L-lactate were characterized thereby demonstrating that the radioimmunoassay permits investigations of the properties of high and low affinity substrate binding sites. (author). refs.; 6 figs.; 2 tabs

  8. Carbamylated albumin is one of the target antigens of anti-carbamylated protein antibodies.

    Science.gov (United States)

    Nakabo, Shuichiro; Hashimoto, Motomu; Ito, Shinji; Furu, Moritoshi; Ito, Hiromu; Fujii, Takao; Yoshifuji, Hajime; Imura, Yoshitaka; Nakashima, Ran; Murakami, Kosaku; Kuramoto, Nobuo; Tanaka, Masao; Satoh, Junko; Ishigami, Akihito; Morita, Satoshi; Mimori, Tsuneyo; Ohmura, Koichiro

    2017-07-01

    Anti-carbamylated protein (anti-CarP) antibodies are detected in RA patients. Fetal calf serum is used as an antigen source in anti-CarP ELISA, and the precise target antigens have not been found. We aimed to identify the target antigens of anti-CarP antibodies. Western blotting of anti-CarP antibodies was conducted. Anti-carbamylated human albumin (CarALB) antibody was detected by in-house ELISA for 493 RA patients and 144 healthy controls (HCs). An inhibition ELISA of anti-CarP antibodies by CarALB and citrullinated albumin (citALB) was performed using eight RA patients' sera. Serum CarALB was detected by liquid chromatography-tandem mass spectroscopy (LC/MS/MS), and the serum MPO concentration was measured by ELISA. We focused on carbamylated albumin because it corresponded to the size of the thickest band detected by western blotting of anti-CarP antibodies. Anti-CarALB antibody was detected in 31.4% of RA patients, and the correlation of the titres between anti-CarALB and anti-CarP was much closer than that between anti-citALB and anti-CCP antibodies (ρ = 0.59 and ρ = 0.16, respectively). The inhibition ELISA showed that anti-CarP antibodies were inhibited by CarALB, but not by citALB. CarALB was detected in sera from RA patients by LC/MS/MS. The serum MPO concentration was correlated with disease activity and was higher in RA patients with anti-CarALB antibody than in those without. We found that carbamylated albumin is a novel target antigen of anti-CarP antibodies, and it is the first reported target antigen that has not been reported as the target of ACPA. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  9. A simplified radioimmunoassay for triiodothyronine (T3) using pre-incubated labelled antigen and antibody

    International Nuclear Information System (INIS)

    Pillai, M.R.A.; Nagvekar, U.H.; Desai, C.N.; Mani, R.S.

    1986-01-01

    The development of a simplified radioimmunoassay for triiodothyronine (T 3 ) using pre-incubated labelled T 3 and antibody is described. The assay is carried out by adding 50 μl of standard or sample to 0.4 ml of pre-incubated reagent dispensed in assay tubes. The reaction was allowed to proceed for about four hours and the antigen-antibody complex precipitated by the addition of 1 cm 3 of 22% polyethylene glycol solution. Due to the high dissociation constant of T 3 -antibody complex at 37 deg C, the labelled antigen-antibody complex dissociates and thereby the unlabelled antigen binds with the antibody. The sensitivity of this assay is comparable to an assay done by the equilibrium method using the same antibody. Sixty serum samples were analyzed using this method and were compared with the equilibrium assay. (author)

  10. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

    Directory of Open Access Journals (Sweden)

    Ondigo Bartholomew N

    2012-12-01

    Full Text Available Abstract Background Multiplex cytometric bead assay (CBA have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Methods Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Results Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from Conclusion With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

  11. Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection.

    Science.gov (United States)

    Vitozzi, Susana; Lapierre, Pascal; Djilali-Saiah, Idriss; Marceau, Gabriel; Beland, Kathie; Alvarez, Fernando

    2004-05-01

    Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.

  12. Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

    Science.gov (United States)

    Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

    2018-02-01

    Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

  13. Antibodies to early EBV, CMV, and HHV6 antigens in systemic lupus erythematosus patients

    DEFF Research Database (Denmark)

    Rasmussen, N S; Draborg, A H; Nielsen, C T

    2015-01-01

    OBJECTIVES: We investigated the antibody levels against early antigens of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV6) in systemic lupus erythematosus (SLE) patients and healthy controls, and further correlated these antibodies to haematology/biochemistry, serol......OBJECTIVES: We investigated the antibody levels against early antigens of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV6) in systemic lupus erythematosus (SLE) patients and healthy controls, and further correlated these antibodies to haematology...

  14. Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. I. Measurement of serum antibodies by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Tsang, R S.W.; Chau, P Y; Lam, S K [Hong Kong Univ.; La Brooy, J T; Rowley, D [Adelaide Univ. (Australia)

    1981-12-01

    Serum antibody responses to the lipopolysaccharide and protein antigens of S. typhi in typhoid patients were studied using a solid-phase radioimmunoassay technique with /sup 125/I labelled anti-immunoglobulin antibody. Sera from 24 adult typhoid patients and 20 non-typhoid adult controls were compared. As a group, sera from typhoid patients showed increased IgA, IgG and IgM immunoglobulin levels and gave significantly higher anti-LPS and anti-protein antibody titres in all three major immunoglobulin classes than did non-typhoid controls. Levels of antibodies against LPS or protein in sera of typhoid patients were highly variable with a skew distribution. A good correlation was found between antibody titres to the LPS antigen and those to a protein antigen. No correlation, however, was found between the anti-LPS antibody titres measured by radioimmunoassay and the anti-O antibody titres measured by the Widal agglutination test. Titration of anti-LPS or anti-protein antibodies by radioimmunoassay was found to be more sensitive and specific than Widal test for the serological diagnosis of typhoid fever. The advantages of measuring antibody response by radioimmunoassay over conventional Widal test are discussed.

  15. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

    Science.gov (United States)

    Ondigo, Bartholomew N; Park, Gregory S; Gose, Severin O; Ho, Benjamin M; Ochola, Lyticia A; Ayodo, George O; Ofulla, Ayub V; John, Chandy C

    2012-12-21

    Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in

  16. Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding

    KAUST Repository

    Di Palma, Francesco; Tramontano, Anna

    2017-01-01

    The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody–antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation.

  17. Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding

    KAUST Repository

    Di Palma, Francesco

    2017-08-03

    The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody–antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation.

  18. Trial of using antibodies as carriers of alkylating agents. Pt. 2. Evaluation of ability to form /sup 32/P-cyclophosphamide + immune antibody complexes with homologous antigen

    Energy Technology Data Exchange (ETDEWEB)

    Trzeciak, J; Felus, E; Nolewajka, E; Szaflarski, J; Dudziak, Z [Slaska Akademia Medyczna, Katowice (Poland)

    1976-01-01

    /sup 32/P-cyclophosphamide was found to combine with ..gamma..-globulin fractions of immune sera. Immune sera incubated with /sup 32/P-cyclophosphamide retained ability to react specifically with homologou antigen in vitro in the system: MN antigens of human erythrocytes + rabbit anti-MN antibody, and probably reacted selectively with target antigens in vivo in the system: antigens of guinea pig kidney tissue + rabbit antibodies against these antigens. Hemagglutination, passive hemagglutination and precipitation in agar gel tests were used in the experiments. Ability to combine of the immune antibody + /sup 32/P-cyclophosphamide complex with homologous antigens was evaluated by measurements of radioactivity of studied materials (erythrocyte agglutinates and organ homogenates). The results indicate feasibility of using immune antibodies as carriers of cytostatic agents.

  19. Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection

    International Nuclear Information System (INIS)

    Tsang, R.S.W.; Chau, P.Y.; Lam, S.K.

    1981-01-01

    Serum antibody responses to the lipopolysaccharide and protein antigens of S. typhi in typhoid patients were studied using a solid-phase radioimmunoassay technique with 125 I labelled anti-immunoglobulin antibody. Sera from 24 adult typhoid patients and 20 non-typhoid adult controls were compared. As a group, sera from typhoid patients showed increased IgA, IgG and IgM immunoglobulin levels and gave significantly higher anti-LPS and anti-protein antibody titres in all three major immunoglobulin classes than did non-typhoid controls. Levels of antibodies against LPS or protein in sera of typhoid patients were highly variable with a skew distribution. A good correlation was found between antibody titres to the LPS antigen and those to a protein antigen. No correlation, however, was found between the anti-LPS antibody titres measured by radioimmunoassay and the anti-O antibody titres measured by the Widal agglutination test. Titration of anti-LPS or anti-protein antibodies by radioimmunoassay was found to be more sensitive and specific than Widal test for the serological diagnosis of typhoid fever. The advantages of measuring antibody response by radioimmunoassay over conventional Widal test are discussed. (author)

  20. Human antibody responses to Schistosoma mansoni: does antigen directed, isotype restriction result in the production of blocking antibodies?

    Directory of Open Access Journals (Sweden)

    David W. Dunne

    1987-01-01

    Full Text Available After treatment young Kenyan schoolchildren are highly susceptible to reinfection with Schistosoma mansoni. Older children and adults are resistant to reinfection. There is no evidence that this age related resistance is due to a slow development of protective immunological mechanisms, rather, it appears that young children are susceptible because of the presence of blocking antibodies which decline with age, thus allowing the expression of protective responses. Correlations between antibody responses to different stages of the parasite life-cycle suggest that, in young children, antigen directed, isotype restriction of the response against cross-reactive polysaccharide egg antigens results in an ineffectual, or even blocking antibody response to the schistosomulum.

  1. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  2. Detection of HBs antigens and antibodies by immunoenzymology and radioimmunology. Comparison of the results

    International Nuclear Information System (INIS)

    Fauchet, R.; Genetet, B.; Phengsavath

    1981-01-01

    The respective sensitivity threshold of immunoenzymology and radioimmunology in HBs antigen and antibody detection were compared and the optical density (o.d.) and counts per minute (cpm) values were correlated with the concentration in ng/ml of a given sample. The sensitivity comparison between RIA and EIA appears in favour of the former technique for both HBs antigen and HBs antibody detection. However the procedure tried here to detect HBs antibodies by immunoenzymology is simple, requires only the choice of neutralising antigenic pool and sensitivity threshold and could usefully be generalized for teams not authorized to handle radioelements. Quantification of the HBs antigen content in ng/ml is a difficult operation, needing great care in the dilution of the sera tested. The reproducibility is not perfect. The semi-quantitative RIA determination expressed in R.U. (radioimmunological units) seems adequate as a mean to follow the progress of an HBs antibody in both liver disease and vaccination [fr

  3. Modeling antigen-antibody nanoparticle bioconjugates and their polymorphs

    Science.gov (United States)

    Desgranges, Caroline; Delhommelle, Jerome

    2018-03-01

    The integration of nanomaterials with biomolecules has recently led to the development of new ways of designing biosensors, and through their assembly, to new hybrid structures for novel and exciting applications. In this work, we develop a coarse-grained model for nanoparticles grafted with antibody molecules and their binding with antigens. In particular, we isolate two possible states for antigen-antibody pairs during the binding process, termed as recognition and anchoring states. Using molecular simulation, we calculate the thermodynamic and structural features of three possible crystal structures or polymorphs, the body-centered cubic, simple cubic, and face-centered cubic phases, and of the melt. This leads us to determine the domain of stability of the three solid phases. In particular, the role played by the switching process between anchoring and recognition states during melting is identified, shedding light on the complex microscopic mechanisms in these systems.

  4. Diagnosis of hypersensitivity pneumonitis by measurement of antibodies against environmental antigens

    International Nuclear Information System (INIS)

    Dewair, M.

    1989-01-01

    Hypersensitivity pneumonitis (HP), an immunologically mediated chronic pulmonary disease, is the result of an inflammatory response of the lung initiated by the inhalation of environmental organic dusts. These organic dusts usually contain substances (antigens) capable of eliciting immune responses in humans. The symptoms of HP generally present as recurrent flu-like episodes which makes it difficult to establish the proper diagnosis. However, detection in patients' sera of high-titer antibodies against the environmental antigens could be of great help in identifying those materials causing the disease and which must be avoided. A highly specific and sensitive serodiagnostic test, a radioimmuno assay (RIA), was developed for measurement of antibodies against antigens relevant to Farmer's Lung Disease (FLD), a type of HP affecting farmers

  5. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.

    Directory of Open Access Journals (Sweden)

    Takayoshi Matsuda

    Full Text Available Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR, so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

  6. ELISA with double antigen sandwich for screening specific serum anti-TP antibody in blood donors

    International Nuclear Information System (INIS)

    Wang Yiqing; Shi Zhixu

    2002-01-01

    Objective: To select a sensitive and specific laboratory examination suitable for screening serum anti-TP antibody in blood donors. Methods: The serum anti-TP antibody in 11271 blood donors were detected using ELISA with double antigen sandwich and the outcomes were compared with those using RPR assay. The conflicting specimen were confirmed by repeating the test with TPHA assay. Results: The positive rates of serum anti-TP antibody by ELISA with double antigen sandwich and RPR was 0.36% (41/11271) and 0.26% (29/11271), respectively. The coincidence of the detecting outcomes by ELISA with double antigen sandwich and RPR with TPHA was 97.5% (40/41) and 63.41%(26/41) respectively. Conclusion: Compared with RPR assay, ELISA with double antigen sandwich has higher sensibility and specificity for screening serum anti-TP antibody in blood donors

  7. Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens.

    Directory of Open Access Journals (Sweden)

    Andrew R Williams

    Full Text Available No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

  8. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies.

    Science.gov (United States)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-11-15

    A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti

    2016-04-01

    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  10. Comparison between an immunochromatographic test with an amplified ELISA for detecting e antigen and anti-e antigen antibodies in chronic Hepatitis B

    International Nuclear Information System (INIS)

    Mainet Gonzalez, Damian; Palenzuela Gardon, Daniel O; Aguilar Rubido; Julio C

    2009-01-01

    The disappearance of the e antigen and the appearance of anti-e antigen antibodies are two biomarkers that indicate favorable prognosis in Hepatitis B. In this study the Advanced QualityTM immunochromatographic test for detecting those biomarkers was compared to the Vidas semi-quantitative ELISA test. Our hypothesis was that it is possible to use these biomarkers measured in a rapid and simple Advanced QualityTM immunochromatographic test for evaluating the therapeutic response in clinical trials with chronic hepatitis B patients. The two methods were done following the manufacturer's instructions. The sera were taken from 69 patients with chronic hepatitis B of the clinical trial of the CIGB 440 therapeutic candidate. The immunochromatographic test and ELISA for detecting e antigen and anti-e antigen antibodies presented from substantial to almost perfect agreement in the evaluation of the sera of chronic Hepatitis B patients in a clinical trial. The immunochromatographic test for detecting e antigen had a low positive average agreement and a high negative average agreement compared to the ELISA. Nevertheless, the immunochromatographic test for detecting anti-e antigen antibodies had a high negative and positive average agreement in comparison to the ELISA. The immunochromagraphic test for the e antigen had a lower positive average agreement compared to the ELISA and some patients infected with Hepatitis B virus could not be detected by the former assay. The immunochromatographic test for anti-e antigen antibodies showed a similar performance to that of ELISA and could therefore be used in clinical trials for chronic Hepatitis B in health institutions without the need of a highly qualified lab technician. (author)

  11. Core antigen and circulating anti-core antibody in hepatitis B infection

    Energy Technology Data Exchange (ETDEWEB)

    Howard, C R; Zuckerman, A J [London School of Hygiene and Tropical Medicine (UK)

    1977-02-01

    Core antigen was obtained from the sera of persistent chronic carriers of hepatitis B virus by centrifugation and treatment with Nonidet P40 and 2-mercaptoethanol. The separated core antigen was radiolabelled and identified as a nucleoprotein structure of buoyant density 1.36 g/cm/sup 3/ and possessing an isoelectric point of 4.4. This material was employed in a radioimmnoassay procedure of high sensitivity for the detection of core antibody. In a series of sera from patients with acute type B hepatitis, core antibody was demonstrated 2 to 3 weeks after the onset of jaundice during the period of surface antigenaemia. The presence of core antibody may therefore provide an accurate serological marker for the detection of active or recent virus replication in future epidemiological studies of hepatitis B infection.

  12. antigen from irradiated Trypanosoma evansi and its correlation with antibody forming

    International Nuclear Information System (INIS)

    Sadi, Suharni; Arifin, Muchson

    1998-01-01

    In this research parasites of T. evansi was weakened by gamma irradiation dose of 300 Gy. This antigen being before being used was coupled/bounded with a carrier (Freund's adjuvant). West star rats of 3 months old were as used as treated animal. These animal were divided into 4 groups contained 5 rats. Group I (Control) was untreated animals, Group II (radiation) the animals were irradiated with a low dose 0.5 Gy. Group III Immunization) the animals were immunized with irradiated antigen, and Group IV (Immunization and radiation) the animal were immunized and then irradiated with a low dose of 0.5 Gy. Immunization were done by intraperitoneal route with irradiated antigen (0.5-1ml). These results were as follows : the polyclonal antibody forming of Group I (control), Group II (Radiation), Group III (Immunization), and Group IV (Immunization and radiation) were 6.34; 5.96; and 5.88 mg/ml, respectively. Group III (Immunization) Yielded polyclonal antibody a little higher than the other treated animals. Even though the antigen was coupled with a carrier, it seemed that it did not influence the parasites variant antigenic types (VTA). (author)

  13. Highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Avraham, H.; Golenser, J.; Gazitt, Y.; Spira, D.T.; Sulitzeanu, D. (Hebrew Univ., Jerusalem (Israel). Hadassah Medical School)

    1982-08-27

    A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10/sup 6/ RBC.

  14. Antibodies to a common outer envelope antigen of Treponema hyodysenteriae with antibacterial activity.

    Science.gov (United States)

    Sellwood, R; Kent, K A; Burrows, M R; Lysons, R J; Bland, A P

    1989-08-01

    Outer envelopes of Treponema hyodysenteriae strains P18A and VS1 were prepared and characterized by SDS-PAGE. In Western blot analysis of eleven strains of T. hyodysenteriae and two intestinal non-pathogenic spirochaetes, polyclonal antiserum raised to the outer envelopes of strain P18A contained antibodies primarily to two polypeptides. A 45 kDa polypeptide was present in only two strains of T. hyodysenteriae, P18A and MC52/80, whereas another antigen of 16 kDa was common to all eleven strains of T. hyodysenteriae but was not present in the two nonpathogens. Immunogold labelling of whole organisms suggested that the 16 kDa antigen was present on the surface of the spirochaetes. In in vitro tests the serum agglutinated and inhibited growth of only the T. hyodysenteriae strains, suggesting that antibodies to the 16 kDa antigen were responsible for these activities. Serum from a gnotobiotic pig infected with T. hyodysenteriae strain P18A had antibodies to the 16 kDa antigen alone and also possessed agglutinating and growth-inhibitory activities.

  15. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

    NARCIS (Netherlands)

    Kreutz, M.; Giquel, B.; Hu, Q.; Abuknesha, R.; Uematsu, S.; Akira, S.; Nestle, F.O.; Diebold, S.S.

    2012-01-01

    Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is

  16. The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens.

    Science.gov (United States)

    Adelman, Miranda K; Schluter, Samuel F; Marchalonis, John J

    2004-02-01

    In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

  17. Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S J

    1982-01-01

    Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

  18. Development of Methods to Isolate Recombinant Antibodies Against Prostate Cancer Antigens

    National Research Council Canada - National Science Library

    Williams, Myron

    2000-01-01

    .... Appropriate materials, which include prostate cancer (PCa) cell lines, tissue specimens from radical prostacetomies, and hybridomas producing antibodies specific for pro static antigens have been procured...

  19. Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Michel Alexandre Yazbek

    2011-01-01

    Full Text Available INTRODUCTION: Epstein-Barr virus exposure appears to be an environmental trigger for rheumatoid arthritis that interacts with other risk factors. Relationships among anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status have been observed in patients with rheumatoid arthritis from different populations. OBJECTIVE: To perform an association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status in Brazilian patients with rheumatoid arthritis. METHODS: In a case-control study, 140 rheumatoid arthritis patients and 143 healthy volunteers who were matched for age, sex, and ethnicity were recruited. Anti-Epstein-Barr nuclear antigen-1 antibodies and anti-cyclic citrullinated peptide antibodies were examined using an enzyme-linked immunosorbent assay, and shared epitope alleles were identified by genotyping. Smoking information was collected from all subjects. A comparative analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status was performed in the patient group. Logistic regression analysis models were used to analyze the risk of rheumatoid arthritis. RESULTS: Anti-Epstein-Barr nuclear antigen-1 antibodies were not associated with anti-cyclic citrullinated peptide antibodies, shared epitope alleles, or smoking status. Anti-cyclic citrullinated peptide antibody positivity was significantly higher in smoking patients with shared epitope alleles (OR = 3.82. In a multivariate logistic regression analysis using stepwise selection, only anti-cyclic citrullinated peptide antibodies were found to be independently associated with rheumatoid arthritis (OR = 247.9. CONCLUSION: Anti-Epstein-Barr nuclear antigen-1 antibodies did not increase the risk of rheumatoid arthritis and were not associated with the rheumatoid arthritis risk factors studied. Smoking

  20. Autologous monoclonal antibodies recognize tumour-associated antigens in X-irradiated C57BL/6 mice

    Energy Technology Data Exchange (ETDEWEB)

    Artus, A; Guillemain, B; Legrand, E; Astier-Gin, T; Mamoun, R; Duplan, J -F

    1986-09-01

    X-irradiation of C57BL/6 mice induces thymic lymphosarcomas which sometimes contain retroviruses which upon injection into normal mice mimic the effect of the irradiation. We examined whether specific antigenicities, viral or cellular, were expressed by tumour cells that could be recognized by antibodies from the irradiated animals. We developed monoclonal antibodies (MAbs) using splenocytes of the diseased animal. The reactivity of such MAbs towards thymoma cell lines established in vitro was investigated by means of an ELISA. At least 10 antibody specificities were detected on the 13 tumours investigated, allowing separation of the MAbs into three classes: (i) those recognizing the autologous tumour, heterologous tumours as well as normal thymic tissue, (ii) those specific for the autologous tumour, and (iii) those specific for one tumour, but not ones of autologous origin. The last two classes corresponded to specific tumour-associated antigens. Our panel of MAbs defined each tumour by the particular pattern of antigens harboured. It is striking that most of the antigens were present in the normal thymus and that only two tumours had additional antigenicities. Additionally, quantitative variations were observed in the levels of expression of these antigens.

  1. A novel merozoite surface antigen of Plasmodium falciparum (MSP-3 identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

    Directory of Open Access Journals (Sweden)

    Claude Oeuvray

    1994-01-01

    Full Text Available We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

  2. RECOMBINATION OF ANTIBODY POLYPEPTIDE CHAINS IN THE PRESENCE OF ANTIGEN

    Science.gov (United States)

    Metzger, Henry; Mannik, Mart

    1964-01-01

    Conditions were developed by which the separated H and L chains of gamma2 globulins recombined to form four-chained molecules in good yields. In the absence of antigen, anti-2,4-dinitrophenyl (anti-DNP) H chains randomly reassociated with a mixture of antibody and non-specific gamma2 globulin L chains. In the presence of a specific hapten, however, the antibody H chains preferentially interacted with the anti-DNP L chains. Antibody H chain-antibody L chain recombinants formed in the presence of hapten were more active than the corresponding recombinants formed in the absence of hapten. Speculations are made regarding the possible mechanisms and biological significance of these effects. PMID:14247718

  3. A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies

    International Nuclear Information System (INIS)

    Avraham, H.; Golenser, J.; Gazitt, Y.; Spira, D.T.; Sulitzeanu, D.

    1982-01-01

    A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10 6 RBC. (Auth.)

  4. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

    Directory of Open Access Journals (Sweden)

    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  5. Radioimmunoassay for antibodies against surface membrane antigens using adhering cells

    Energy Technology Data Exchange (ETDEWEB)

    Tax, A; Manson, L A [Wistar Inst. of Anatomy and Biology, Philadelphia, Pa. (USA)

    1976-07-01

    A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit /sup 125/I-antimouse-Fab. The bound radioactive material was eluted with glycine-HCl buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolic or noncytolic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface.

  6. Antigen recognition by IgG4 antibodies in human trichinellosis

    Directory of Open Access Journals (Sweden)

    Pinelli E.

    2001-06-01

    Full Text Available The antibody isotype response to Trichinella spiralis excretory/secretory (ES products of muscle larva was examined using sera from patients with confirmed trichinellosis. Using Western blots we identify components of the ES antigen that are recognized by IgM and IgG antibodies. A 45 kDa component was strongly recognized by different antibody classes and subclasses. We observed a 45 kDa-specific lgG4 response that was detected exclusively using sera of patients with trichinellosis and not of patients with echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis. These results are relevant for the diagnosis of human trichinellosis.

  7. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

  8. Heritability of antibody isotype and subclass responses to Plasmodium falciparum antigens.

    Directory of Open Access Journals (Sweden)

    Nancy O Duah

    2009-10-01

    Full Text Available It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types and MSP3 (two allelic types. Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h(2 estimates for 48% (20/42 of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested among the adults, 48% (20/42 among the children in the dry season and 31% (13/42 among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20 of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation.

  9. Characterisation of antibody responses in pigs induced by recombinant oncosphere antigens from Taenia solium.

    Science.gov (United States)

    Jayashi, César M; Gonzalez, Armando E; Castillo Neyra, Ricardo; Kyngdon, Craig T; Gauci, Charles G; Lightowlers, Marshall W

    2012-12-14

    Recombinant antigens cloned from the oncosphere life cycle stage of the cestode parasite Taenia solium (T. solium) have been proven to be effective as vaccines for protecting pigs against infections with T. solium. Previous studies have defined three different host protective oncosphere antigens, TSOL18, TSOL16 and TSOL45. In this study, we evaluated the potential for combining the antigens TSOL16 and TSOL18 as a practical vaccine. Firstly, in a laboratory trial, we compared the immunogenicity of the combined antigens (TSOL16/18) versus the immunogenicity of the antigens separately. Secondly, in a field trial, we tested the ability of the TSOL16/18 vaccine to induce detectable antibody responses in animals living under environmental stress and traditionally reared in areas where T. solium cysticercosis is endemic; and finally, we characterised the immune response of the study population. Pigs of 8-16 weeks of age were vaccinated with 200 μg each of TSOL16 and TSOL18, plus 5mg of Quil-A. Specific total IgG, IgG(1) and IgG(2) antibody responses induced by TSOL16 and TSOL18 were determined with ELISA. The immunogenicity of both antigens was retained in the combined TSOL16/18 vaccine. The combined vaccine TSOL16/18 induced detectable specific anti-TSOL18 antibody responses in 100% (113/113) and specific anti-TSOL16 in 99% (112/113) of the vaccinated animals measured at 2 weeks following the booster vaccination. From the two IgG antibody subtypes analysed we found there was stronger response to IgG(2). Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

    Science.gov (United States)

    Lin, Margaret; Krawitz, Denise; Callahan, Matthew D.; Deperalta, Galahad; Wecksler, Aaron T.

    2018-05-01

    We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. [Figure not available: see fulltext.

  11. Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

    Science.gov (United States)

    Lin, Margaret; Krawitz, Denise; Callahan, Matthew D.; Deperalta, Galahad; Wecksler, Aaron T.

    2018-03-01

    We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. [Figure not available: see fulltext.

  12. Rationalization and Design of the Complementarity Determining Region Sequences in an Antibody-Antigen Recognition Interface

    Science.gov (United States)

    Chen, Ing-Chien; Lee, Yu-Ching; Chen, Jun-Bo; Tsai, Keng-Chang; Chen, Ching-Tai; Chang, Jeng-Yih; Yang, Ei-Wen; Hsu, Po-Chiang; Jian, Jhih-Wei; Hsu, Hung-Ju; Chang, Hung-Ju; Hsu, Wen-Lian; Huang, Kai-Fa; Ma, Alex Che; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes. PMID:22457753

  13. Evaluation of an Antigen-Antibody “Combination” Enzyme Linked ...

    African Journals Online (AJOL)

    Conclusion: We conclude that although this assay depicts high sensitivity and specificity in detecting antibodies to HCV, it seems not to add further benefit in our study population to detect HCV infections by enhanced sensitivity due the potential contingency to trace viral capsid antigens. Keywords: Ag-Ab Combination assay ...

  14. Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization

    International Nuclear Information System (INIS)

    Horenstein, A.L.; Feinstein, R.E.

    1985-01-01

    Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody. (orig.)

  15. Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

    International Nuclear Information System (INIS)

    Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

    2016-01-01

    In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders

  16. Purification of antibodies to bacterial antigens by an immunoadsorbent and a method to quantify their reaction with insoluble bacterial targets

    International Nuclear Information System (INIS)

    Mathews, H.L.; Minden, P.

    1979-01-01

    A combination of procedures was employed to develop a radioimmunoassay which quantified the binding of antibodies to antigens of either intact Propionibacterium acnes or to antigens of insoluble extracts derived from the bacteria. Reactive antibody populations were purified by use of bacterial immunoadsorbents which were prepared by coupling P. acnes to diethylaminoethyl cellulose. Binding of antibodies was detected with [ 125 I]staphylococcal protein A ([ 125 I]SpA) and optimal conditions for the assay defined by varying the amounts of antibodies, bacterial antigenic targets and [ 125 I]SpA. In antibody excess, 100% of available [ 125 I]SpA was bound by the target-antibody complexes. However, when antibody concentration was limiting, a linear relationship was demonstrated between per cent specific binding of[ 125 I]SpA and antibodies bound to bacterial targets. These results were achieved only with immunoadsorbent-purified antibody populations and not with hyperimmune sera or IgG. The radioimmunoassay detected subtle antigenic differences and similarities between P. acnes, P. acnes extracts and a variety of unrelated microorganisms. (Auth.)

  17. Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

    Science.gov (United States)

    Lynch, D M; Howe, S E

    1985-11-01

    Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

  18. Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

    Science.gov (United States)

    He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Yang, Xiqin; Bai, Guanzhong; Ling, Shigan; Zhang, Heqiu

    2011-08-01

    Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

  19. Immunoglobulin M antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi in chronic chagasic patients.

    Science.gov (United States)

    Vasconcelos, Romero H T; Azevedo, Elisa A N; Cavalcanti, Maria G A M; Silva, Edimilson D; Ferreira, Antonio G P; Morais, Clarice N L; Gomes, Yara M

    2011-05-01

    Previous works of our research group have demonstrated aspects of the humoral immune response of chronic Chagas disease using the cytoplasmatic repetitive antigen (CRA) and the flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The aim of this work was to analyze the presence of specific immunoglobulin M (IgM) antibodies in chronic chagasic patients using these recombinant antigens of T. cruzi. The positivity of IgM in chronic chagasic patients against CRA and FRA antigens was determined by indirect enzyme-linked immunosorbent assay. We reported no statistical significant differences between the levels of IgM for both recombinant antigens and the different chronic clinical forms of Chagas disease. However, a small proportion of chronic chagasic patients analyzed in this study was positive for this antibody isotype. The findings of this study indicate that the IgM antibodies cannot be used to elucidate the differences in the profile of humoral immune response among chronic chagasic patients with different clinical forms using the CRA and FRA recombinant antigens of T. cruzi. Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  20. Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects.

    Directory of Open Access Journals (Sweden)

    Mauro Tognon

    Full Text Available Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18-65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses.

  1. OptMAVEn--a new framework for the de novo design of antibody variable region models targeting specific antigen epitopes.

    Directory of Open Access Journals (Sweden)

    Tong Li

    Full Text Available Antibody-based therapeutics provides novel and efficacious treatments for a number of diseases. Traditional experimental approaches for designing therapeutic antibodies rely on raising antibodies against a target antigen in an immunized animal or directed evolution of antibodies with low affinity for the desired antigen. However, these methods remain time consuming, cannot target a specific epitope and do not lead to broad design principles informing other studies. Computational design methods can overcome some of these limitations by using biophysics models to rationally select antibody parts that maximize affinity for a target antigen epitope. This has been addressed to some extend by OptCDR for the design of complementary determining regions. Here, we extend this earlier contribution by addressing the de novo design of a model of the entire antibody variable region against a given antigen epitope while safeguarding for immunogenicity (Optimal Method for Antibody Variable region Engineering, OptMAVEn. OptMAVEn simulates in silico the in vivo steps of antibody generation and evolution, and is capable of capturing the critical structural features responsible for affinity maturation of antibodies. In addition, a humanization procedure was developed and incorporated into OptMAVEn to minimize the potential immunogenicity of the designed antibody models. As case studies, OptMAVEn was applied to design models of neutralizing antibodies targeting influenza hemagglutinin and HIV gp120. For both HA and gp120, novel computational antibody models with numerous interactions with their target epitopes were generated. The observed rates of mutations and types of amino acid changes during in silico affinity maturation are consistent with what has been observed during in vivo affinity maturation. The results demonstrate that OptMAVEn can efficiently generate diverse computational antibody models with both optimized binding affinity to antigens and reduced

  2. Acute HIV Discovered During Routine HIV Screening With HIV Antigen-Antibody Combination Tests in 9 US Emergency Departments.

    Science.gov (United States)

    White, Douglas A E; Giordano, Thomas P; Pasalar, Siavash; Jacobson, Kathleen R; Glick, Nancy R; Sha, Beverly E; Mammen, Priya E; Hunt, Bijou R; Todorovic, Tamara; Moreno-Walton, Lisa; Adomolga, Vincent; Feaster, Daniel J; Branson, Bernard M

    2018-01-05

    Newer combination HIV antigen-antibody tests allow detection of HIV sooner after infection than previous antibody-only immunoassays because, in addition to HIV-1 and -2 antibodies, they detect the HIV-1 p24 antigen, which appears before antibodies develop. We determine the yield of screening with HIV antigen-antibody tests and clinical presentations for new diagnoses of acute and established HIV infection across US emergency departments (EDs). This was a retrospective study of 9 EDs in 6 cities with HIV screening programs that integrated laboratory-based antigen-antibody tests between November 1, 2012, and December 31, 2015. Unique patients with newly diagnosed HIV infection were identified and classified as having either acute HIV infection or established HIV infection. Acute HIV infection was defined as a repeatedly reactive antigen-antibody test result, a negative HIV-1/HIV-2 antibody differentiation assay, or Western blot result, but detectable HIV ribonucleic acid (RNA); established HIV infection was defined as a repeatedly reactive antigen-antibody test result and a positive HIV-1/HIV-2 antibody differentiation assay or Western blot result. The primary outcomes were the number of new HIV diagnoses and proportion of patients with laboratory-defined acute HIV infection. Secondary outcomes compared reason for visit and the clinical presentation of acute HIV infection. In total, 214,524 patients were screened for HIV and 839 (0.4%) received a new diagnosis, of which 122 (14.5%) were acute HIV infection and 717 (85.5%) were established HIV infection. Compared with patients with established HIV infection, those with acute HIV infection were younger, had higher RNA and CD4 counts, and were more likely to have viral syndrome (41.8% versus 6.5%) or fever (14.3% versus 3.4%) as their reason for visit. Most patients with acute HIV infection displayed symptoms attributable to acute infection (median symptom count 5 [interquartile range 3 to 6]), with fever often

  3. Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

    NARCIS (Netherlands)

    Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

    1985-01-01

    T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

  4. Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

    Science.gov (United States)

    Onodera, Rie; Kurita, Emi; Taniguchi, Kikuyo; Karakawa, Shuhei; Okada, Satoshi; Kihara, Hirotaka; Fujii, Teruhisa; Kobayashi, Masao

    2017-11-01

    Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN. © 2017 AABB.

  5. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    International Nuclear Information System (INIS)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-01-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with 111 In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity

  6. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    Energy Technology Data Exchange (ETDEWEB)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-05-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with /sup 111/In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

  7. The effect of high antigen density on solid-phase radioimmunoassays for antibody regardless of immunoglobulin class

    International Nuclear Information System (INIS)

    Rubin, R.L.; Hardtke, M.A.; Carr, R.I.

    1980-01-01

    Human sera containing antibody to casein or to bovine serum albumin were used to assess the validity and utility of a solid-phase assay for quantitating antibody activity. Rabbit anti-human immunoglobulin radiolabeled with 125 I and capable of reacting with all human immunoglobulin classes was used to detect antibody bound to antigen immobilized to polystyrene tubes by a new covalent technique. This method results in very high antigen concentrations in highly stable association with polystyrene tubes. Kinetic and absorption studies demonstrated that low avidity antibodies are better detected when antigen is immobilized by the covalent method than when passively adsorbed. Conditions are described for minimizing artifactual interactions and for obtaining results similar to those obtained with conventional, liquid-phase assays. Failure to reach equilibrium in solid-phase assays and other problems are proposed to explain, in part, the inability to obtain a better correlation between solid- and liquid-phase immunoassays. (Auth.)

  8. A radioimmunoassay for antibodies against surface membrane antigens using adhering cells

    International Nuclear Information System (INIS)

    Tax, A.; Manson, L.A.

    1976-01-01

    A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit 125 I-antimouse-Fab. The bound radioactive material was eluted with glycine-HCl buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolic or noncytolic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface

  9. Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

    Science.gov (United States)

    Reyes, L; Hartikka, J; Bozoukova, V; Sukhu, L; Nishioka, W; Singh, G; Ferrari, M; Enas, J; Wheeler, C J; Manthorpe, M; Wloch, M K

    2001-06-14

    Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-gamma or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 -/- mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells.

  10. An analytical approach to reduce between-plate variation in multiplex assays that measure antibodies to Plasmodium falciparum antigens.

    Science.gov (United States)

    Fang, Rui; Wey, Andrew; Bobbili, Naveen K; Leke, Rose F G; Taylor, Diane Wallace; Chen, John J

    2017-07-17

    Antibodies play an important role in immunity to malaria. Recent studies show that antibodies to multiple antigens, as well as, the overall breadth of the response are associated with protection from malaria. Yet, the variability and reliability of antibody measurements against a combination of malarial antigens using multiplex assays have not been well characterized. A normalization procedure for reducing between-plate variation using replicates of pooled positive and negative controls was investigated. Sixty test samples (30 from malaria-positive and 30 malaria-negative individuals), together with five pooled positive-controls and two pooled negative-controls, were screened for antibody levels to 9 malarial antigens, including merozoite antigens (AMA1, EBA175, MSP1, MSP2, MSP3, MSP11, Pf41), sporozoite CSP, and pregnancy-associated VAR2CSA. The antibody levels were measured in triplicate on each of 3 plates, and the experiments were replicated on two different days by the same technician. The performance of the proposed normalization procedure was evaluated with the pooled controls for the test samples on both the linear and natural-log scales. Compared with data on the linear scale, the natural-log transformed data were less skewed and reduced the mean-variance relationship. The proposed normalization procedure using pooled controls on the natural-log scale significantly reduced between-plate variation. For malaria-related research that measure antibodies to multiple antigens with multiplex assays, the natural-log transformation is recommended for data analysis and use of the normalization procedure with multiple pooled controls can improve the precision of antibody measurements.

  11. Development of a immunochromatographic test with avidin-biotin for the detection of antibodies against antigen e of hepatitis B in human plasma

    International Nuclear Information System (INIS)

    Mainet Gonzalez, Damian; Palenzuela Gardon, Daniel O; Diaz Argudin, Tamara

    2007-01-01

    The disappearance of antigen e of hepatitis B in the presence of the plasmatic antibodies against antigen e may indicate a satisfactory therapeutic response in patients with chronic hepatitis B. The immuno-chromatographic test carried out in the diagnosis of diseases use different antibody combinations and may employ the avidin or streptavidin-biotin technology to develop a rapid immuno-chromatographic test for the detection of antibodies anti-antigen e in the plasma. They were detected in the laboratory by means of two fast immuno-chromatographic tests when using in one of them the avidin-biotin technology. These tests are carried out with a one-step competitive inhibition format and amplified or not with avidin-biotin. Monoclonal antibodies against antigen e obtained by cellular hybridization were used. Forty-six plasmatic samples classified as positive and negative to the anti-antigen antibodies were evaluated with a reference immunochromatographic test Advanced QualityTM. The possible expiry time of the biological reagents forming part of these tests were studied with accelerated thermal-stability experiments. The possible interference in the plasma of some of the biochemical compounds used in these trials was analyzed. Four murine monoclonal antibodies anti-antigen e were obtained and only one of them was used in these immunochromatographic tests with an anti-antigen polyclonal antibody conjugated with gold. Both tests and their stable biological reagents discriminated the positive and negative samples to the antibodies anti-antigen e, as well as the commercial test. There was no interference in the biochemical compounds studied in these tests. Both immuno-chromatographic tests made in the laboratory are useful to detect antibodies anti-antigen e in the plasma. The avidin-biotin increased the analytical sensitivity of this type of fast immuno-chromatographic test without altering its performance features. (Author)

  12. Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens

    International Nuclear Information System (INIS)

    Stempfer, René; Weinhäusel, Andreas; Syed, Parvez; Vierlinger, Klemens; Pichler, Rudolf; Meese, Eckart; Leidinger, Petra; Ludwig, Nicole; Kriegner, Albert; Nöhammer, Christa

    2010-01-01

    The simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate. Candidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients. Methods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly. Protein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto-antibody

  13. A simple assay for the detection of antibodies to endocrine islet cell surface antigens

    International Nuclear Information System (INIS)

    Contreas, G.; Madsen, O.D.; Vissing, H.; Lernmark, Aa.

    1986-01-01

    A simple and sensitive immunoradiometric assay for the detection of islet cell surface antibodies (CIRMA) has been developed. Live, transformed islet cells derived from a liver metastasis of a transplantable islet cell tumor were grown in removable microtiter wells and incubated with antibody. Cell-bound antibodies were quantitated using 125 I-labelled second antibodies. The assay was used to detect islet cell antibodies present in sera from non-diabetic and diabetic BB rats and proved to be particularly effective for screening hybridoma supernatants in order to identify monoclonal antibodies recognizing islet cell surface antigens. (Auth.)

  14. Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

    International Nuclear Information System (INIS)

    Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R.; Medeiros-Neto, G.; Iacona, A.; Lima, N.

    1989-01-01

    We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in 51 Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of 51 Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. When tested in CMAC against eye muscle cells, one of 9 mouse and 5 of 8 human monoclonal antibodies showed significant activity while tests were positive in one of 9 and one of 10 monoclonal antibodies

  15. Definition of a virulence-related antigen of Neisseria gonorrhoeae with monoclonal antibodies and lectins.

    Science.gov (United States)

    Demarco de Hormaeche, R; Bundell, C; Chong, H; Taylor, D W; Wildy, P

    1986-03-01

    Variants of one strain of Neisseria gonorrhoeae, grown in vivo or in vitro, that have been previously shown to differ in infectivity, serum resistance, and capsule production were compared with use of monoclonal antibodies and lectins. Monoclonal antibodies to virulent gonococci recognized an antigenic site of the lipopolysaccharide (LPS) produced in large amounts by gonococci grown in vivo but present only in a small proportion of in vitro-grown gonococci. This antigen (C-LPS) was found in all 85 different gonococcal isolates studied but not among nonpathogenic neisseriae. It was shared by group B and C meningococci but not by groups A and D. Enzyme-linked immunosorbent assay and Western blot analysis showed that N-acetylglucosamine and N-acetylgalactosamine form part of the epitope. The C-LPS antigen was shown by immunofluorescence to be present on the surface of the gonococci and also free as slime. This antigen appears to confer resistance to killing by normal sera.

  16. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  17. Intravenous IgA complexed with antigen reduces primary antibody response to the antigen and anaphylaxis upon antigen re-exposure by inhibiting Th1 and Th2 activation in mice.

    Science.gov (United States)

    Yamaki, Kouya; Miyatake, Kenji; Nakashima, Takayuki; Morioka, Ayumi; Yamamoto, Midori; Ishibashi, Yuki; Ito, Ayaka; Kuranishi, Ayu; Yoshino, Shin

    2014-10-01

    Serum IgG, IgE and IgM have been shown to enhance the primary antibody responses upon exposure to the soluble antigens recognized by those antibodies. However, how IgA affects these responses remains unknown. We investigated the effects of intravenously administered monoclonal IgA on the immune responses in mice. DBA/1J mice were immunized with ovalbumin in the presence or absence of anti-ovalbumin monoclonal IgA. The Th1 and Th2 immune responses to ovalbumin and the anaphylaxis induced by re-exposure to ovalbumin were measured. IgA complexed with antigen attenuated the primary antibody responses to the antigen in mice, in contrast to IgG2b and IgE. The primary antibody responses, i.e. the de novo synthesis of anti-ovalbumin IgG2a, IgG1 and IgE in the serum, and the subsequent anaphylaxis induced with re-exposure to ovalbumin were reduced by the co-injection of anti-ovalbumin monoclonal IgA at ovalbumin immunization. The Th1, Th2 and Tr1 cytokines interferon-γ, interleukin-4 and interleukin-10, respectively, released from ovalbumin-restimulated cultured splenocytes collected from allergic mice were also reduced by the treatment. The induction of interferon-γ and interleukin-4 secretion by splenocytes from ovalbumin-immunized mice stimulated in vitro with ovalbumin was also significantly reduced by the antigen complexed with anti-ovalbumin IgA. These data suggest that the direct inhibition of Th1 and Th2 activation by anti-ovalbumin monoclonal IgA participates in the inhibition of the primary antibody responses. IgA plays important immunosuppressive roles under physiological and pathological conditions and is a promising candidate drug for the treatment of immune disorders.

  18. Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

    2004-01-01

    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

  19. Radioimmunoassay and some properties of human antibodies to hepatitis B core antigen

    Energy Technology Data Exchange (ETDEWEB)

    Neurath, A R; Szmuness, W; Stevens, C E; Strick, N; Harley, E J [New York Blood Center, N.Y. (USA)

    1978-03-01

    A solid-phase radioimmunoassay for antibodies to hepatitis B core antigen (anti-HBsub(c)) is described. Polystyrene beads coated with anti-HBsub(c), hepatitis B core antigen prepared from pooled sera of humans infected with hepatitis B virus (HBV) and /sup 125/I-labelled anti-HBsub(c) were used for the test. Distinct patterns of development and changes of anti-HBsub(c) and their immunological properties are all related to variations of other markers specific for HBV infections. Knowledge concerning the detailed features of the immune response to hepatitis B core antigen may provide deeper insight into the pathogenesis of HBV infections.

  20. Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R. (Thyroid Research Unit, The Montreal General Hospital Research Institute, Montreal, Quebec (Canada)); Medeiros-Neto, G.; Iacona, A.; Lima, N. (Thyroid Clinic, Hospital das Clinicas, Sao Paulo (Brazil))

    1989-01-01

    We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in {sup 51}Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of {sup 51}Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. (Abstract Truncated)

  1. Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

    Directory of Open Access Journals (Sweden)

    Masato Kiyoshi

    Full Text Available The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1. We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101 is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody. Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu. The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

  2. Antigen-specific immature dendritic cell vaccine ameliorates anti-dsDNA antibody-induced renal damage in a mouse model.

    Science.gov (United States)

    Xia, Yumin; Jiang, Shan; Weng, Shenhong; Lv, Xiaochun; Cheng, Hong; Fang, Chunhong

    2011-12-01

    Dendritic cells (DCs) can inhibit immune response by clonal anergy when immature. Recent studies have shown that immature DCs (iDCs) may serve as a live cell vaccine after specific antigen pulse based on its potential of blocking antibody production. In this study, we aimed to investigate the effects of nuclear antigen-pulsed iDCs in the treatment of lupus-like renal damages induced by anti-dsDNA antibodies. iDCs were generated from haemopoietic stem cells in bone marrow and then pulsed in vitro with nuclear antigen. The iDC vaccine and corresponding controls were injected into mice with lupus-like renal damages. The evaluation of disease was monitored by biochemical parameters and histological scores. Anti-dsDNA antibody isotypes and T-lymphocyte-produced cytokines were analysed for elucidating therapeutic mechanisms. RESULTS; The mice treated with antigen-pulsed iDCs had a sustained remission of renal damage compared with those injected with non-pulsed iDCs or other controls, including decreased anti-dsDNA antibody level, less proteinuria, lower blood urea nitrogen and serum creatinine values, and improved histological evaluation. Analysis on isotypes of anti-dsDNA antibody showed that iDC vaccine preferentially inhibited the production of IgG3, IgG2b and IgG2a. Furthermore, administration of antigen-treated iDCs to mice resulted in significantly reduced IL-2, IL-4 and IL-12 and IFN-γ produced by T-memory cells. Conversely, the vaccination of antigen-pulsed mature DCs led to increased anti-dsDNA antibody production and an aggravation of lupus-like disease in the model. CONCLUSIONS; These results suggested the high potency of iDC vaccine in preventing lupus-like renal injuries induced by pathogenic autoantibodies.

  3. Strong Antibody Responses Induced by Protein Antigens Conjugated onto the Surface of Lecithin-Based Nanoparticles

    Science.gov (United States)

    Sloat, Brian R.; Sandoval, Michael A.; Hau, Andrew M.; He, Yongqun; Cui, Zhengrong

    2009-01-01

    An accumulation of research over the years has demonstrated the utility of nanoparticles as antigen carriers with adjuvant activity. Herein we defined the adjuvanticity of a novel lecithin-based nanoparticle engineered from emulsions. The nanoparticles were spheres of around 200 nm. Model protein antigens, bovine serum albumin (BSA) or Bacillus anthracis protective antigen (PA) protein, were covalently conjugated onto the nanoparticles. Mice immunized with the BSA-conjugated nanoparticles developed strong anti-BSA antibody responses comparable to that induced by BSA adjuvanted with incomplete Freund's adjuvant and 6.5-fold stronger than that induced by BSA adsorbed onto aluminum hydroxide. Immunization of mice with the PA-conjugated nanoparticles elicited a quick, strong, and durable anti-PA antibody response that afforded protection of the mice against a lethal dose of anthrax lethal toxin challenge. The potent adjuvanticity of the nanoparticles was likely due to their ability to move the antigens into local draining lymph nodes, to enhance the uptake of the antigens by antigen-presenting cells (APCs), and to activate APCs. This novel nanoparticle system has the potential to serve as a universal protein-based vaccine carrier capable of inducing strong immune responses. PMID:19729045

  4. Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

    Science.gov (United States)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

    2004-01-01

    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

  5. Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo

    Directory of Open Access Journals (Sweden)

    Yakov Lomakin

    2017-07-01

    Full Text Available Multiple sclerosis (MS is an autoimmune chronic inflammatory disease of the central nervous system (CNS. Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV latent membrane protein 1 (LMP1. In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

  6. Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo

    Science.gov (United States)

    Lomakin, Yakov; Arapidi, Georgii Pavlovich; Chernov, Alexander; Ziganshin, Rustam; Tcyganov, Evgenii; Lyadova, Irina; Butenko, Ivan Olegovich; Osetrova, Maria; Ponomarenko, Natalia; Telegin, Georgy; Govorun, Vadim Markovich; Gabibov, Alexander; Belogurov, Alexey

    2017-01-01

    Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading. PMID:28729867

  7. Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo.

    Science.gov (United States)

    Lomakin, Yakov; Arapidi, Georgii Pavlovich; Chernov, Alexander; Ziganshin, Rustam; Tcyganov, Evgenii; Lyadova, Irina; Butenko, Ivan Olegovich; Osetrova, Maria; Ponomarenko, Natalia; Telegin, Georgy; Govorun, Vadim Markovich; Gabibov, Alexander; Belogurov, Alexey

    2017-01-01

    Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo . We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

  8. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

    Directory of Open Access Journals (Sweden)

    Isabel Correa

    2018-03-01

    Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

  9. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

    Science.gov (United States)

    Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

    2018-01-01

    Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

  10. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

    Science.gov (United States)

    Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

    2018-01-01

    Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

  11. Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

    Directory of Open Access Journals (Sweden)

    Stewart Nuttall

    2013-01-01

    Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

  12. The antibody response to well-defined malaria antigens after acute malaria in individuals living under continuous malaria transmission

    DEFF Research Database (Denmark)

    Petersen, E; Høgh, B; Dziegiel, M

    1992-01-01

    The IgG and IgM antibody responses to the C-terminal 783 amino acids of the P. falciparum glutamate-rich protein, GLURP489-1271, expressed as an E. coli fusion protein, the IgG response to a 18-mer synthetic peptide EDKNEKGQHEIVEVEEIL (GLURP899-916) representing the C-terminal repeats of GLURP......, and a synthetic peptide (EENV)6 representing the C-terminal repeats from Pf155/RESA, were investigated longitudinally in 13 children and 7 adults living under conditions of continuous, intense malaria transmission. Some subjects did not recognize the antigens after malaria infection, and in subjects recognizing...... the antigens, the responses were often short-lived. In adults, the antibody responses to the GLURP489-1271 fusion protein and the (EENV)6 peptide peaked after 2 weeks, and not all individuals responded to all antigens. The antibody response, even against large fragments of conserved antigens, is not uniformly...

  13. Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

  14. Use of monoclonal antibodies against Hendra and Nipah viruses in an antigen capture ELISA

    Directory of Open Access Journals (Sweden)

    Spiropoulou Christina F

    2010-06-01

    Full Text Available Abstract Background Outbreaks of Hendra (HeV and Nipah (NiV viruses have been reported starting in 1994 and 1998, respectively. Both viruses are capable of causing fatal disease in humans and effecting great economical loss in the livestock industry. Results Through screening of hybridomas derived from mice immunized with γ-irradiated Nipah virus, we identified two secreted antibodies; one reactive with the nucleocapsid (N protein and the other, the phosphoprotein (P of henipaviruses. Epitope mapping and protein sequence alignments between NiV and HeV suggest the last 14 amino acids of the carboxyl terminus of the N protein is the target of the anti-N antibody. The anti-P antibody recognizes an epitope in the amino-terminal half of P protein. These monoclonal antibodies were used to develop two antigen capture ELISAs, one for virus detection and the other for differentiation between NiV and HeV. The lower limit of detection of the capture assay with both monoclonal antibodies was 400 pfu. The anti-N antibody was used to successfully detect NiV in a lung tissue suspension from an infected pig. Conclusion The antigen capture ELISA developed is potentially affordable tool to provide rapid detection and differentiation between the henipaviruses.

  15. Concurrence of hepatitis B surface antibodies and surface antigen: implications for postvaccination control of health care workers

    NARCIS (Netherlands)

    Zaaijer, Hans L.; Lelie, P. N.; Vandenbroucke-Grauls, C. M. J. E.; Koot, M.

    2002-01-01

    Among 1081 persons testing positive for hepatitis B surface antigen, 106 (10%) tested positive for antibodies to surface antigen (anti-HBs) in the same blood sample. Thirty of these persons were studied in detail: seven tested positive for hepatitis B e-antigen, nine were apparently healthy blood

  16. Quantitative relationship between antibody affinity and antibody avidity

    International Nuclear Information System (INIS)

    Griswold, W.R.

    1987-01-01

    The relationship between antibody avidity, measured by the dissociation of the antigen-antibody bond in antigen excess, and antibody affinity was studied. Complexes of radiolabelled antigen and antibody of known affinity were prepared in vitro and allowed to stand for seven days to reach equilibrium. Then nonlabelled antigen in one hundred fold excess was added to dissociate the complexes. After an appropriate incubation the fraction of antigen bound to antibody was measured by the ammonium sulfate precipitation method. The dissociation index was the fraction bound in the experimental sample divided by the fraction bound in the control. The correlation coefficient between the dissociation index and the antibody binding constant was 0.92 for early dissociation and 0.98 for late dissociation. The regression equation relating the binding constant to the dissociation index was K = 6.4(DI) + 6.25, where DI is the late dissociation index and K is the logarithm to the base 10 of the binding constant. There is a high correlation between avidity and affinity of antibody. Antibody affinity can be estimated from avidity data. The stability of antigen-antibody complexes can be predicted from antibody affinity

  17. Antigen-decorated shell cross-linked nanoparticles: synthesis, characterization, and antibody interactions.

    Science.gov (United States)

    Joralemon, Maisie J; Smith, Norah L; Holowka, David; Baird, Barbara; Wooley, Karen L

    2005-01-01

    Antigen-decorated shell cross-linked knedel-like nanoparticles (SCKs) were synthesized and studied as multivalent nanoscale surfaces from which antibody-binding units were presented in a manner that was designed to approach virus particle surfaces. The SCK nanostructures were fabricated with control over the number of antigenic groups, from mixed micellization of amphiphilic diblock copolymer building blocks that contained either an antigen (2,4-dinitrophenyl) or an ethylpropionate group at the hydrophilic alpha-chain terminus. Amphiphilic diblock copolymers were synthesized by atom transfer radical polymerization of tert-butyl acrylate and methyl acrylate sequentially from either a 2,4-dinitrophenyl-functionalized initiator or ethyl 2-bromopropionate, followed by selective removal of the tert-butyl groups to afford 2,4-dinitrophenyl-poly(acrylic acid)60-b-poly(methyl acrylate)60 (DNP-PAA(60)-b-PMA60) and poly(acrylic acid)70-b-poly(methyl acrylate) (PAA70-b-PMA70). Micelles were assembled via addition of water to THF solutions of the polymers in 0:1, 1:1, and 1:0 molar ratios of DNP-PAA60-b-PMA60 to PAA70-b-PMA70, followed by dialysis against water. The acrylic acid groups of the micelle coronas were partially cross-linked (nominally 50%) with 2,2'-(ethylenedioxy)bis(ethylamine), in the presence of 1-(3'-dimethylaminopropyl)-3-ethylcarbodiimide methiodide. Following extensive dialysis against water, the 0%, 50%, and 100% dinitrophenylated shell cross-linked nanoparticles (DNP-SCKs) were characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared and UV-vis spectroscopies, and analytical ultracentrifugation (AU). The surface accessibility and bioavailability of the DNP units upon the DNP-SCKs were investigated by performing quenching titrations of fluorescein-labeled IgE antibody in solution and degranulation of Ig

  18. An optimized antibody-chelator conjugate for imaging of carcinoembryonic antigen with indium-111

    International Nuclear Information System (INIS)

    Sumerdon, G.A.; Rogers, P.E.; Lombardo, C.M.; Schnobrich, K.E.; Melvin, S.L.; Tribby, I.I.E.; Stroupe, S.D.; Johnson, D.K.; Hobart, E.D.

    1990-01-01

    A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10 -4 M, gave radiochemical yields of indium labeled antibody of ≥ 95% and was stable for 1 yr. (author)

  19. Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Brown, P J; Molloy, C M; Johnson, P M [Liverpool Univ. (UK). Dept. of Immunology

    1983-11-01

    Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Msub(r) of 68000. H310 and H316 both recognise an antigen with a subunit Msub(r) of 34000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Msub(r) 62000. It is unknown whether this 62000 dalton component is a dimer of the 34000 dalton protein with either itself or a second protein chain of presumed Msub(r) around 28000. H315 recognises an antigen with subunit Msub(r) of 36000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.

  20. Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen

    NARCIS (Netherlands)

    ter Borg, F.; ten Kate, F. J.; Cuypers, H. T.; Leentvaar-Kuijpers, A.; Oosting, J.; Wertheim-van Dillen, P. M.; Honkoop, P.; Rasch, M. C.; de Man, R. A.; van Hattum, J.; Chamuleau, R. A.; Reesink, H. W.; Jones, E. A.

    1998-01-01

    BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA

  1. Systems Analysis Reveals High Genetic and Antigen-Driven Predetermination of Antibody Repertoires throughout B Cell Development

    Directory of Open Access Journals (Sweden)

    Victor Greiff

    2017-05-01

    Full Text Available Antibody repertoire diversity and plasticity is crucial for broad protective immunity. Repertoires change in size and diversity across multiple B cell developmental stages and in response to antigen exposure. However, we still lack fundamental quantitative understanding of the extent to which repertoire diversity is predetermined. Therefore, we implemented a systems immunology framework for quantifying repertoire predetermination on three distinct levels: (1 B cell development (pre-B cell, naive B cell, plasma cell, (2 antigen exposure (three structurally different proteins, and (3 four antibody repertoire components (V-gene usage, clonal expansion, clonal diversity, repertoire size extracted from antibody repertoire sequencing data (400 million reads. Across all three levels, we detected a dynamic balance of high genetic (e.g., >90% for V-gene usage and clonal expansion in naive B cells and antigen-driven (e.g., 40% for clonal diversity in plasma cells predetermination and stochastic variation. Our study has implications for the prediction and manipulation of humoral immunity.

  2. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  3. Native Mass Spectrometry, Ion mobility, and Collision-Induced Unfolding Categorize Malaria Antigen/Antibody Binding

    Science.gov (United States)

    Huang, Yining; Salinas, Nichole D.; Chen, Edwin; Tolia, Niraj H.; Gross, Michael L.

    2017-09-01

    Plasmodium vivax Duffy Binding Protein (PvDBP) is a promising vaccine candidate for P. vivax malaria. Recently, we reported the epitopes on PvDBP region II (PvDBP-II) for three inhibitory monoclonal antibodies (2D10, 2H2, and 2C6). In this communication, we describe the combination of native mass spectrometry and ion mobility (IM) with collision induced unfolding (CIU) to study the conformation and stabilities of three malarial antigen-antibody complexes. These complexes, when collisionally activated, undergo conformational changes that depend on the location of the epitope. CIU patterns for PvDBP-II in complex with antibody 2D10 and 2H2 are highly similar, indicating comparable binding topology and stability. A different CIU fingerprint is observed for PvDBP-II/2C6, indicating that 2C6 binds to PvDBP-II on an epitope different from 2D10 and 2H2. This work supports the use of CIU as a means of classifying antigen-antibody complexes by their epitope maps in a high throughput screening workflow. [Figure not available: see fulltext.

  4. Dynamic interaction of 111indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy

    International Nuclear Information System (INIS)

    Hwang, K.M.; Keenan, A.M.; Frincke, J.; David, G.; Pearson, J.; Oldham, R.K.; Morgan, A.C. Jr.

    1986-01-01

    Two 111 indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases

  5. Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

    DEFF Research Database (Denmark)

    Kirkeby, Svend; André, Sabine; Gabius, Hans-Joachim

    2004-01-01

    OBJECTIVES: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic alphagalactose (Galalpha) antigens in the doner tissue. DESIGN AND METHODS: We have measured the binding potentials of Galalpha detecting lectins and antibodies, including...

  6. Prevalence of Diego blood group antigen and the antibody in three ethnic population groups in Klang valley of Malaysia

    Directory of Open Access Journals (Sweden)

    Cheong Tar Wei

    2013-01-01

    Full Text Available Background: Diego blood group antigen, Di(a, is very rare among Caucasians and Blacks, but relatively common among the South American Indians and Asians of Mongolian origin. The antibody to Di(a is clinically significant to cause hemolytic disease in a new-born or hemolytic transfusion reaction. Objectives: This study was designed to determine the prevalence of Di(a antigen among the blood donors from the three major ethnic groups in Klang Valley of Malaysia as well as to find an incidence of an antibody of the Diego antigen, anti-Di(a, in a tertiary care hospital to ascertain the need to include Di(a+ red cells for an antibody screen cell panel. Materials and Methods: Serological tests were performed by column agglutination technique using commercial reagents and following instruction as per kit insert. Results: Di(a antigen was found with a frequency of 2.1% among the Malaysians donors in three ethnic groups viz, Malay, Chinese and Indian. It was present among 1.25% of 401 Malay, 4.01% of Chinese and 0.88% of 114 Indian origin donors. None of the 1442 patients, including 703 antenatal outpatients, had anti-Di(a in serum. Conclusion: The prevalence of Di(a antigen was found among the donors of all the three ethnic background with varying frequency. Inclusion of Di(a+ red cells in routine antibody screening program would certainly help in detection of this clinically significant antibody and to provide safe blood transfusion in the Klang Valley, though the incidence of antibody appears to be very low in the region.

  7. Prevalence of Diego blood group antigen and the antibody in three ethnic population groups in Klang valley of Malaysia.

    Science.gov (United States)

    Wei, Cheong Tar; Al-Hassan, Faisal Muti; Naim, Norris; Knight, Aishah; Joshi, Sanmukh R

    2013-01-01

    Diego blood group antigen, Di(a), is very rare among Caucasians and Blacks, but relatively common among the South American Indians and Asians of Mongolian origin. The antibody to Di(a) is clinically significant to cause hemolytic disease in a new-born or hemolytic transfusion reaction. This study was designed to determine the prevalence of Di(a) antigen among the blood donors from the three major ethnic groups in Klang Valley of Malaysia as well as to find an incidence of an antibody of the Diego antigen, anti-Di(a), in a tertiary care hospital to ascertain the need to include Di(a+) red cells for an antibody screen cell panel. Serological tests were performed by column agglutination technique using commercial reagents and following instruction as per kit insert. Di(a) antigen was found with a frequency of 2.1% among the Malaysians donors in three ethnic groups viz, Malay, Chinese and Indian. It was present among 1.25% of 401 Malay, 4.01% of Chinese and 0.88% of 114 Indian origin donors. None of the 1442 patients, including 703 antenatal outpatients, had anti-Di(a) in serum. The prevalence of Di(a) antigen was found among the donors of all the three ethnic background with varying frequency. Inclusion of Di(a+) red cells in routine antibody screening program would certainly help in detection of this clinically significant antibody and to provide safe blood transfusion in the Klang Valley, though the incidence of antibody appears to be very low in the region.

  8. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    International Nuclear Information System (INIS)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-01-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

  9. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    Energy Technology Data Exchange (ETDEWEB)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  10. Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

    Science.gov (United States)

    Magaña-Ortíz, Denis; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

    2018-01-01

    Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104 kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

  11. Distribution of 125I-monoclonal antibodies to antigen Ly 2.1 of T-lymphocytes in mice under the influence of immunomodulators

    International Nuclear Information System (INIS)

    Mirolyubova, Sh.Yu.; Fadeev, N.P.; Serzhanina, V.A.; Klimovich, V.B.; Makarenko, M.V.; Korsakova, L.N.

    1991-01-01

    A study was made of the distribution of 125 I (a chloramine method of labelling) monoclonal antibodies to the surface antigen Ly 2.1 T-lymphocytes during action of immunomodulators (tactivin, hydrocortisone, tactivin administered after hydrocortisone) on ACR mice. These antibodies were shown to retain antigen binding capacity, permitting monitoring of the redistribution of the antigen in the body exposed to immunomodulators

  12. Temporal relation of antigenaemia and loss of antibodies to core antigens to development of clinical disease in HIV infection

    DEFF Research Database (Denmark)

    Pedersen, C; Nielsen, C M; Vestergaard, B F

    1987-01-01

    and 16 months after the estimated time of seroconversion. These results show that the late stages of HIV infection are characterised by increased production of antigen and a decrease in antibodies directed against the core protein. Antigenaemia indicates a poor prognosis; and as the antigen test......A total of 276 sequential serum samples from 34 men with antibodies to the human immunodeficiency virus (HIV) followed up for two to seven years were analysed for HIV antigen and antibodies to the viral core and envelope proteins. Results were correlated with clinical outcome and CD4 T lymphocyte...... count. Both antigenaemia and the disappearance of antibodies to the core protein were associated with development of the acquired immune deficiency syndrome (AIDS) or AIDS related complex and depletion of CD4 cells. Thus AIDS or AIDS related complex developed in eight out of 16 patients...

  13. Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

    Directory of Open Access Journals (Sweden)

    Fleur S. van de Bovenkamp

    2018-04-01

    Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

  14. Acquired Antibody Responses against Plasmodium vivax Infection Vary with Host Genotype for Duffy Antigen Receptor for Chemokines (DARC)

    Science.gov (United States)

    Maestre, Amanda; Muskus, Carlos; Duque, Victoria; Agudelo, Olga; Liu, Pu; Takagi, Akihide; Ntumngia, Francis B.; Adams, John H.; Sim, Kim Lee; Hoffman, Stephen L.; Corradin, Giampietro; Velez, Ivan D.; Wang, Ruobing

    2010-01-01

    Background Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are ‘resistant’ to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. Methodology/Findings We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. Conclusion/Significance Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the

  15. Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC.

    Directory of Open Access Journals (Sweden)

    Amanda Maestre

    2010-07-01

    Full Text Available Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens.We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1 and Duffy binding protein (PvDBP varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B. The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion.Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades

  16. Identification of a surface antigen on Theileria parva sporozoites by monoclonal antibody.

    OpenAIRE

    Dobbelaere, D A; Shapiro, S Z; Webster, P

    1985-01-01

    A mouse monoclonal antibody (mAbD1) that neutralizes sporozoites of different stocks of the protozoan parasite Theileria parva has been used to localize and identify a sporozoite antigen. Protein A-colloidal gold was used to localize bound mAbD1 in immunoelectron microscopic studies. mAbD1 bound to sporozoite antigen, which was evenly spread over the surface of all sporozoites. Immune complexes were obtained by incubation of sporozoite suspensions with mAbD1 followed by Zwittergent 3-14 extra...

  17. Greek rheumatoid arthritis patients have elevated levels of antibodies against antigens from Proteus mirabilis.

    Science.gov (United States)

    Christopoulos, Georgios; Christopoulou, V; Routsias, J G; Babionitakis, A; Antoniadis, C; Vaiopoulos, G

    2017-03-01

    Patients with rheumatoid arthritis (RA) from different ethnic groups present elevated levels of antibodies against Proteus mirabilis. This finding implicates P. mirabilis in the development of RA. The aim of this study was to investigate the importance of P. mirabilis in the etiopathogenesis of RA in Greek RA patients. In this study, 63 patients with RA and 38 healthy controls were included. Class-specific antibodies IgM, IgG, and IgA against three human cross-reactive and non-cross-reactive synthetic peptides from P. mirabilis-hemolysin (HpmB), urease C (UreC), and urease F (UreF)-were performed in all subjects, using the ELISA method. RA patients had elevated levels of IgM, IgG, and IgA antibodies against HpmB and UreC Proteus peptide which are significantly different compared to healthy controls: p = 0.005, p Proteus peptide-which are non-cross-reactive with human tissue antigens-were observed and their significant difference compared to healthy controls (p = 0.007, p mirabilis antigenic epitopes, such as in North European populations, albeit Greek RA patients presenting the cross-reaction antigen in a low percentage. These results indicate that P. mirabilis through the molecular mimicry mechanism leads to inflammation and damage of the joints in RA.

  18. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    Science.gov (United States)

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  19. Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    Sofía Duque-Beltrán

    2002-12-01

    Full Text Available The present study developed and standardized an enzime-linked immunosorbent assay (ELISA to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate. One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%; specificity, 95% (95% CI: 88.6-97.6%; positive predictive value, 91% (95% CI: 81.4-95.9%; and negative predictive value, 100% (95% CI: 96.1-100%. This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

  20. Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies

    Directory of Open Access Journals (Sweden)

    Katherine E. Harris

    2018-04-01

    Full Text Available We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1. This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

  1. Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

    Science.gov (United States)

    Paredes, Adriana; Sáenz, Patricia; Marzal, Miguel W; Orrego, Miguel A; Castillo, Yesenia; Rivera, Andrea; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E

    2016-07-01

    Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. A new application of scanning electrochemical microscopy for the label-free interrogation of antibody-antigen interactions

    Energy Technology Data Exchange (ETDEWEB)

    Holmes, Joanne L.; Davis, Frank; Collyer, Stuart D. [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom); Higson, Seamus P.J., E-mail: s.p.j.higson@cranfield.ac.uk [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom)

    2011-03-18

    Within this work we present a 'proof of principle' study for the use of scanning electrochemical microscopy (SECM) to detect and image biomolecular interactions in a label-free assay as a potential alternative to current fluorescence techniques. Screen-printed carbon electrodes were used as the substrate for the deposition of a dotted array, where the dots consist of biotinylated polyethyleneimine. These were then further derivatised, first with neutravidin and then with a biotinylated antibody to the protein neuron specific enolase (NSE). SECM using a ferrocene carboxylic acid mediator showed clear differences between the array and the surrounding unmodified carbon. Imaging of the arrays before and following exposure to various concentrations of the antigen showed clear evidence for specific binding of the NSE antigen to the antibody derivatised dots. Non-specific binding was quantified. Control experiments with other proteins showed only non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen at the surface of the dots. Binding of the antigen was accompanied by a measured increase in current response, which may be explained in terms of protein electrostatic interaction and hydrophobic interactions to the mediator, thereby increasing the localised mediator flux. A calibration curve was obtained between 500 fg mL{sup -1} to 200 pg mL{sup -1} NSE which demonstrated a logarithmic relationship between the current change upon binding and antigen concentration without the need for any labelling of the substrate.

  3. Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

    Science.gov (United States)

    Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

    2017-09-02

    The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. New monoclonal antibodies to rat testicular antigen, TEC-21

    Czech Academy of Sciences Publication Activity Database

    Hálová, Ivana; Dráberová, Lubica; Dráber, Petr

    2001-01-01

    Roč. 47, č. 5 (2001), s. 180-182 ISSN 0015-5500 R&D Projects: GA ČR GV312/96/K205; GA ČR GA204/00/0204; GA ČR GA310/00/0205; GA AV ČR IAA5052005; GA AV ČR IAA7052006; GA MŠk LN00A026 Institutional research plan: CEZ:AV0Z5052915 Keywords : monoclonal antibody * GPI-anchored * testicular antigen Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  5. In vivo localization of radiolabeled monoclonal antibody to carcinoembryonic antigen (CEA) in a CEA-producing tumor

    International Nuclear Information System (INIS)

    Kamei, Tetsuya; Seto, Hikaru; Taki, Kuniyasu; Soya, Toshio; Kakishita, Masao; Maeda, Masatoshi; Honda, Takashi; Koshimura, Saburou.

    1987-01-01

    To compare accumulation of the 125 I-labeled antibodies(anti-carcinoembryonic antigen(CEA) monoclonal antibody and polyclonal antibody) to a CEA-producing tumor (SC-2-JCK), an in vivo localization study was performed in nude mice. The tumor-to-blood ratio at 120 hours after injection rose to 4.6 for the monoclonal antibody, but remained at 1.3 for the polyclonal antibody. However, no differences were noted between the antibodies up to 72 hours after injection. In autoradiograms, selective accumulation of the tracer was noted in the tumor for both antibodies. However, no superiority or inferiority of imaging for either of the antibodies could be definitely determined. (author)

  6. Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients.

    Science.gov (United States)

    Ayat, Hoda; Burrone, Oscar R; Sadghizadeh, Majid; Jahanzad, Eissa; Rastgou, Nasrin; Moghadasi, Sarrira; Arbabi, Mehdi

    2013-11-01

    Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These 'immune' libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning. Copyright © 2013 The International Alliance for Biological Standardization. All rights reserved.

  7. Enhanced acquired antibodies to a chimeric Plasmodium falciparum antigen; UB05-09 is associated with protective immunity against malaria.

    Science.gov (United States)

    Dinga, J N; Gamua, S D; Titanji, V P K

    2017-08-01

    It has been shown that covalently linking two antigens could enhance the immunogenicity of the chimeric construct. To prioritize such a chimera for malaria vaccine development, it is necessary to demonstrate that naturally acquired antibodies against the chimera are associated with protection from malaria. Here, we probe the ability of a chimeric construct of UB05 and UB09 antigens (UB05-09) to better differentiate between acquired immune protection and susceptibility to malaria. In a cross-sectional study, recombinant UB05-09 chimera and the constituent antigens were used to probe for specific antibodies in the plasma from children and adults resident in a malaria-endemic zone, using the enzyme-linked immunosorbent assay (ELISA). Anti-UB05-09 antibody levels doubled that of its constituent antigens, UB09 and UB05, and this correlated with protection against malaria. The presence of enhanced UB05-09-specific antibody correlated with the absence of fever and parasitaemia, which are the main symptoms of malaria infection. The chimera is more effective in detecting and distinguishing acquired protective immunity against malaria than any of its constituents taken alone. Online B-cell epitope prediction tools confirmed the presence of B-cell epitopes in the study antigens. UB05-09 chimera is a marker of protective immunity against malaria that needs to be studied further. © 2017 John Wiley & Sons Ltd.

  8. Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

    Science.gov (United States)

    Tipton, Thomas R W; Roghanian, Ali; Oldham, Robert J; Carter, Matthew J; Cox, Kerry L; Mockridge, C Ian; French, Ruth R; Dahal, Lekh N; Duriez, Patrick J; Hargreaves, Philip G; Cragg, Mark S; Beers, Stephen A

    2015-03-19

    Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. © 2015 by The American Society of Hematology.

  9. Antibodies to variable Plasmodium falciparum-infected erythrocyte surface antigens are associated with protection from novel malaria infections

    DEFF Research Database (Denmark)

    Giha, H A; Staalsoe, T; Dodoo, D

    2000-01-01

    is maintained at low densities. Here, we test the hypothesis that the presence or absence of antibodies against variant antigens on the surface of P. falciparum-infected erythrocytes protect individuals against some infectious challenges and render them susceptible to others. Plasma collected in Daraweesh...... susceptible and protected individuals. Together, the results indicate that pre-existing anti-PfEMP1 antibodies can reduce the risk of contracting clinical malaria when challenged by novel parasite clones expressing homologous, but not heterologous variable surface antigens. The results also confirm...

  10. An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies.

    Science.gov (United States)

    Dekker, Lennard; Wu, Si; Vanduijn, Martijn; Tolić, Nikolai; Stingl, Christoph; Zhao, Rui; Luider, Theo; Paša-Tolić, Ljiljana

    2014-05-01

    We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

    Directory of Open Access Journals (Sweden)

    Ana Teresa B.F. Antonangelo

    2012-09-01

    Full Text Available Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC. The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

  12. Effect of praziquantel treatment of Schistosoma mansoni during pregnancy on intensity of infection and antibody responses to schistosome antigens

    DEFF Research Database (Denmark)

    Tweyongyere, Robert; Mawa, Patrice A.; Emojong, Nicholas O.

    2009-01-01

    with those first treated after delivery (undetected (88.5%), light (10.6%), moderate (0.9%) and heavy (0%), p = 0.16). Parasite specific antibody levels were lower during pregnancy than after delivery. Praziquantel treatment during pregnancy boosted anti-worm IgG isotypes and to a lesser extent Ig......E, but these boosts were less pronounced than in women whose treatment was delayed until after delivery. Praziquantel had limited effects on antibodies against egg antigens. Conclusion S mansoni antigen-specific antibody levels and praziquantel-induced boosts in antibody levels were broadly suppressed during...

  13. Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

    Science.gov (United States)

    Igawa, Tomoyuki

    2017-01-01

    Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

  14. Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

    OpenAIRE

    Alderete, J F

    1984-01-01

    An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses durin...

  15. Differential Killing of Salmonella enterica Serovar Typhi by Antibodies Targeting Vi and Lipopolysaccharide O:9 Antigen.

    Directory of Open Access Journals (Sweden)

    Peter J Hart

    Full Text Available Salmonella enterica serovar Typhi expresses a capsule of Vi polysaccharide, while most Salmonella serovars, including S. Enteritidis and S. Typhimurium, do not. Both S. Typhi and S. Enteritidis express the lipopolysaccharide O:9 antigen, yet there is little evidence of cross-protection from anti-O:9 antibodies. Vaccines based on Vi polysaccharide have efficacy against typhoid fever, indicating that antibodies against Vi confer protection. Here we investigate the role of Vi capsule and antibodies against Vi and O:9 in antibody-dependent complement- and phagocyte-mediated killing of Salmonella. Using isogenic Vi-expressing and non-Vi-expressing derivatives of S. Typhi and S. Typhimurium, we show that S. Typhi is inherently more sensitive to serum and blood than S. Typhimurium. Vi expression confers increased resistance to both complement- and phagocyte-mediated modalities of antibody-dependent killing in human blood. The Vi capsule is associated with reduced C3 and C5b-9 deposition, and decreased overall antibody binding to S. Typhi. However, purified human anti-Vi antibodies in the presence of complement are able to kill Vi-expressing Salmonella, while killing by anti-O:9 antibodies is inversely related to Vi expression. Human serum depleted of antibodies to antigens other than Vi retains the ability to kill Vi-expressing bacteria. Our findings support a protective role for Vi capsule in preventing complement and phagocyte killing of Salmonella that can be overcome by specific anti-Vi antibodies, but only to a limited extent by anti-O:9 antibodies.

  16. Differential Killing of Salmonella enterica Serovar Typhi by Antibodies Targeting Vi and Lipopolysaccharide O:9 Antigen.

    Science.gov (United States)

    Hart, Peter J; O'Shaughnessy, Colette M; Siggins, Matthew K; Bobat, Saeeda; Kingsley, Robert A; Goulding, David A; Crump, John A; Reyburn, Hugh; Micoli, Francesca; Dougan, Gordon; Cunningham, Adam F; MacLennan, Calman A

    2016-01-01

    Salmonella enterica serovar Typhi expresses a capsule of Vi polysaccharide, while most Salmonella serovars, including S. Enteritidis and S. Typhimurium, do not. Both S. Typhi and S. Enteritidis express the lipopolysaccharide O:9 antigen, yet there is little evidence of cross-protection from anti-O:9 antibodies. Vaccines based on Vi polysaccharide have efficacy against typhoid fever, indicating that antibodies against Vi confer protection. Here we investigate the role of Vi capsule and antibodies against Vi and O:9 in antibody-dependent complement- and phagocyte-mediated killing of Salmonella. Using isogenic Vi-expressing and non-Vi-expressing derivatives of S. Typhi and S. Typhimurium, we show that S. Typhi is inherently more sensitive to serum and blood than S. Typhimurium. Vi expression confers increased resistance to both complement- and phagocyte-mediated modalities of antibody-dependent killing in human blood. The Vi capsule is associated with reduced C3 and C5b-9 deposition, and decreased overall antibody binding to S. Typhi. However, purified human anti-Vi antibodies in the presence of complement are able to kill Vi-expressing Salmonella, while killing by anti-O:9 antibodies is inversely related to Vi expression. Human serum depleted of antibodies to antigens other than Vi retains the ability to kill Vi-expressing bacteria. Our findings support a protective role for Vi capsule in preventing complement and phagocyte killing of Salmonella that can be overcome by specific anti-Vi antibodies, but only to a limited extent by anti-O:9 antibodies.

  17. Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

    OpenAIRE

    Nam, Ho-Woo; Kang, Seung-Won; Choi, Won-Young

    1998-01-01

    Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kD...

  18. The hypervariable region of Streptococcus pyogenes M protein escapes antibody attack by antigenic variation and weak immunogenicity

    DEFF Research Database (Denmark)

    Lannergård, Jonas; Gustafsson, Caj Ulrik Mattias; Waldemarsson, Johan

    2011-01-01

    Sequence variation of antigenic proteins allows pathogens to evade antibody attack. The variable protein commonly includes a hypervariable region (HVR), which represents a key target for antibodies and is therefore predicted to be immunodominant. To understand the mechanism(s) of antibody evasion...

  19. Genotoxic effect and antigen binding characteristics of SLE auto-antibodies to peroxynitrite-modified human DNA.

    Science.gov (United States)

    Khan, Md Asad; Alam, Khursheed; Mehdi, Syed Hassan; Rizvi, M Moshahid A

    2017-12-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by auto-antibodies against native deoxyribonucleic acid after modification and is one of the reasons for the development of SLE. Here, we have evaluated the structural perturbations in human placental DNA by peroxynitrite using spectroscopy, thermal denaturation and high-performance liquid chromatography (HPLC). Peroxynitrite is a powerful potent bi-functional oxidative/nitrative agent that is produced both endogenously and exogenously. In experimental animals, the peroxynitrite-modified DNA was found to be highly immunogenic. The induced antibodies showed cross-reactions with different types of DNA and nitrogen bases that were modified with peroxynitrite by inhibition ELISA. The antibody activity was inhibited by approximately 89% with its immunogen as the inhibitor. The antigen-antibodies interaction between induced antibodies with peroxynitrite-modified DNA showed retarded mobility as compared to the native form. Furthermore, significantly increased binding was also observed in SLE autoantibodies with peroxynitrite-modified DNA than native form. Moreover, DNA isolated from lymphocyte of SLE patients revealed significant recognition of anti-peroxynitrite-modified DNA immunoglobulin G (IgG). Our data indicates that DNA modified with peroxynitrite presents unique antigenic determinants that may induce autoantibody response in SLE. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

    DEFF Research Database (Denmark)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-01-01

    microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 oC without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies...

  1. Seroprevalence of Antibodies against Plasmodium falciparum Sporozoite Antigens as Predictive Disease Transmission Markers in an Area of Ghana with Seasonal Malaria Transmission.

    Directory of Open Access Journals (Sweden)

    Kwadwo A Kusi

    Full Text Available As an increasing number of malaria-endemic countries approach the disease elimination phase, sustenance of control efforts and effective monitoring are necessary to ensure success. Mathematical models that estimate anti-parasite antibody seroconversion rates are gaining relevance as more sensitive transmission intensity estimation tools. Models however estimate yearly seroconversion and seroreversion rates and usually predict long term changes in transmission, occurring years before the time of sampling. Another challenge is the identification of appropriate antigen targets since specific antibody levels must directly reflect changes in transmission patterns. We therefore investigated the potential of antibodies to sporozoite and blood stage antigens for detecting short term differences in malaria transmission in two communities in Northern Ghana with marked, seasonal transmission.Cross-sectional surveys were conducted during the rainy and dry seasons in two communities, one in close proximity to an irrigation dam and the other at least 20 Km away from the dam. Antibodies against the sporozoite-specific antigens circumsporozoite protein (CSP and Cell traversal for ookinetes and sporozoites (CelTOS and the classical blood stage antigen apical membrane antigen 1 (AMA1 were measured by indirect ELISA. Antibody levels and seroprevalence were compared between surveys and between study communities. Antibody seroprevalence data were fitted to a modified reversible catalytic model to estimate the seroconversion and seroreversion rates.Changes in sporozoite-specific antibody levels and seroprevalence directly reflected differences in parasite prevalence between the rainy and dry seasons and hence the extent of malaria transmission. Seroconversion rate estimates from modelled seroprevalence data did not however support the above observation.The data confirms the potential utility of sporozoite-specific antigens as useful markers for monitoring short term

  2. Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

    Directory of Open Access Journals (Sweden)

    Fred Luciano Neves Santos

    Full Text Available The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6, demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

  3. High-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination.

    Science.gov (United States)

    Tan, Yann-Chong; Blum, Lisa K; Kongpachith, Sarah; Ju, Chia-Hsin; Cai, Xiaoyong; Lindstrom, Tamsin M; Sokolove, Jeremy; Robinson, William H

    2014-03-01

    We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination. Published by Elsevier Inc.

  4. Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A P; Rezapkin, G V; Dzagurova, T K; Tkachenko, E A [Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow

    1984-05-01

    Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

  5. Use of Monoclonal Antibodies to Lipopolysaccharide for Antigenic Analysis of Coxiella burnetii

    OpenAIRE

    Hotta, Akitoyo; Kawamura, Midori; To, Ho; Andoh, Masako; Yamaguchi, Tsuyoshi; Fukushi, Hideto; Amano, Ken-Ichi; Hirai, Katsuya

    2003-01-01

    Antigenic differences among Coxiella burnetii strains were analyzed. The monoclonal antibodies against the lipopolysaccharide outer core did not react with the strains containing a QpRS plasmid or with plasmidless strains, whereas they reacted with strains containing a QpH1 or QpDV plasmid. C. burnetii isolates could be divided into two groups immunologically.

  6. High prevalence of HIV p24 antigen among HIV antibody negative prospective blood donors in Ile-Ife, Nigeria.

    Science.gov (United States)

    Japhet, Margaret Oluwatoyin; Adewumi, Moses Olubusuyi; Adesina, Olufisayo Adeyemi; Donbraye, Emmanuel

    2016-01-01

    Blood transfusion service centers in Nigeria screen donated blood for markers of HIV infection using antibody- (Ab) based rapid test and in some centers, positives are re-tested using Ab-based ELISA. Paucity of data exists on p24 antigen prevalence among HIV Ab-negative donors in Nigeria. This study aims at detecting HIV p24 antigen among prospective blood donors in Osun State, Nigeria. Prospective blood donors negative for HIV antibodies using Determine test kit were re-tested using BIORAD GENSCREEN Ultra Ag-Ab ELISA kit, a fourth-generation ELISA kit that detects HIV antibodies/p24 antigen. Of the 169 HIV Ab-negative prospective donors, 10 (5.9%) were positive for HIV p24 antigen and 70% (7/10) of them were in the age range 18-30 years. Results of this study show that blood transfusion is still one of the major routes of HIV transmission in Nigeria and a higher proportion is among youth. Inclusion of p24 antigen testing into the blood donor screening will help reduce transfusion associated HIV in Nigeria if Nucleic Acid Testing (NAT) of all blood donor samples is not affordable; also, HIV enlightenment programs tailored toward youth may help reduce this rate among donors since more young people donate blood in low/middle-income countries than in high-income countries.

  7. Deletion of Dock10 in B Cells Results in Normal Development but a Mild Deficiency upon In Vivo and In Vitro Stimulations

    Directory of Open Access Journals (Sweden)

    Eva Severinson

    2017-05-01

    Full Text Available We sought to identify genes necessary to induce cytoskeletal change in B cells. Using gene expression microarray, we compared B cells stimulated with interleukin-4 (IL-4 and anti-CD40 antibodies that induce B cell spreading, cell motility, tight aggregates, and extensive microvilli with B cells stimulated with lipopolysaccharide that lack these cytoskeletal changes. We identified 84 genes with 10-fold or greater expression in anti-CD40 + IL-4 stimulated B cells, one of these encoded the guanine nucleotide exchange factor (GEF dedicator of cytokinesis 10 (Dock10. IL-4 selectively induced Dock10 expression in B cells. Using lacZ expression to monitor Dock10 promoter activity, we found that Dock10 was expressed at all stages during B cell development. However, specific deletion of Dock10 in B cells was associated with a mild phenotype with normal B cell development and normal B cell spreading, polarization, motility, chemotaxis, aggregation, and Ig class switching. Dock10-deficient B cells showed lower proliferation in response to anti-CD40 and IL-4 stimulation. Moreover, the IgG response to soluble antigen in vivo was lower when Dock10 was specifically deleted in B cells. Together, we found that most B cell responses were intact in the absence of Dock10. However, specific deletion of Dock10 in B cells was associated with a mild reduction in B cell activation in vitro and in vivo.

  8. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    International Nuclear Information System (INIS)

    Bujak, Emil; Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah; Neri, Dario

    2014-01-01

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  9. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Bujak, Emil [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland); Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah [Philochem AG, Libernstrasse 3, CH-8112 Otelfingen (Switzerland); Neri, Dario, E-mail: neri@pharma.ethz.ch [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)

    2014-09-10

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  10. Use of Monoclonal Antibodies to Lipopolysaccharide for Antigenic Analysis of Coxiella burnetii

    Science.gov (United States)

    Hotta, Akitoyo; Kawamura, Midori; To, Ho; Andoh, Masako; Yamaguchi, Tsuyoshi; Fukushi, Hideto; Amano, Ken-Ichi; Hirai, Katsuya

    2003-01-01

    Antigenic differences among Coxiella burnetii strains were analyzed. The monoclonal antibodies against the lipopolysaccharide outer core did not react with the strains containing a QpRS plasmid or with plasmidless strains, whereas they reacted with strains containing a QpH1 or QpDV plasmid. C. burnetii isolates could be divided into two groups immunologically. PMID:12682176

  11. Detection of antibodies to the extractable nuclear antigens by enzyme linked immunosorbent assay

    International Nuclear Information System (INIS)

    Aziz, Khalil A.; Fzizal, Abul A.

    2005-01-01

    Anti-extractable nuclear antigen (ENA) antibodies are a group of autoantibodies that are directed against various components of the cell nucleus. Antibodies to these antigens are closely associated with connective tissue disease. Early diagnosis of these diseases can prove very difficult and therefore clinicians rely on the use of anti-ENA antibody testing for the exclusion. Old methods of testing are time consuming and require great skills. For these reasons clinical immunology laboratories are switching to testing for anti-ENA antibodies by enzyme linked immunosorbent assay (ELISA). The latter assays are more sensitive and require little skills. In the present study we have investigated a number of different ELISA preparations. The study was conducted at Birmingham Heartlands Hospital during the period 2003. We tested a number of ENA-positive and negative samples using 3 different commercial ELISA preparations and compared the results with traditional CCIE-assay. The present study revealed that some ELISA preparations can be more sensitive than CCIE method. Laboratories still using later method should switch to ELISA. However it is important that laboratories evaluate a long range of different ELISA preparations before selecting the most optimal one. In addition it is recommended that laboratories then audit results in order to determine true significance of such results. Finally until the true significance of ELISA generated results is known, positive ENA-results should be interpreted in conjunction with the clinical picture and this would require close liaison in between the clinical immunology laboratory and clinicians

  12. Radioimmunoassays of hidden viral antigens

    International Nuclear Information System (INIS)

    Neurath, A.R.; Strick, N.; Baker, L.; Krugman, S.

    1982-01-01

    Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure

  13. Adoptive transfer of natural antibodies to non-immunized chickens affects subsequent antigen-specific humoral and cellular immune responses

    NARCIS (Netherlands)

    Lammers, A.; Klomp, M.E.V.; Nieuwland, M.G.B.; Savelkoul, H.F.J.; Parmentier, H.K.

    2004-01-01

    To determine a regulatory function of natural antibodies in the immune response of chickens, pooled plasma obtained from non-immunized (naive) 15 months old hens was subjected to keyhole limpet hemocyanin (KLH) antigen-affinity chromatography. Purified KLH-binding antibodies were adoptively

  14. DOCK8 is critical for the survival and function of NKT cells.

    Science.gov (United States)

    Crawford, Greg; Enders, Anselm; Gileadi, Uzi; Stankovic, Sanda; Zhang, Qian; Lambe, Teresa; Crockford, Tanya L; Lockstone, Helen E; Freeman, Alexandra; Arkwright, Peter D; Smart, Joanne M; Ma, Cindy S; Tangye, Stuart G; Goodnow, Christopher C; Cerundolo, Vincenzo; Godfrey, Dale I; Su, Helen C; Randall, Katrina L; Cornall, Richard J

    2013-09-19

    Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper-immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1(+) NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease.

  15. DOCK8 is critical for the survival and function of NKT cells

    Science.gov (United States)

    Crawford, Greg; Enders, Anselm; Gileadi, Uzi; Stankovic, Sanda; Zhang, Qian; Lambe, Teresa; Crockford, Tanya L.; Lockstone, Helen E.; Freeman, Alexandra; Arkwright, Peter D.; Smart, Joanne M.; Ma, Cindy S.; Tangye, Stuart G.; Goodnow, Christopher C.; Cerundolo, Vincenzo; Godfrey, Dale I.; Su, Helen C.; Randall, Katrina L.

    2013-01-01

    Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper–immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1+ NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease. PMID:23929855

  16. Means for use in radioimmunodetection of an antigen-antibody reaction

    International Nuclear Information System (INIS)

    Reynolds, J.H.

    1983-01-01

    A unitized, solid phase kit for radioimmunoassay is described. All of the necessary assay reagents are incorporated in a single tube. Requiring only the addition of the patient's sample, all phases of the assay procedure are performed in this tube. Antibodies are bound to the tube surface, while labelled antigens are also present but unbound. Storage in the absence of air and water results in the stabilization of the reagents such that the system can be stored for long periods

  17. Systemic and Mucosal Antibody Responses to Soluble and Nanoparticle-Conjugated Antigens Administered Intranasally

    Directory of Open Access Journals (Sweden)

    Savannah E. Howe

    2016-10-01

    Full Text Available Nanoparticles (NPs are increasingly being used for drug delivery, as well as antigen carriers and immunostimulants for the purpose of developing vaccines. In this work, we examined how intranasal (i.n. priming followed by i.n. or subcutaneous (s.c. boosting immunization affects the humoral immune response to chicken ovalbumin (Ova and Ova conjugated to 20 nm NPs (NP-Ova. We show that i.n. priming with 20 mg of soluble Ova, a dose known to trigger oral tolerance when administered via gastric gavage, induced substantial systemic IgG1 and IgG2c, as well as mucosal antibodies. These responses were further boosted following a s.c. immunization with Ova and complete Freund’s adjuvant (Ova+CFA. In contrast, 100 µg of Ova delivered via NPs induced an IgG1-dominated systemic response, and primed the intestinal mucosa for secretion of IgA. Following a secondary s.c. or i.n. immunization with Ova+CFA or NP-Ova, systemic IgG1 titers significantly increased, and serum IgG2c and intestinal antibodies were induced in mice primed nasally with NP-Ova. Only Ova- and NP-Ova-primed mice that were s.c.-boosted exhibited substantial systemic and mucosal titers for up to 6 months after priming, whereas the antibodies of i.n.-boosted mice declined over time. Our results indicate that although the amount of Ova delivered by NPs was 1000-fold less than Ova delivered in soluble form, the antigen-specific antibody responses, both systemic and mucosal, are essentially identical by 6 months following the initial priming immunization. Additionally, both i.n.- and s.c.-boosting strategies for NP-Ova-primed mice were capable of inducing a polarized Th1/Th2 immune response, as well as intestinal antibodies; however, it is only by using a heterogeneous prime-boost strategy that long-lasting antibody responses were initiated. These results provide valuable insight for future mucosal vaccine development, as well as furthering our understanding of mucosal antibody responses.

  18. Development of a Novel, Ultra-rapid Biosensor for the Qualitative Detection of Hepatitis B Virus-associated Antigens and Anti-HBV, Based on “Membrane-engineered” Fibroblast Cells with Virus-Specific Antibodies and Antigens

    Directory of Open Access Journals (Sweden)

    Antonios Perdikaris

    2009-03-01

    Full Text Available A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV-associated antigens and anti-HBV is described. The biosensor is based on “membrane-engineered” Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe or antigens (HBsAg in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, respectively, of the antibody to the electroinserted antigen triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA. The sensor was used for screening 133 clinical blood serum samples according to a double-blind protocol. Considerably higher sensor responses were observed against HBV-positive samples, compared with responses against negative samples or samples positive for heterologous hepatitis viruses such as Hepatitis C (HCV virus. Detection of anti-HBs antibodies was made possible by using a biosensor based on immobilized Vero cells bearing the respective antigen (HBsAg. The observed response was rapid (45 sec and quite reproducible. Fluorescence microscopy observations showed that attachment of HBV particles to cells membrane-engineered with anti-HBs was associated with a decrease of [Ca2+]cyt. The perspectives for using the novel biosensor as a qualitative, rapid screening, high throughput assay for HBV antigens and anti-HBs in clinical samples is discussed.

  19. A solid phase radio immunoassay on hydrophobic membrane filters: detection of antibodies to gonocal surface antigens

    International Nuclear Information System (INIS)

    Lambden, P.R.; Watt, P.J.

    1978-01-01

    A solid phase radioimmunoassay (SPRIA) has been developed for detection of IgG antibodies to gonococcal outer membrane components. Gonococcal antigens was immobilised on a solid support by covalent coupling to CNBr-activated Sepharose in the presence of the detergent Triton X-100. Binding of specific antibody to the Sepharose-antigen complex was detected using radiolabelled Protein A as the antiglobulin. Protein A was labelled by radioacetylation with tritiated acetic anhydride, yielding a product of high specific activity and high stability. No detectable loss of activity was observed over a ten month period. The entire assay was performed on Mitex teflon hydrophobic membrane filters which held the Sepharose beads and aqueous supernatant as a discrete drop of liquid. The supernatants and incubation were easily and rapidly removed from the beads by suction on a specially-designed manifold system. This procedure removed the need for repeated and time-consuming centrifugations. Titres were obtained graphically from double log plots of cpm bound versus antiserum dilution by extrapolation of the straight line to a point corresponding to twice the control level of radioactivity binding. The assay proved to be a very reliable and simple procedure for the detection of IgG antibodies to gonococcal surface antigens. (Auth.)

  20. Chlorphenesin: an antigen-associated immunosuppressant.

    Science.gov (United States)

    Whang, H Y; Neter, E

    1970-07-01

    Chlorphenesin (3-p-chlorophenoxy-1,2-propanediol), when injected intravenously together with either of two common bacterial antigens, inhibits the antibody response of the rabbit. The antigens studied are those common to Enterobacteriaceae and to gram-positive bacteria. The immunosuppression is contingent upon incubation of chlorphenesin and antigen in vitro prior to administration, since separate injection of antigen and inhibitor or of mixtures without prior incubation yields undiminished antibody response. Chlorphenesin, as shown by hemagglutination-inhibition tests, does not alter the antigenic determinants, because antibody neutralization occurs in the presence or absence of the drug. The immunosuppressive effect is reversible, since precipitation of chlorphenesin at 4 C substantially restores immunogenicity. Animals immunized with antigen-drug mixtures, which fail to respond with significant antibody production, nonetheless are immunologically primed. It is concluded that chlorphenesin represents another example of antigen-associated immunosuppressants.

  1. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    International Nuclear Information System (INIS)

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-01-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

  2. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    Directory of Open Access Journals (Sweden)

    Nan Zhong

    Full Text Available We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP protocols.

  3. Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans

    1985-01-01

    Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera...... chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide....

  4. Glycosphingolipid antigens from Leishmania (L. amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    A.H. Straus

    1997-03-01

    Full Text Available Specific glycosphingolipid antigens of Leishmania (L. amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC. Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L. amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

  5. Identification of an antigenic domain on Mycobacterium leprae protein antigen 85B, which is specifically recognized by antibodies from patients with leprosy

    NARCIS (Netherlands)

    Filley, E.; Thole, J. E.; Rook, G. A.; Nagai, S.; Waters, M.; Drijfhout, J. W.; Rinke de Wit, T. F.; de Vries, R. R.; Abou-Zeid, C.

    1994-01-01

    Sixty-three overlapping 15-oligomer peptides covering the 30-kDa protein antigen 85B of Mycobacterium leprae were tested by ELISA to identify epitopes recognized by human antibodies. Serum samples from patients with lepromatous leprosy (LL) reacted mainly with peptides comprising amino acid regions

  6. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China)

    2017-06-15

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC{sub 50} value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  7. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    International Nuclear Information System (INIS)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin; Yu, Ruijin; Lai, Weihua; Xiong, Yonghua

    2017-01-01

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC_5_0 value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  8. A new technique to detect antibody-antigen reaction (biological interactions) on a localized surface plasmon resonance (LSPR) based nano ripple gold chip

    Energy Technology Data Exchange (ETDEWEB)

    Saleem, Iram, E-mail: iiram.qau@gmail.com [Department of Physics and Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States); Widger, William, E-mail: widger@uh.edu [Department of Biology and Biochemistry and Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States); Chu, Wei-Kan, E-mail: wkchu@uh.edu [Department of Physics and Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States)

    2017-07-31

    Highlights: • The nano ripple LSPR chip has monolayer molecule-coating sensitivity and specific selectivity. • Gold nano-ripple sensing chip is a low cost, and a label-free method for detecting the antibody-antigen reaction. • The plasmonic resonance shift depends upon the concentration of the biomolecules attached on the surface of the nano ripple pattern. - Abstract: We demonstrate that the gold nano-ripple localized surface plasmon resonance (LSPR) chip is a low cost and a label-free method for detecting the presence of an antigen. A uniform stable layer of an antibody was coated on the surface of a nano-ripple gold pattern chip followed by the addition of different concentrations of the antigen. A red shift was observed in the LSPR spectral peak caused by the change in the local refractive index in the vicinity of the nanostructure. The LSPR chip was fabricated using oblique gas cluster ion beam (GCIB) irradiation. The plasmon-resonance intensity of the scattered light was measured by a simple optical spectroscope. The gold nano ripple chip shows monolayer scale sensitivity and high selectivity. The LSPR substrate was used to detect antibody-antigen reaction of rabbit X-DENTT antibody and DENTT blocking peptide (antigen).

  9. Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

    Science.gov (United States)

    Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh

    2017-02-01

    Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately

  10. Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response

    International Nuclear Information System (INIS)

    Rodak, L.

    1975-01-01

    An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125 I-labelled IgG 2 anti CSA (for demonstration of antigen) or with 125 I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection

  11. Insecticide-treated bed nets reduce plasma antibody levels and limit the repertoire of antibodies to Plasmodium falciparum variant surface antigens

    DEFF Research Database (Denmark)

    Askjaer, N; Maxwell, C; Chambo, W

    2001-01-01

    The use of insecticide-treated bed nets (ITN) has been documented to reduce malaria morbidity and mortality in areas with endemic malaria, but concerns have been raised that ITN usage could affect the acquisition of malaria immunity. Several lines of evidence have indicated that antibodies against...... variant surface antigens (VSA) are important in the development of naturally acquired immunity to Plasmodium falciparum malaria and may thus be good indicators of immune status. We have compared the levels of VSA antibodies in plasma from children who have used ITN for 4 years to levels in plasma from...

  12. [Study of anti-idiotype antibodies to human monoclonal antibody].

    Science.gov (United States)

    Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

    1992-02-01

    A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

  13. Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

    Science.gov (United States)

    Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

    2009-11-17

    The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

  14. Exploring the antigenic response to multiplexed immunizations in a chicken model of antibody production

    DEFF Research Database (Denmark)

    Kousted, Tina Mostrup; Kalliokoski, Otto; Christensen, Sofie Kjellerup

    2017-01-01

    that the competing effects of co-administering multiple immunogens effectively limit the antibody specificities that can be raised in a single individual to a fairly low number. Two potent model immunogens, KLH and CRM197, were administered together with competing antigens in various concentrations and complexities...

  15. A novel IgE antibody targeting the prostate-specific antigen as a potential prostate cancer therapy

    International Nuclear Information System (INIS)

    Daniels-Wells, Tracy R; Nicodemus, Christopher F; Penichet, Manuel L; Helguera, Gustavo; Leuchter, Richard K; Quintero, Rafaela; Kozman, Maggie; Rodríguez, José A; Ortiz-Sánchez, Elizabeth; Martínez-Maza, Otoniel; Schultes, Birgit C

    2013-01-01

    Prostate cancer (PCa) is the second leading cause of cancer deaths in men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 and CD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal was to take advantage of the unique properties of IgE in order to trigger immune activation against PCa. Binding characteristics of the antibody were determined by ELISA and flow cytometry. In vitro degranulation was determined by the release of β-hexosaminidase from effector cells. In vivo degranulation was monitored in human FcεRIα transgenic mice using the passive cutaneous anaphylaxis assay. These mice were also used for a vaccination study to determine the in vivo anti-cancer effects of this antibody. Significant differences in survival were determined using the Log Rank test. In vitro T-cell activation was studied using human dendritic cells and autologous T cells. The anti-PSA IgE, expressed in murine myeloma cells, is properly assembled and secreted, and binds the antigen and FcεRI. In addition, this antibody is capable of triggering effector cell degranulation in vitro and in vivo when artificially cross-linked, but not in the presence of the natural soluble antigen, suggesting that such an interaction will not trigger systemic anaphylaxis. Importantly, the anti-PSA IgE combined with PSA also triggers immune activation in vitro and in vivo and significantly prolongs the survival of human FcεRIα transgenic mice challenged with PSA-expressing tumors in a prophylactic vaccination setting. The anti-PSA IgE exhibits

  16. Binding of hydrophobic antigens to surfaces

    DEFF Research Database (Denmark)

    2017-01-01

    A first aspect of the present invention is a method of detecting antibodies comprising the steps of: i) providing a first group of beads comprising a surface modified with C1-C10 alkyl groups comprising amine, ammonium, ether and/or hydroxyl groups, ii) contacting said first group of beads......-antigen-antibody conjugates, and v) detecting said bead-antigen-antibody conjugates. Further aspects include an antibody detection kit, a bead-antigen conjugate and a composition comprising at least two different groups of bead-antigen-conjugates....

  17. Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis.

    Science.gov (United States)

    Omar, Noorsharmimi; Hamidon, Nurul Hamizah; Yunus, Muhammad Hafiznur; Noordin, Rahmah; Choong, Yee Siew; Lim, Theam Soon

    2018-05-01

    Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10 9 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  18. Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ponzetto, A; Cote, P J; Ford, E C; Engle, R; Gerin, J L; Cicmanec, J; Shapiro, M; Purcell, R H

    1985-06-01

    Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy and light cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.

  19. The relationship of HBsub(s) antigen and antibody to atomic bomb radiation in the adult health study sample, 1975-77

    International Nuclear Information System (INIS)

    Kato, Hiroo; Mayumi, Makoto; Nishioka, Kusuya; Hamilton, H.B.

    1983-01-01

    A study was conducted on the frequency of HBsub(s) positive antigen and antibody reactions as an index for determining whether there is any change in the immune competence as a late observable effect of atomic bomb exposure in Hiroshima and Nagasaki. There was no difference between the two comparison groups in the occurrence of positive HBsub(s) antibody reactions (prevalence of HB virus). However, the frequency of positive HBsub(s) antigen reactions (79% remained persistently antigen-positive) was significantly higher in those exposed to 100 rad or more than in the controls. The same tendency was apparent for the two exposure groups when classified by sex, city, and age, though by age the difference between exposure groups among the younger age groups (age 20 or less at the time of the bomb) was more marked. On the other hand, contrary to expectation, there was no difference between exposure groups in the distribution by high vs low titers among the antigen-positive individuals. There is no difference in the prevalence of positive HBsub(s) antibody reactions between Nagasaki and Hiroshima, but the frequency of positive antigen reactions is higher in Nagasaki. With respect to HBsub(s) antigen subtypes, adr occurs in a higher and adw in a lower frequency in Nagasaki than in Hiroshima, corresponding to earlier reports for the Japan archipelago of reverse and complementary distributions of these two subtypes. (author)

  20. Highly sensitive radioimmunoassay technique for subtyping the antibody to hepatitis B surface antigen

    Energy Technology Data Exchange (ETDEWEB)

    Fang, C T; Nath, N; Berberian, H; Dodd, R Y [American Red Cross, Blood Research Laboratory, Bethesda, MD, USA

    1978-12-01

    A highly sensitive technique for determining the subtype specificity of antibody to hepatitis B surface antigen (anti-HBs) is described. Immunoadsorbent consisting of controlled pore glass coated with subtype specific HBsAg was used to remove homologous antibody from the test samples before testing them for residual antibody by a commercially available radioimmunoassay (RIA). A total of 73 anti-HBs-positive samples from asymptomatic blood donors were tested. In nearly 80% of these samples the subtype reactivity could be determined by this technique. Only 67% could be typed by conventional liquid phase absorption RIA and 22% by passive hemagglutination inhibition techniques. Among the samples with low anti-HBs titer, ad and ay subtypes were found with equal frequency; however, with the increase in anti-HBs titer, considerably higher proportion of ad specificity was detected.

  1. Antibodies reactive to Plasmodium falciparum serine repeat antigen in children with Burkitt lymphoma from Ghana.

    Science.gov (United States)

    Guech-Ongey, Mercy; Yagi, Masanori; Palacpac, Nirianne Marie Q; Emmanuel, Benjamin; Talisuna, Ambrose O; Bhatia, Kishor; Stefan, D Cristina; Biggar, Robert J; Nkrumah, Francis; Neequaye, Janet; Tougan, Takahiro; Horii, Toshihiro; Mbulaiteye, Sam M

    2012-04-15

    The role of protective immunity to Plasmodium falciparum (Pf) malaria in Burkitt lymphoma (BL) is unknown. We investigated the association between BL and antibodies reactive to SE36 antigen, a recombinant protein based on P. falciparum serine repeat antigen 5 gene, targeted by protective malaria immune responses. Cases were children (0-14 years) enrolled at the Korle-Bu Teaching Hospital, Accra, Ghana, during 1965-1994 with BL confirmed by histology or cytology (92% of cases). Controls were apparently healthy children enrolled contemporaneous to the cases from the nearest neighbor house to the case house and were age,- sex-frequency-matched to the cases. Anti-SE36 IgG antibodies were measured using enzyme-linked absorbent immunoassays (ELISAs). SE36 titers were estimated by extrapolating ELISA optical density readings to a standard fitting curve. Anti-SE36 titers were log-transformed for analysis. Odds ratios (ORs) and two-sided 95% confidence intervals (95% CIs) were estimated using unconditional logistic regression. The mean log endpoint dilution titers were 0.63 logs lower in cases than in controls (8.26 [SD 1.68] vs. 8.89 [SD 1.75], Student's t-test, p = 0.019). Lower titers were observed in cases than controls aged 0-4 years (p = 0.05) and in those aged 5-14 years (p = 0.06). Low and medium tertiles of anti-SE36 IgG antibodies were associated with increased OR for BL ([OR 1.67, 95% CI 1.21-2.31] and [OR 1.33, 95% CI 0.96-1.86], respectively, p(trend) = 0.002) in analyses adjusting for age, sex, calendar period and test plate. Our findings suggest that compared to similarly aged children enrolled from the same community, children with BL in Ghana have lower antibodies to SE36 antigen. Copyright © 2011 UICC.

  2. The effect of circulating antigen on the biodistribution of the engineered human antibody hCTM01 in a nude mice model

    International Nuclear Information System (INIS)

    Davies, Q.; Perkins, A.C.; Frier, M.; Watson, S.; Lalani, E.N.; Symonds, E.M.

    1997-01-01

    Clinical studies are currently underway to assess the biodistribution and therapeutic potential of the genetically engineered human antibody hCTM01 directed against polymorphic epithelial mucin (PEM) in patients with ovarian carcinoma. The present study was undertaken to assess the effect of circulating PEM antigen on the biodistribution of the anti-PEM antibody in mice bearing MUC-1 transfected adenocarcinoma cell lines. Tumour xenografts were established from three cell lines: 413-BCR, which expressed antigen on the cell surface and also shed antigen into the circulation, E3P23, which expressed the antigen but did not shed into the circulation, and a negative control (410.4 MUCI). Groups of five mice were injected with 1.0 mg/kg antibody, imaged after 72 h and then sacrificed, followed by assay of tissue uptake. The results showed a clear difference in the tumour and liver uptake, with the non-secreting cell line showing almost twice the tumour uptake and approximately 20% of the liver uptake of the secreting cell line. (orig.). With 4 figs., 1 tab

  3. Analysis of a solid-phase radioimmunoassay for antibodies to cytoplasmic antigen fractions of Candida albicans

    International Nuclear Information System (INIS)

    Mauch, H.; Bromback, J.

    1981-01-01

    An indirect solid-phase radioimmunoassay (SPRIA) in individual polystyrene microtiter cups has been adapted for measurement of antibody to various cytoplasmic and carbohydrate antigen fractions of Candida albicans. The assay was optimized for sensitivity, precision and linearization of serum dilution curves. The optimized procedure allows computerized measurement of anti-Candida antibodies and can be used for measurement of antibody over a wide concentration range. The procedure obviates variation due to changes in day-to-day counts as a result of isotope decay and end-point antibody dilutions. The assay has been used to demonstrate a Poisson-like distribution of antibody levels in the sera of persons showing no symptoms of candidiasis. The minimum antibody level detectable by the assay is about two orders of magnitude lower than the lowest level found in human serum and 4 orders of magnitude lower than the most sensitive test used hitherto, the hemagglutination test. (Auth.)

  4. Ovarian carcinoma glyco-antigen targeted by human IgM antibody.

    Directory of Open Access Journals (Sweden)

    Yi Chen

    Full Text Available Epithelial Ovarian Cancer (EOC cells expression of a novel carbohydrate antigen was defined using a human VH4-34 encoded IgM monoclonal antibody (mAb216. MAb216 binds to a poly N-acetyllactosamine epitope expressed on B cells and kills normal and malignant B cells in vitro and in vivo. EOC patient ascites and EOC cell lines were used to study the anti tumor effect of mAb216. Various assays were used to characterize the epitope and demonstrate antibody-mediated binding and cytotoxicity in EOC. Drug and antibody combination effects were determined by calculating the combination index values using the Chou and Talalay method. MAb216 displays direct antibody mediated cytotoxicity on a population of human EOC tumor and ascites samples and EOC cell lines, which express high amounts of poly N-acetyllactosamine epitope, carried by CD147/CD98. Eighty four percent of patient samples, including platin resistant, had a tumor population that bound the monoclonal antibody. The binding pattern of mAb216 and mechanism of cytotoxicity was similar to that seen on normal and malignant B cells with unique general membrane disruption and "pore" formation. In vitro incubation with mAb216 and cisplatin enhanced killing of OVCAR3 cell line. In EOC cell lines percent cytotoxicity correlated with percent expression of epitope. Although in vitro data shows specific EOC cytotoxicity, for possible treatment of EOC MAb216 would need to be evaluated in a clinical trial with or without chemotherapy.

  5. A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers

    International Nuclear Information System (INIS)

    Oram, J.D.; Crooks, A.J.

    1979-01-01

    A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods. (Auth.)

  6. Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

    Science.gov (United States)

    Alderete, J F

    1984-06-01

    An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses during the development of lesions in animals inoculated subcutaneously and it reproducibly measured the individual classes immunoglobulins directed at T vaginalis. The colorimetric assay was also suitable for showing cross reactivity between trichomonal species as well as between different strains of T vaginalis. Conditions established for monitoring antibody to trichomanads in immunised rabbits or infected mice were equally effective for human materials, such as serum or vaginal washes. Serum from experimental animals or infected people showed high concentrations of IgG, IgA, and IgM antibody to trichomonads. Only antibodies of the IgG and IgA class were detected in vaginal washes from women with acute trichomoniasis. No IgE antibody to trichomonads was found under a variety of conditions in serum samples from patients or experimental animals.

  7. Use of thermodynamic coupling between antibody-antigen binding and phospholipid acyl chain phase transition energetics to predict immunoliposome targeting affinity.

    Science.gov (United States)

    Klegerman, Melvin E; Zou, Yuejiao; Golunski, Eva; Peng, Tao; Huang, Shao-Ling; McPherson, David D

    2014-09-01

    Thermodynamic analysis of ligand-target binding has been a useful tool for dissecting the nature of the binding mechanism and, therefore, potentially can provide valuable information regarding the utility of targeted formulations. Based on a consistent coupling of antibody-antigen binding and gel-liquid crystal transition energetics observed for antibody-phosphatidylethanolamine (Ab-PE) conjugates, we hypothesized that the thermodynamic parameters and the affinity for antigen of the Ab-PE conjugates could be effectively predicted once the corresponding information for the unconjugated antibody is determined. This hypothesis has now been tested in nine different antibody-targeted echogenic liposome (ELIP) preparations, where antibody is conjugated to dipalmitoylphosphatidylethanolamine (DPPE) head groups through a thioether linkage. Predictions were satisfactory (affinity not significantly different from the population of values found) in five cases (55.6%), but the affinity of the unconjugated antibody was not significantly different from the population of values found in six cases (66.7%), indicating that the affinities of the conjugated antibody tended not to deviate appreciably from those of the free antibody. While knowledge of the affinities of free antibodies may be sufficient to judge their suitability as targeting agents, thermodynamic analysis may still provide valuable information regarding their usefulness for specific applications.

  8. Radioimmunoassay for Epstein-Barr Virus (EBV)-associated Nuclear Antigen (EBNA). Binding of iodinated antibodies to antigen immobilized in polyacrylamide gel

    International Nuclear Information System (INIS)

    Dolken, G.; Klein, G.

    1977-01-01

    A solid-phase radioimmunoassay was developed for the EBV-associated nuclear antigen (EBNA). Total homogenates of EBV-DNA and EBNA positive or negative cells were polymerized in polyacrylamide gel and compared for their ability to bind 125 I-IgG prepared from anti-EBNA positive and anti-EBNA negative sera. EBNA specific binding was demonstrated and confirmed by serological and cellular specificity controls. The assay allows the quantitation of antigen or antibody even in the presence of detergents and is suitable for biochemical characterization of the antigen. Reciprocal blocking studies with extracts from different cell lines showed quantitative and qualitative differences. One part of the EBNA specificiti(es) present in the human Burkitt lymphoma derived lines RAJI, DAUDI and AW-RAMOS was lacking in B96-8, a marmoset line carrying EBV derived from a human infectious mononucleosis line. This result may reflect differences in the viral genomes derived from Burkitt lymphoma and infectious mononucleosis lines or differences in the host cells. (author)

  9. Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs.

    Science.gov (United States)

    Juhl, David; Marget, Matthias; Hallensleben, Michael; Görg, Siegfried; Ziemann, Malte

    2017-03-01

    Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Radioimmunolocalisation of tumours by external scintigraphy after administration of 131I antibody to carcinoembryonic antigen

    International Nuclear Information System (INIS)

    Searle, F.; Bagshawe, K.D.; Begent, R.H.J.; Jewkes, R.F.; Jones, B.E.; Keep, P.A.; Lewis, J.; Vernon, P.

    1980-01-01

    Investigations of 131 I-labelled antibody to carcinoembryonic antigen (CEA) were performed in nude mice bearing human colonic carcinoma xenografts and in external scintigraphy of patients with various tumours. In mice, the activities of 131 I (antiCEA) and 125 I(normal γ globulin) were measured in the human colon carcinoma xenografts. The results were expressed as a ratio of uptake of specific to non-specific antibody showing that antiCEA was retained in the tumours with a maximum specificity index of 2.2 at 7 days after antibody administration. Palpable carcinomas of the colon were localised by scintiscanning in patients given 131 I-labelled antibody to CEA. However, uptake of antiCEA was also demonstrated in apparently normal colon due to non-specific uptake of antibody and the fact that some CEA is present in normal colon. Thus further development of the technique particularly as regards antibody specificity, is necessary before radioimmunolocalisation could be used as a means of detecting tumours in clinical practice. (UK)

  11. Assessment of specific IgM antibodies to core antigen of hepatitis B virus in acute and chronic hepatitis B using immunoradiometric assay

    International Nuclear Information System (INIS)

    Zichova, M.; Vodak, M.; Kostrhun, L.; Nadvornik, V.; Stransky, J.

    1986-01-01

    A group of 24 patients with acute viral hepatitis B was assessed for specific antibodies against the ''core'' antigen class IgM (HB c AB IgM) during 1st-4th week of the illness. These specific antibodies were positive in all patients, the mean titre being 10 -5 . The high content of these antibodies persisted for 1-2 months after the onset of the disease. The assessment of specific antibodies against ''core'' antigen class IgM was also made in a group of 39 patients with chronic hepatitis. In these patients positive HB c Ab IgM with a lower content were found (titre 10 -3 ) than in the group with acute viral hepatitis B. Based on the results the conclusion is made that specific antibodies HB c Ab class IgM are, in addition to the estimation of the surface antigen of the hepatitis B virus (HB s Ag), one more indicator of acute viral hepatitis B. The assessment is diagnostically valuable, in particular in acute hepatitis of obscure etiology, in acute jaundice of obscure etiology for the period of low and short-term antigenemia. (author). 6 figs., 1 tab., 14 refs

  12. Effect of alterations in glomerular charge on deposition of cationic and anionic antibodies to fixed glomerular antigens in the rat.

    Science.gov (United States)

    Adler, S; Baker, P; Pritzl, P; Couser, W G

    1985-07-01

    Reduction of the negative charge of the glomerular capillary wall alters its charge- and size-selective properties. To investigate the effect of alteration in glomerular charge properties on antibody localization, we prepared cationic and anionic fractions of antibodies to subepithelial and glomerular basement membrane (GBM) antigens, and compared their deposition in normal rats and rats treated with protamine sulfate or aminonucleoside of puromycin to reduce capillary wall charge. IgG antibodies were eluted from kidneys of rats with active Heymann's nephritis (AICN), passive Heymann's nephritis (PHN), or anti-GBM nephritis (NTN), separated into cationic and anionic fractions, and radiolabeled with iodine 125 or iodine 131. Relative antibody content of each fraction was determined by incubation with an excess of glomerular antigen. Varying amounts of cationic and anionic IgG eluted from kidneys of rats with AICN or PHN were injected into 24 normal or protamine sulfate-treated rats. Glomerular binding of all antibodies was highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 4 hours was 1.08 +/- 0.07 for AICN eluate and 0.37 +/- 0.04 for PHN eluate. The ratios were not significantly different in animals pretreated with protamine sulfate (1.15 +/- 0.06 and 0.44 +/- 0.06, respectively; P greater than 0.05). Varying amounts of cationic and anionic IgG eluted from kidneys of rats with NTN were injected into 10 normal rats and four rats treated with aminonucleoside of puromycin. Glomerular binding of antibody was again highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 1 hour was 1.03 +/- 0.06, and was not significantly altered in rats treated with aminonucleoside of puromycin (1.05 +/- 0.03, P greater than 0.5). Proteinuria in PHN rats was also unaffected by treatment with protamine sulfate for

  13. Platelet transfusion refractoriness attributable to HLA antibodies produced by donor-derived cells after allogeneic bone marrow transplantation from one HLA-antigen-mismatched mother.

    Science.gov (United States)

    Hatakeyama, Naoki; Hori, Tsukasa; Yamamoto, Masaki; Inazawa, Natsuko; Iesato, Kotoe; Miyazaki, Toru; Ikeda, Hisami; Tsutsumi, Hiroyuki; Suzuki, Nobuhiro

    2011-12-01

    PTR is a serious problem in patients being treated for hematologic disorders. Two patients with acute leukemia developed PTR after allogeneic BMT from one HLA-antigen-mismatched mother attributable to HLA antibodies, which could not be detected in their serum before BMT. HLA antibodies, whose specificity resembled that of each patient, were detected in each donor's serum. Each donor had probably been immunized during pregnancy by their partner's HLA antigens expressed by the fetus, consequently, transplanted donor-derived cells provoked HLA antibodies in each recipient early after BMT, and those HLA antibodies induced PTR. If the mothers are selected as donors for their children, they should be tested for the presence of HLA antibodies. © 2010 John Wiley & Sons A/S.

  14. Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

    Science.gov (United States)

    Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

    2005-09-01

    Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

  15. Antibody guided irradiation of brain glioma by arterial infusion of radioactive monoclonal antibody against epidermal growth factor receptor and blood group A antigen

    Energy Technology Data Exchange (ETDEWEB)

    Epenetos, A.A.; Courtenay-Luck, N.; Pickering, D.; Hooker, G.; Lavender, J.P.; McKenzie, C.G. (Hammersmith Hospital, London (UK)); Durbin, H. (Imperial Cancer Research Fund, London (UK). Labs.)

    1985-05-18

    In a patient with recurrent grade IV glioma of the brain resistant to conventional treatment an antibody guided isotopic scan showed uptake by the tumour of a monoclonal antibody (9A) that was developed against epidermal growth factor receptor but cross reacted with blood group A antigen. As a therapeutic attempt antibody labelled with 1665 MBq (45.0 mCi) iodine-131 was delivered to the tumour area by infusion into the internal carotid artery. Computed tomography showed regression of the tumour after treatment, and an appreciable and sustained clinical improvement was noted without any toxicity. Delivery of irradiation guided by monoclonal antibody delivered by arterial infusion of the tumour area may be of clinical value in the treatment of brain gliomas resistant to conventional forms of treatment.

  16. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    International Nuclear Information System (INIS)

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji

    1991-01-01

    A 13 C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V H , V L , and C L domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with 13 C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with [1- 13 C]Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how 13 C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule

  17. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  18. Characterisation of monoclonal antibodies for human luteinising hormone, and mapping of antigenic determinants on the hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1983-01-01

    Twelve mouse monoclonal antibodies for human luteinising hormone were produced. The affinities varied from 4 X 10 7 to 1 X 10 10 l/mol. The specificity of each antibody was assessed by determining the relative reactivities with luteinising hormone, thyroid stimulating hormone, follicle stimulating hormone and chorionic gonadotrophin. Six antibodies bound to the α-subunit as shown by similar reactivity with all hormones, and the remainder to the β-subunit as shown by specificity for luteinising hormone. This latter group of antibodies cross-reacted only weakly with thyroid stimulating hormone (approximately 10%) and follicle stimulating hormone (approximately 3%). Three of these antibodies also showed low reactivity towards chorionic gonadotrophin (<10%), though the others did not (80-300%). The ability of different antibodies to bind simultaneously to luteinising hormone was examined and it was shown that several distinct antigenic determinants existed on both subunits. The characterisation of monoclonal binding sites is discussed in relation to the use of antibodies in two-site immunoradiometric assays. (Auth.)

  19. News and views on tumor markers: The use of radioactive antibodies against cell-bound antigens

    International Nuclear Information System (INIS)

    Kleist, S. von

    1984-01-01

    It was doubtless due to the phenomenal progress in the field of tumor immunology that took place during the last 20 years, that today we dispose not only of highly sensitive immunological tests like the RIA or EIA, but also of most specific reagents like monospecific polyclonal and monoclonal antibodies. In this context the discovery in human carcinomas of tumor-associated antigens was of prime importance, especially since some of them were found to have clinical relevance as so-called tumor markers. It has been shown that there is a direct correlation between the absolute tumor burden and the blood concentration of these substances. Based on animal models a new technology for tumor and metastases detection was developed in recent years, that used polyvalent or monoclonal antibodies prepared against tumor-associated antigens. This technique called radioimmuno-detection (RAID), especially in the hands of experts, may be superior in many instances to conventional radiology, radionuclide scanning or ultra-sonographic techniques. (orig.) [de

  20. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting Ab......SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti......-polysaccharide antibodies aggregation of secreted monomeric antibody (IgG) is critical for plaque formation and increases the avidity of binding to target cells....

  1. A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules

    Czech Academy of Sciences Publication Activity Database

    Zhang, A.X.; Murelli, R.P.; Bařinka, Cyril; Michel, J.; Cocleaza, A.; Jorgensen, W.L.; Lubkowski, J.; Spiegel, D.A.

    2010-01-01

    Roč. 132, č. 36 (2010), s. 12711-12716 ISSN 0002-7863 Institutional research plan: CEZ:AV0Z50520701 Keywords : Prostate -specific membrane antigen * antibody recruiting molecules * Structure-activity relationship Subject RIV: CE - Biochemistry Impact factor: 9.019, year: 2010

  2. Allosensibilisation to erythrocyte antigens (literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2015-01-01

    Full Text Available In this article literature review of the causes of allosensibilisation to erythrocyte antigens are presented. It is shown that the ability to produce antierythrocyte antibodies is affected by many factors, principal of whom it is difficult to identify. For the allosensibilisation development requires genetically determined differences in erythrocyte antigens phenotypes of donor and recipient, mother and fetus, which can lead to immune response and antibodies production. The biochemical nature of erythrocyte antigens, antigen dose (the amount of transfused doses, the number of antigens determinants on donor and fetus erythrocytes, the number of pregnancies are important. Individual patient characteristics: age, gender, diseases, the use of immunosuppressive therapy and the presence of inflammatory processes, are also relevant. Note that antibody to one erythrocyte antigens have clinical value, and to the other – have no. The actual data about frequency of clinically significant antibodies contribute to the development of post-transfusion hemolytic complications prophylaxis as well as the improvement of laboratory diagnosis of hemolytic disease of the newborn in the presence of maternal antierythrocyte antibodies.

  3. A highly sensitive radioimmunoassay technique for subtyping the antibody to hepatitis B surface antigen

    International Nuclear Information System (INIS)

    Fang, C.T.; Nath, N.; Berberian, H.; Dodd, R.Y.

    1978-01-01

    A highly sensitive technique for determining the subtype specificity of antibody to hepatitis B surface antigen (anti-HBs) is described. Immunoadsorbent consisting of controlled pore glass coated with subtype specific HBsAg was used to remove homologous antibody from the test samples before testing them for residual antibody by a commercially available radioimmunoassay (RIA). A total of 73 anti-HBs-positive samples from asymptomatic blood donors were tested. In nearly 80% of these samples the subtype reactivity could be determined by this technique. Only 67% could be typed by conventional liquid phase absorption RIA and 22% by passive hemagglutination inhibition techniques. Among the samples with low anti-HBs titer, ad and ay subtypes were found with equal frequency; however, with the increase in anti-HBs titer, considerably higher proportion of ad specificity was detected. (Auth.)

  4. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  5. Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

    Science.gov (United States)

    Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

    2015-09-01

    The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo (Japan))

    1991-07-02

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with {sup 13}C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with (1-{sup 13}C)Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how {sup 13}C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.

  7. Comparison of serum and salivary antibodies in children vaccinated with oral live or parenteral inactivated poliovirus vaccines of different antigen concentrations.

    Science.gov (United States)

    Zaman, S; Carlsson, B; Jalil, F; Mellander, L; Van Wezel, A L; Böttiger, M; Hanson, L A

    1991-12-01

    A new antigen-rich inactivated poliovirus vaccine (IPV) in ordinary (IPV1), double (IPV2) and quadruple (IPV4) antigen concentrations was given in 2 doses to 6 and 18 week old Pakistani infants. The immune responses to poliovirus types 1 and 3 were compared to those in infants given three doses of oral poliovirus vaccine (OPV) at 6, 12 and 18 weeks of age. Enzyme-linked immunosorbent assay, ELISA, was used to estimate IgG and IgA in serum and secretory IgA (SIgA) in saliva. Two to three years later, a follow-up of the serum antibody response was carried out in the same infants using a microneutralization test. Serum IgG antibody responses to poliovirus type 1 antigen after two doses of IPV1, IPV2 and IPV4 were not significantly higher than the response after three doses of OPV at 21 weeks of age (p greater than 0.05). The serum IgG responses to poliovirus type 3 were similar to those against type 1 in all the groups. Mean neutralizing antibody titres to poliovirus type 1 was significantly higher in the IPV2 group than the rest of the groups (p less than 0.01). For type 3, these titres were highest but not significantly, in the IPV4 group (p greater than 0.05). This study shows that two doses of a new antigen-rich IPV can give similar immediate serum antibody responses as OPV but higher late responses. SIgA antibodies in saliva were more efficiently induced by OPV after three doses than after 2 doses of IPV (p less than 0.05).

  8. Assessment of specific IgM antibodies to core antigen of hepatitis B virus in acute and chronic hepatitis B using immunoradiometric assay

    Energy Technology Data Exchange (ETDEWEB)

    Zichova, M; Vodak, M; Kostrhun, L; Nadvornik, V; Stransky, J

    1987-12-31

    A group of 24 patients with acute viral hepatitis B was assessed for specific antibodies against the ''core'' antigen class IgM (HB/sub c/AB IgM) during 1st-4th week of the illness. These specific antibodies were positive in all patients, the mean titre being 10/sup -5/. The high content of these antibodies persisted for 1-2 months after the onset of the disease. The assessment of specific antibodies against ''core'' antigen class IgM was also made in a group of 39 patients with chronic hepatitis. In these patients positive HB/sub c/Ab IgM with a lower content were found (titre 10/sup -3/) than in the group with acute viral hepatitis B. Based on the results the conclusion is made that specific antibodies HB/sub c/Ab class IgM are, in addition to the estimation of the surface antigen of the hepatitis B virus (HB/sub s/Ag), one more indicator of acute viral hepatitis B. The assessment is diagnostically valuable, in particular in acute hepatitis of obscure etiology, in acute jaundice of obscure etiology for the period of low and short-term antigenemia. (author). 6 figs., 1 tab., 14 refs.

  9. Development of a double-antibody radioimmunoassay for detecting ovarian tumor-associated antigen fraction OCA in plasma

    International Nuclear Information System (INIS)

    Knauf, S.; Urbach, G.I.

    1978-01-01

    Ovarian tumor-associated antigen isolated from human tumor tissue was shown to have a different mobility from that of carcinoembryonic antigen (CEA) in both acrylamide gel electrophoresis and immunoelectrophoresis in agarose. The ovarian tumor antigen is composed of six species with different electrophoretic mobility in acrylamide gel electrophoresis. Three of these species were detected in Sephadex G-100 ovarian fraction OCA (from the void volume peak) and the other three species of lower apparent molecular weight were detected in fraction OCD (from the second peak). Fractions OCA and OCD did not share common antigenic determinants as determined by immunodiffusion. CEA was shown to share antigenic determinants with both OCA and OCD. A double-antibody radioimmunoassay capable of detecting nanogram quantities of plasma OCA was developed. In a preliminary study of ovarian cancer patients, OCA appeared to be a more sensitive marker for ovarian cancer than CEA. There was virtually no correlation (r 2 = 0.1) between OCA and CEA levels in these patients, as determined by radioimmunoassay

  10. Spleen vagal denervation inhibits the production of antibodies to circulating antigens.

    Directory of Open Access Journals (Sweden)

    Ruud M Buijs

    Full Text Available BACKGROUND: Recently the vagal output of the central nervous system has been shown to suppress the innate immune defense to pathogens. Here we investigated by anatomical and physiological techniques the communication of the brain with the spleen and provided evidence that the brain has the capacity to stimulate the production of antigen specific antibodies by its parasympathetic autonomic output. METHODOLOGY/PRINCIPAL FINDINGS: This conclusion was reached by successively demonstrating that: 1. The spleen receives not only sympathetic input but also parasympathetic input. 2. Intravenous trinitrophenyl-ovalbumin (TNP-OVA does not activate the brain and does not induce an immune response. 3. Intravenous TNP-OVA with an inducer of inflammation; lipopolysaccharide (LPS, activates the brain and induces TNP-specific IgM. 4. LPS activated neurons are in the same areas of the brain as those that provide parasympathetic autonomic information to the spleen, suggesting a feed back circuit between brain and immune system. Consequently we investigated the interaction of the brain with the spleen and observed that specific parasympathetic denervation but not sympathetic denervation of the spleen eliminates the LPS-induced antibody response to TNP-OVA. CONCLUSIONS/SIGNIFICANCE: These findings not only show that the brain can stimulate antibody production by its autonomic output, it also suggests that the power of LPS as adjuvant to stimulate antibody production may also depend on its capacity to activate the brain. The role of the autonomic nervous system in the stimulation of the adaptive immune response may explain why mood and sleep have an influence on antibody production.

  11. Increased levels of IgA antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi differentiate digestive forms of Chagas disease.

    Science.gov (United States)

    Vasconcelos, Romero H T; Amaral, Fábio N; Cavalcanti, Maria G A M; Silva, Edimilson D; Ferreira, Antonio G P; Morais, Clarice N L; Gomes, Yara M

    2010-10-01

    In the chronic phase of Chagas disease, individuals infected by Trypanosoma cruzi may be asymptomatic or may present cardiac and/or digestive complications. Our aim here was to analyze the relationship between the presence of specific immunoglobulin A antibodies and the different chronic clinical forms of Chagas disease using two recombinant antigens of Trypanosoma cruzi, cytoplasmatic repetitive antigen and flagellar repetitive antigen. The association of this immunoglobulin isotype with the digestive and cardio-digestive forms of the disease determined by indirect enzyme-linked immunosorbent assay, strongly suggests that IgA antibodies against these recombinant antigens of T. cruzi can be used as an immunological marker of the digestive alterations caused by Chagas disease. The tests performed in this study show that it is possible to differentiate digestive forms of Chagas disease. The knowledge provided by these results may help physicians to manage early alterations in the digestive tract of patients with the indeterminate or cardiac forms of Chagas disease. Prospective studies, however, with follow-up of the patients that presenting with high levels of immunoglobulin A against cytoplasmatic repetitive antigen and flagellar repetitive antigen recombinant antigens, need to be conducted to confirm this hypothesis. 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  12. Solid phase double-antibody radioimmunoassay procedure

    International Nuclear Information System (INIS)

    Niswender, G.D.

    1977-01-01

    The present invention is concerned with the radioimmunoassay (RIA) procedure for assaying body fluid content of an antigenic substance which may either be an antigen itself or a hapten capable of being converted, such as by means of reaction with a protein, to an antigenic material. The present invention is concerned with a novel and improved modification of a double-antibody RIA technique in which there is a first antibody that is specific to the antigenic substance suspected to be present in a body fluid from which the assay is intended. The second antibody, however, is not specific to the antigenic substance or analyte, but is an antibody against the first antibody

  13. Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

    Science.gov (United States)

    Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

    2004-10-01

    Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

  14. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  15. Anti-N-methyl-D-aspartate receptor encephalitis with serum anti-thyroid antibodies and IgM antibodies against Epstein-Barr virus viral capsid antigen: a case report and one year follow-up

    Directory of Open Access Journals (Sweden)

    Xu Chun-Ling

    2011-11-01

    Full Text Available Abstract Background Anti-N-methyl-D-aspartate receptor encephalitis is an increasingly common autoimmune disorder mediated by antibodies to certain subunit of the N-methyl-D-aspartate receptor. Recent literatures have described anti-thyroid and infectious serology in this encephalitis but without follow-up. Case presentation A 17-year-old Chinese female patient presented with psychiatric symptoms, memory deficits, behavioral problems and seizures. She then progressed through unresponsiveness, dyskinesias, autonomic instability and central hypoventilation during treatment. Her conventional blood work on admission showed high titers of IgG antibodies to thyroglobulin, thyroid peroxidase and IgM antibodies to Epstein-Barr virus viral capsid antigen. An immature ovarian teratoma was found and removal of the tumor resulted in a full recovery. The final diagnosis of anti-N-methyl-D-aspartate receptor encephalitis was made by the identification of anti-N-methyl-D-aspartate receptor antibodies in her cerebral spinal fluid. Pathology studies of the teratoma revealed N-methyl-D-aspartate receptor subunit 1 positive ectopic immature nervous tissue and Epstein-Barr virus latent infection. She was discharged with symptoms free, but titers of anti-thyroid peroxidase and anti-thyroglobulin antibodies remained elevated. One year after discharge, her serum remained positive for anti-thyroid peroxidase and anti-N-methyl-D-aspartate receptor antibodies, but negative for anti-thyroglobulin antibodies and IgM against Epstein-Barr virus viral capsid antigen. Conclusions Persistent high titers of anti-thyroid peroxidase antibodies from admission to discharge and until one year later in this patient may suggest a propensity to autoimmunity in anti- N-methyl-D-aspartate receptor encephalitis and support the idea that neuronal and thyroid autoimmunities represent a pathogenic spectrum. Enduring anti-N-methyl-D-aspartate receptor antibodies from admission to one year

  16. Analysis of antibodies to newly described Plasmodium falciparum merozoite antigens supports MSPDBL2 as a predicted target of naturally acquired immunity.

    Science.gov (United States)

    Tetteh, Kevin K A; Osier, Faith H A; Salanti, Ali; Kamuyu, Gathoni; Drought, Laura; Failly, Marilyne; Martin, Christophe; Marsh, Kevin; Conway, David J

    2013-10-01

    Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in Plasmodium falciparum schizonts and merozoites: MSPDBL1 (also termed MSP3.4) and MSPDBL2 (MSP3.8), which possess Duffy binding-like (DBL) domains, and SURFIN4.2, encoded by a member of the surface-associated interspersed (surf) multigene family. After testing the antigenicities of these reagents by murine immunization and parasite immunofluorescence, we analyzed naturally acquired antibody responses to the antigens in two cohorts in coastal Kenya in which the parasite was endemic (Chonyi [n = 497] and Ngerenya [n = 461]). As expected, the prevalence and levels of serum antibodies increased with age. We then investigated correlations with subsequent risk of clinical malaria among children <11 years of age during 6 months follow-up surveillance. Antibodies to the polymorphic central region of MSPDBL2 were associated with reduced risk of malaria in both cohorts, with statistical significance remaining for the 3D7 allelic type after adjustment for individuals' ages in years and antibody reactivity to whole-schizont extract (Chonyi, risk ratio, 0.51, and 95% confidence interval [CI], 0.28 to 0.93; Ngerenya, risk ratio, 0.38, and 95% CI, 0.18 to 0.82). For the MSPDBL1 Palo Alto allelic-type antigen, there was a protective association in one cohort (Ngerenya, risk ratio, 0.53, and 95% CI, 0.32 to 0.89), whereas the other antigens showed no protective associations after adjustment. These findings support the prediction that antibodies to the polymorphic region of MSPDBL2 contribute to protective immunity.

  17. Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).

    Science.gov (United States)

    Golsaz-Shirazi, Forough; Amiri, Mohammad Mehdi; Farid, Samira; Bahadori, Motahareh; Bohne, Felix; Altstetter, Sebastian; Wolff, Lisa; Kazemi, Tohid; Khoshnoodi, Jalal; Hojjat-Farsangi, Mohammad; Chudy, Michael; Jeddi-Tehrani, Mahmood; Protzer, Ulrike; Shokri, Fazel

    2017-08-01

    Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The "a" determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Design of indirect solid-phase immunosorbent methods for detecting arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A P; Rezapkin, G V; Dzagurova, T K; Tkachenko, E A

    1984-05-01

    Specifications have been elaborated for formulating indirect solid-phase enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (SPRIA) methods that employ anti-human and anti-mice G class immunoglobulin (IgG), conjugated with horseradish peroxidase and /sup 125/I for detecting the arenaviruses Junin, Machupo, Tacaribe, Amalpari, Tamiami, Lassa, and LCM (lymphocytic choriomeningitis). These methods make it possible to identify with a high degree of sensitivity arenavirus antigens and antibodies in various kinds of material.

  19. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    Science.gov (United States)

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

  20. Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

    Science.gov (United States)

    Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

    1984-09-01

    By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

  1. Adult T-cell leukemia-associated antigen (ATLA) and anti-ATLA antibodies in patients with Hodgkin's disease in the Nagasaki District.

    Science.gov (United States)

    Kinoshita, K; Amagasaki, T; Yamada, Y; Ikeda, S; Momita, S; Toriya, K; Kamihira, S; Ichimaru, M

    1983-01-01

    Seven patients with Hodgkin's disease in the Nagasaki district were examined for adult T-cell leukemia-associated antigen (ATLA), a human retrovirus-associated antigen, and anti-ATLA antibodies. Anti-ATLA antibody reactivity with the ATLA-positive cultured cells from an ATL patient was demonstrated in four (57.1%) of seven patients. This suggests that infection by a human retrovirus may be closely associated with Hodgkin's disease in the Nagasaki district. However, ATLA could not be induced in the cultured mononuclear cells taken from biopsied lymph nodes of the three patients examined. Hence, it is necessary to collect more direct evidence in the search for a viral etiology of Hodgkin's disease.

  2. Antibodies against high frequency Gerbich 2 antigen (anti-Ge2: A real challenge in cross matching lab

    Directory of Open Access Journals (Sweden)

    Ravindra P Singh

    2013-01-01

    Full Text Available Transfusion management of patients′ alloimmunized against high-prevalence erythrocyte antigens is often problematic in emergency situations. Gerbich (Ge is very common blood group system and Gerbich-2 (Ge-2 antigen present in high frequency and outside Papua New Guinea population, Ge-2 negative population almost nil. To manage such kind of problems with real emergencies, implementation of rare donor registry program, cryopreservation of red cells of rare donors and biological cross matching to assess significance of these antibodies is warranted.

  3. Docking of Antibodies into Cavities in DNA Origami

    DEFF Research Database (Denmark)

    Quyang, X; Stefano, Mattia De; Krissanaprasit, Abhichart

    2017-01-01

    -selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA) modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysines. Atomic force...... microscopy (AFM) and transmission electron microscopy (TEM) validated efficient antibody immobilization in the origami structures. The increased ability to control the orientation of antibodies in nanostructures and at surfaces has potential for directing the interactions of antibodies with targets...

  4. Characterization and standardization of Sabin based inactivated polio vaccine: proposal for a new antigen unit for inactivated polio vaccines.

    Science.gov (United States)

    Westdijk, Janny; Brugmans, Debbie; Martin, Javier; van't Oever, Aart; Bakker, Wilfried A M; Levels, Lonneke; Kersten, Gideon

    2011-04-18

    GMP-batches of Sabin-IPV were characterized for their antigenic and immunogenic properties. Antigenic fingerprints of Sabin-IPV reveal that the D-antigen unit is not a fixed amount of antigen but depends on antibody and assay type. Instead of the D-antigen unit we propose standardization of IPV based on a combination of protein amount for dose and D-antigenicity for quality of the vaccine. Although Sabin-IPV type 2 is less immunogenic than regular wild type IPV type 2, the immunogenicity (virus neutralizing titers) per microgram antigen for Sabin-IPV type 2 is in the same order as for wild type serotypes 1 and 3. The latter observations are in line with data from human trials. This suggests that a higher dose of Sabin-IPV type 2 to compensate for the lower rat immunogenicity may not be necessary. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. In vitro and in vivo properties of human/mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen

    International Nuclear Information System (INIS)

    Saga, T.; Endo, K.; Koizumi, M.; Kawamura, Y.; Watanabe, Y.; Konishi, J.; Ueda, R.; Nishimura, Y.; Yokoyama, M.; Watanabe, T.

    1990-01-01

    A human/mouse chimeric monoclonal antibody specific for a common acute lymphocytic leukemia antigen was efficiently obtained by ligating human heavy-chain enhancer element to the chimeric heavy- and light-chain genes. Cell binding and competitive inhibition assays of both radioiodine and indium-111- (111In) labeled chimeric antibodies demonstrated in vitro immunoreactivity identical with that of the parental murine monoclonal antibodies. The biodistribution of the radiolabeled chimeric antibody in tumor-bearing nude mice was similar to that of the parental murine antibody. Tumor accumulation of radioiodinated parental and chimeric antibodies was lower than that of 111 In-labeled antibodies, probably because of dehalogenation of the radioiodinated antibodies. Indium-111-labeled chimeric antibody clearly visualized xenografted tumor. These results suggest that a human/mouse chimeric antibody can be labeled with 111 In and radioiodine without the loss of its immunoreactivity, and that chimeric antibody localizes in vivo in the same way as the parental murine antibody

  6. Augmentation of the antibody response of Atlantic salmon by oral administration of alginate-encapsulated IPNV antigens.

    Directory of Open Access Journals (Sweden)

    Lihan Chen

    Full Text Available The objective of the present study was to assess the effect of alginate-encapsulated infectious pancreatic necrosis virus antigens in inducing the immune response of Atlantic salmon as booster vaccines. One year after intraperitoneal injection with an oil-adjuvanted vaccine, post-smolts were orally boosted either by 1 alginate-encapsulated IPNV antigens (ENCAP; 2 soluble antigens (UNENCAP or 3 untreated feed (control. This was done twice, seven weeks apart. Sampling was done twice, firstly at 7 weeks post 1st oral boost and the 2nd, at 4 weeks after the 2nd oral boost. Samples included serum, head kidney, spleen and hindgut. Serum antibodies were analyzed by ELISA while tissues were used to assess the expression of IgM, IgT, CD4, GATA3, FOXP3, TGF-β and IL-10 genes by quantitative PCR. Compared to controls, fish fed with ENCAP had a significant increase (p<0.04 in serum antibodies following the 1st boost but not after the 2nd boost. This coincided with significant up-regulation of CD4 and GATA3 genes. In contrast, serum antibodies in the UNENCAP group decreased both after the 1st and 2nd oral boosts. This was associated with significant up-regulation of FOXP3, TGF-β and IL-10 genes. The expression of IgT was not induced in the hindgut after the 1st oral boost but was significantly up-regulated following the 2nd one. CD4 and GATA3 mRNA expressions exhibited a similar pattern to IgT in the hindgut. IgM mRNA expression on the other hand was not differentially regulated at any of the times examined. Our findings suggest that 1 Parenteral prime with oil-adjuvanted vaccines followed by oral boost with ENCAP results in augmentation of the systemic immune response; 2 Symmetrical prime and boost (mucosal with ENCAP results in augmentation of mucosal immune response and 3 Symmetrical priming and boosting (mucosal with soluble antigens results in the induction of systemic immune tolerance.

  7. Experimental radioimmunotherapy with I-131-antibody against a differentiation antigen

    International Nuclear Information System (INIS)

    Badger, C.C.; Krohn, K.A.; Bernstein, I.D.

    1985-01-01

    The authors have previously shown that I-131-labeled antibodies (Ab) against the Thyl.l antigen can care AKR/Cu (Thyl.2+) mice bearing the AKR/J (Thy 1l.1+) SL2 T-cell lymphoma. The authors have now extended these studies to therapy with I-131-anti-Thyl.1 of SL2 lymphoma in AKR/J mice where Ab reacts with both tumor and normal cells. A 25 μg bolus was rapidly cleared from serum by binding to spleen cells (75% with Tl/2 <60 min.) and only low concentrations of Ab(<2% ID/gm) were present in tumor after infusion. Therapy of AKR/J mice bearing established s.c. lymphoma nodules with 1500 μCi I-131-anti-Thyl.1 resulted in complete regression of the nodule in 6/6 animals although tumor eventually regrew and all animals died of metastatic lymphoma. In contrast, I-131-irrelevant Ab given to produce the same amount of whole body radiation (750 μCi) did not affect tumor growth. These studies suggest that radiolabeled-AB against differentiation antigens may be useful for therapy in spite of binding to normal cell populations

  8. Monoclonal anti-melanoma antibodies and their possible clinical use

    International Nuclear Information System (INIS)

    Hellstroem, K.E.; Hellstroem, Ingegerd; Washington Univ., Seattle; Washington Univ., Seattle

    1985-01-01

    Cell surface antigens of human melanoma, as defined by monoclonal antibodies, are discussed and in particular the three antigens p97, a GD3 ganglioside and a proteoglycan. The potential diagnostic uses of antibodies to melanoma antigens are reviewed including in vitro diagnosis by immuno-histology, in vitro diagnosis by serum assays and in vivo diagnosis by tumour imaging using radioactively labelled antibodies. The potential therapeutic uses of monoclonal antibodies to melanoma antigens are also reviewed including targets for antibody therapy, the use of antibodies alone, radiolabelled antibodies, antibody-toxin conjugates, antibody-drug conjugates, anti-idiotypic antibodies and vaccines. (UK)

  9. Designing two-in-one antibodies.

    Science.gov (United States)

    Valladares, Ignacio Garcia; Espinoza, Luis R

    2009-09-01

    Evaluation of: Bostrom J, Shang-Fan Y, Kan D et al.: Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610-1614 (2009). The longstanding held notion that one antibody equals one antigen and, hence, one function has been challenged in recent years. Improved technology in antibody production, especially the accumulation of sequence data of immunoglobulin genes and the advent of PCR have made it possible to clone antibody gene repertoires. The current paper provides further challenge to the notion of one antibody = one antigen by developing 'two-in-one' antibodies with an antigen-binding site that binds two distinct proteins with high affinity. A therapeutic variant antibody of Herceptin (Genentech, CA, USA) was isolated that binds the human EGF receptor (HER)2 and also to VEGF. This development may represent a breakthrough discovery and may have significant implications in the therapy of malignant, infectious, allergic and autoimmune disorders.

  10. Diagnostic significance of DNA and antibodies against capsid antigens of anti-Epstein–Barr virus antibodies levels in blood plasma of nasopharyngeal carcinoma patients from non-endemic region

    Directory of Open Access Journals (Sweden)

    V. E. Gurtsevich

    2015-01-01

    Full Text Available Epstein–Barr virus (EBV, a representative of the herpesvirus family, is the etiological agent for a number of benign and malignant human neoplasms. Among the latter, the nasopharyngeal carcinoma (NPC occupies a special place. In NPC development EBV plays a key role stimulating the progression of the pathological process from precancerous lesions to the cancer development. For most NPC patients, elevated levels of humoral IgG and IgA antibodies against capsid and early EBV antigens are characteristic and their antibody titers rise to high levels long before the diagnosis of cancer. Using this phenomenon, virus-specific antibodies are used for many years as markers for NPC screening, especially in cases of undiagnosed primary lesion. In recent years, in endemic for NPC regions (South China, South-East Asia a great attention has been paid to the use of quantitative determination of EBV DNA copies in the blood plasma of patients with NPC as a method of early cancer detection and monitoring.The aim of this study was to compare clinical significance of EBV DNA and humoral antibodies levels in blood plasma of NPC patients in non-endemic region, Russia. The results obtained indicate that both markers DNA / EBV and IgA antibodies against capsid EBV antigens can be successfully used for diagnosis of NPC in non-endemic region. However, in comparison with the virus-specific antibody titers, the viral DNA levels in the patients plasma are more sensitive and specific as NPC marker reflecting the efficacy of the therapy, and the state of remission or relapse.

  11. Molecular definition of multiple sites of antibody inhibition of malaria transmission-blocking vaccine antigen Pfs25.

    Science.gov (United States)

    Scally, Stephen W; McLeod, Brandon; Bosch, Alexandre; Miura, Kazutoyo; Liang, Qi; Carroll, Sean; Reponen, Sini; Nguyen, Ngan; Giladi, Eldar; Rämisch, Sebastian; Yusibov, Vidadi; Bradley, Allan; Lemiale, Franck; Schief, William R; Emerling, Daniel; Kellam, Paul; King, C Richter; Julien, Jean-Philippe

    2017-11-16

    The Plasmodium falciparum Pfs25 protein (Pfs25) is a leading malaria transmission-blocking vaccine antigen. Pfs25 vaccination is intended to elicit antibodies that inhibit parasite development when ingested by Anopheles mosquitoes during blood meals. The Pfs25 three-dimensional structure has remained elusive, hampering a molecular understanding of its function and limiting immunogen design. We report six crystal structures of Pfs25 in complex with antibodies elicited by immunization via Pfs25 virus-like particles in human immunoglobulin loci transgenic mice. Our structural findings reveal the fine specificities associated with two distinct immunogenic sites on Pfs25. Importantly, one of these sites broadly overlaps with the epitope of the well-known 4B7 mouse antibody, which can be targeted simultaneously by antibodies that target a non-overlapping site to additively increase parasite inhibition. Our molecular characterization of inhibitory antibodies informs on the natural disposition of Pfs25 on the surface of ookinetes and provides the structural blueprints to design next-generation immunogens.

  12. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

    Science.gov (United States)

    Alcorn, S.W.; Pascho, R.J.

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

  13. Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies.

    Science.gov (United States)

    Aboklaish, Ali F; Ahmed, Shatha; McAllister, Douglas; Cassell, Gail; Zheng, Xiaotian T; Spiller, Owen B

    2016-08-01

    Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis

    OpenAIRE

    Rigopoulou, Eirini I; Mytilinaiou, Maria; Romanidou, Ourania; Liaskos, Christos; Dalekos, George N

    2007-01-01

    Background Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody – the serological marker of type 2 autoimmune hepatitis (AIH-2)- is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1) – either in association with anti-LKM1, or in isolation- and anti-soluble liver antigen antibodies (anti-SLA) have been considered as us...

  15. Involvement of lymphocyte function-associated antigen-1 (LFA-1) in HIV infection: inhibition by monoclonal antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Mathiesen, Lars Reinhardt

    1991-01-01

    Monoclonal antibodies (MAbs) against the alpha- and beta-chain of lymphocyte-associated antigen-1 (LFA-1) were examined for inhibition of HIV-1 infection in vitro. Infection of the T cell line MT4 and the monocytic cell line U937 by isolates HTLVIIIB and SSI-002, respectively was inhibited...

  16. Docking of Antibodies into Cavities in DNA Origami

    DEFF Research Database (Denmark)

    Quyang, X; Stefano, Mattia De; Krissanaprasit, Abhichart

    2017-01-01

    microscopy (AFM) and transmission electron microscopy (TEM) validated efficient antibody immobilization in the origami structures. The increased ability to control the orientation of antibodies in nanostructures and at surfaces has potential for directing the interactions of antibodies with targets...

  17. Development of a Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay Using a Synthetic Peptide Antigen for Detection of Avian Metapneumovirus Antibodies in Turkey Sera

    Science.gov (United States)

    Alvarez, Rene; Njenga, M. Kariuki; Scott, Melissa; Seal, Bruce S.

    2004-01-01

    Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes. PMID:15013970

  18. Evaluation of antigen and antibody ELISA's for epidemiological surveys of tsetse-transmitted trypanosomosis in cattle

    International Nuclear Information System (INIS)

    Eisler, M.C.; Hopkins, J.S.; Machila, N.; Bossche, P. van den; Peregrine, A.S.; Luckins, A.G.

    2000-01-01

    Sensitivity and specificity of the FAO/IAEA antigen-detection enzyme-linked immunosorbent assay (ELISA) kits for diagnosis of bovine trypanosomosis were investigated using sera from experimental cattle infected by tsetse challenge with cloned populations of Trypanosoma congolense (3 populations) or T. vivax (1 population). The kits are based on monoclonal antibodies that recognise internal antigens of tsetse-transmitted trypanosomes. Ten cattle were infected with each trypanosome population for at least 60 days, and in combination with uninfected cohorts (n=16) were used in a double-blind study design. Sensitivity and specificity of the tests depended on the choice of positive-negative thresholds expressed as percent positivity with respect to the median OD of 4 replicates of the strong positive reference serum provided with the kit. In general, while overall specificities were high, sensitivities of the antigen-ELISA's were poor. For example, at a cut-off of 5% positivity, the sensitivities of the antigen-ELISA's were 11% for samples (n=1162) from T. congolense infected cattle (n=30), and 24% for samples (n=283) from T. vivax infected cattle (n=10). The corresponding specificity values were 95% and 79%, respectively. There were no values of the positive-negative threshold at which both sensitivity and specificity were satisfactory. Trypanosome species-specificities of the antigen-ELISA's were also poor. Sensitivity and species-specificity of the antigen-ELISA for T. brucei infections were not investigated. The indirect ELISA for the detection of anti-trypanosomal antibodies in bovine serum was adapted for use with dried blood spots on filter paper, and standardised using a strong positive reference serum and the percent positivity system of data expression. The antibody-ELISA was evaluated in Zambia for use in epidemiological surveys of the prevalence of tsetse-transmitted bovine trypanosomosis. Known negative samples (sera, n=209; blood spots, n=466) were

  19. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  20. Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

    DEFF Research Database (Denmark)

    Owens, T; Fazekas de St Groth, B

    1987-01-01

    two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4......The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used...

  1. RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

    Science.gov (United States)

    Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Weitzner, Brian D; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R; Dunbrack, Roland L

    2018-04-01

    A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native

  2. Antibody Maturation in Trypanosoma cruzi-Infected Rats

    Science.gov (United States)

    Marcipar, Iván S.; Risso, Marikena G.; Silber, Ariel M.; Revelli, Silvia; Marcipar, Alberto J.

    2001-01-01

    The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens in Trypanosome cruzi-infected rats are presented. A Western blotting technique, combined with avidity analysis to identify antigens that elicit high-avidity antibodies, is suggested. In this system, antibodies showed high avidity values only during the chronic phase of infection and only in relation to antibodies against 21-, 33-, 41-, 42-, 56-, 58-, 66-, and 72-kDa antigens. Finally, a 97-kDa T. cruzi antigen, which was recognized by high-avidity antibodies and occurred in noninfected rats, was identified. These results allow us to evaluate the different antigens in chagasic infection. Our results show that with the correct choice of antigen it is possible to detect differences in maturation of antibodies and to discriminate, in an experimental model, between recent (acute) and chronic infections. PMID:11427430

  3. Evaluation of performance of human immunodeficiency virus antigen/antibody combination assays in Taiwan.

    Science.gov (United States)

    Chang, Chun-Kai; Kao, Cheng-Feng; Lin, Pi-Han; Huang, Hui-Lin; Ho, Shu-Yuan; Wong, Kuo-Chen; Lin, Bo-Chang; Yeh, Chang-Ching; Lee, Chia-Yeh; Kao, Chuan-Liang; Lee, Chun-Nan; Chang, Sui-Yuan; Yang, Jyh-Yuan

    2017-08-01

    The fourth-generation human immunodeficiency virus (HIV) combination assay, which can simultaneously detect the presence of anti-HIV antibody and HIV antigen, has been shown to shorten the window period in HIV diagnosis compared with the third-generation HIV antibody immunoassay. This study was aimed to determine the performance of HIV combination assays in Taiwan, where the HIV-1 seroprevalence is 0.007% and HIV-2 infection has never been reported. Performance of three fourth-generation HIV Ag/Ab combination assays (Dia.Pro, Wantai, and Bio-Rad) and one third-generation HIV Ab immunoassay (AxSYM HIV 1/2 gO) was assessed. A total of 152 specimens, including 86 confirmed HIV-seropositive and 66 HIV-seronegative samples, were used in the study. The sensitivity of four assays varied from 98.8% to 100%, and specificity varied from 98.5% to 100%. Performance of the 75 equivocal samples, the HIV status of which was confirmed later, in terms of negative prediction varied from 81.8% to 87.5%. The Bio-Rad and Dia.Pro assays exhibited higher sensitivity for the detection of p24 antigen among the three fourth-generation HIV combination assays. The three fourth-generation HIV Ag/Ab combination assays exhibited better sensitivity, specificity, and negative prediction than the third-generation HIV Ab immunoassay. Copyright © 2015. Published by Elsevier B.V.

  4. Anti-Chol-1 antigen, GQ1bα, antibodies are associated with Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Toshio Ariga

    Full Text Available The interaction of amyloid β-proteins (Aβ with membrane gangliosides has been reported to be an early event in Aβ fibril formation in Alzheimer's disease (AD. Neuronal degeneration in AD has been postulated to be associated with the presence of anti-ganglioside antibodies in patient sera. Using an enzyme-linked immunosorbent assay (ELISA and high-performance thin-layer chromatography (HPTLC immunostaining, sera from 27 individuals (10 with AD, 6 with vascular dementia (VD, and 11 non-demented age-matched pathological controls were examined in order to detect anti-glycosphingolipid (GSL antibodies, including anti-cholinergic-specific antigen (Chol-1α; GQ1bα antibodies. All sera had natural antibodies against ganglio-N-tetraosyl gangliosides (brain-type gangliosides. However, sera of demented patients with AD and VD had significantly higher titers of anti-GSL antibodies than those in age-matched pathological controls. Although most serum antibodies, including anti- GM1, -GT1b, -GQ1b, -GQ1bα, were of the IgM type, the presence of the IgG type antibodies was also significantly elevated in the sera of demented patients with AD. Anti-GT1b antibodies of the IgG type were elevated in AD (90%, 9 of 10 cases and VD (100%, respectively. Most surprisingly, anti-GQ1bα antibodies (IgM were found in 90% (9/10 and 100% (6/6 in the sera of patients with AD and VD, respectively. Since GQ1bα is present in the cerebral cortex and hippocampus, the presence of anti-GQ1bα antibodies may play an important role in disrupting cholinergic synaptic transmission and may participate in the pathogenesis of dementia. We conclude that elevated anti-GSL antibody titers may be useful as an aid for clinical diagnosis of those dementias.

  5. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    International Nuclear Information System (INIS)

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J.

    1990-01-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins

  6. Development of a solid-phase radioimmunoassay for antibody to antigens of Babesia bovis infected bovine erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kahl, L.P.; Anders, R.F.; Mitchell, G.F. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia)); Callow, L.L.; Rodwell, B.J. (Animal Research Inst. Wacol (Australia). Tick Fever Research Centre)

    1982-06-01

    A quantitative solid-phase radioimmunoassay (RIA) has been developed for the purpose of ranking sera from exposed animals according to their titre of anti-B. bovis antibody. The antigen was a sonicate of lysed infected blood cells, and antibody binding was detected with /sup 125/I-labelled anti-bovine IgG. The assay was tested using sera from experimentally infected splenectomized and intact cattle and from animals resident in an endemic area. A high specificity for B. bovis was demonstrated. There was good agreement in identifying exposed cattle when the test was compared with an indirect fluorescent antibody (IFA) test although no correlation was seen between titres obtained in the two tests. Analysis of the effects of challenge infection with B. bovis on IFA and RIA titres in previously exposed animals illustrated that the RIA was a more sensitive test for detecting changes in antibody titre.

  7. Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

    Directory of Open Access Journals (Sweden)

    Christine Bee

    Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

  8. Characterization of sporozoite surface antigens of Plasmodium falciparum, using monoclonal antibodies. Part of a coordinated programme on the preparation of irradiated vaccines against some human diseases

    International Nuclear Information System (INIS)

    Groot, M.

    1982-10-01

    Sporozoites are considered as a source of potential vaccine. Characterization of their antigens is therefore important and can be achieved by monoclonal antibodies. The purpose of this project is to study the production of monoclonal antibodies against sporozoites of P. falciparum. Various infections of mosquitoes were carried out during the period 1981-1982 to obtain antigens for the production of hybridomas. Hybridomas were produced from mice immunized through the bites of infected mosquitoes and by intravenous inoculation. The anti-sporozoite activity of the hybridomas was tested by an immunofluorescent antibody test using P. falciparum sporozoites as antigens. Positive immunofluorescence was seen in hybridoma cell lines tested with P. falciparum, whereas negative results were obtained when the cell lines were cross-reacted with other human species (P. vivax) and with a rodent malaria parasite (P. berghei)

  9. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...... and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps...

  10. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

    Directory of Open Access Journals (Sweden)

    Kristensen Peter

    2009-01-01

    Full Text Available Abstract Background Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. Results Here we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA. Conclusion These results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications.

  11. Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required?

    Science.gov (United States)

    Staudacher, Alexander H; Brown, Michael P

    2017-12-05

    Antibody drug conjugates (ADCs) employ the exquisite specificity of tumour-specific monoclonal antibodies (mAb) for the targeted delivery of highly potent cytotoxic drugs to the tumour site. The chemistry of the linker, which connects the drug to the mAb, determines how and when the drug is released from the mAb. This, as well as the chemistry of the drug, can dictate whether the drug can diffuse into surrounding cells, resulting in 'bystander killing'. Initially, any bystander killing mechanism of action of an ADC was understood to involve an essential sequence of steps beginning with surface antigen targeting, internalisation, intracellular linker cleavage, drug release, and diffusion of drug away from the targeted cell. However, recent studies indicate that, depending on the linker and drug combination, this mechanism may not be essential and ADCs can be cleaved extracellularly or via other mechanisms. In this minireview, we will examine the role of bystander killing by ADCs and explore the emerging evidence of how this can occur independently of internalisation.

  12. Immunization of rhesus macaques with Echinococcus multilocularis recombinant 14-3-3 antigen leads to specific antibody response.

    Science.gov (United States)

    Lampe, Karen; Gottstein, B; Becker, T; Stahl-Hennig, C; Kaup, F-J; Mätz-Rensing, K

    2017-01-01

    E. multilocularis (Em) is the etiologic agent of alveolar echinococcosis (AE), a severe and potentially fatal disease, primarily affecting the liver of and occurring in aberrant intermediate hosts, e.g., humans and non-human primates. Due to increasing numbers of spontaneous cases of AE in the Old World monkey colonies of the German Primate Center, the question arose as to whether vaccination of non-human primates may represent a useful prophylactic approach. In this pilot study, the recombinant antigen Em14-3-3, which has provided a 97 % protection against E. multilocularis challenge infection in rodent models, was used for the first time to immunize rhesus macaques. In order to increase immunogenicity, the antigen was formulated with different adjuvants including Quil A®, aluminum hydroxide (alum), and muramyl dipeptide (MDP). Also, different vaccination regimens were tested. All vaccinated animals developed antigen-specific antibodies. While Quil A® induced a local adverse reaction, alum proved to be the most potent adjuvant in terms of induced antibody levels, longevity as well as tolerability. In conclusion, our pilot study demonstrated that recombinant Em14-3-3 is safe and immunogenic in rhesus monkeys. As a next step, efficacy of the vaccination remains to be explored.

  13. Relationship between natural and heme-mediated antibody polyreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Hadzhieva, Maya; Vassilev, Tchavdar [Stephan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113 (Bulgaria); Bayry, Jagadeesh; Kaveri, Srinivas; Lacroix-Desmazes, Sébastien [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France); Dimitrov, Jordan D., E-mail: jordan.dimitrov@crc.jussieu.fr [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France)

    2016-03-25

    Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.

  14. Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody.

    OpenAIRE

    Bezeaud, A; Rosenswajg, M; Guillin, M C

    1989-01-01

    Five commercially available assays for hepatitis delta (HD) virus markers were compared for sensitivity, specificity, and reproducibility: three assays for antibody (anti-HD), provided by Diagnostics Pasteur, Organon Teknika, and Abbott Laboratories, and two assays for antigen (HD Ag), from Pasteur and Organon Teknika. The assay from Organon Teknika is the less sensitive assay for anti-HD detection. Although the sensitivities of the Pasteur and Abbott assays for anti-HD detection are similar,...

  15. Dengue NS1 Antigen - for Early Detection of Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Amol Hartalkar

    2015-08-01

    Full Text Available Objectives: To evaluate the efficacy of NS1 antigen assay for early diagnosis of dengue virus infection in a tertiary care hospital. Methods: This cross sectional study was carried out in department of Medicine from August to December 2013. Total 100 patients with dengue fever were included. Complete blood count, alanine aminotransferase (ALT, aspartate aminotransferase (AST, Dengue NS1 antigen and IgM and IgG antibodies of dengue virus were done in all cases. Results: Of the 100 sera tested, 75% were positive for dengue virus infection based on dengue NS1 antigen, IgM antibody and IgG antibody. Dengue NS1 antigen and IgM, IgG antibody were able to detect dengue virus infection between day 1 to day 8 in 92% of samples, 86.7% of samples and 82.6% of samples respectively. Sixty nine percent (69% were found positive for dengue NS1 antigen, 65% were IgM positive and 62% were IgG positive. Based on the dengue NS1 antigen and IgM antibody combination, 74% were positive for dengue virus infections. Sensitivity of Dengue NS1 antigen was 92.3% and specificity of 74.28% in comparison to IgM antibody. Detection rate increased to 75%, based on the antigen and IgG antibody combination. Sensitivity of dengue NS1 antigen was 90.3% and specificity of 65.8% in comparison to IgG antibody. Conclusion: Dengue NS1 antigen is a useful, sensitive and specific test for early diagnosis of dengue virus infection and it improves diagnostic efficiency in combination with antibody test. Key words: Dengue fever, NS1 antigen. Introduction: Dengue fever (DF is the most common arboviral illness in humans. Each year, an estimated 50-100 million cases of dengue fever and 500,000 cases of dengue hemorrhagic fever occur worldwide, with 30000 deaths (mainly in children. Globally 2.5-3 billion people in approximately 112 tropical and subtropical countries are at risk of dengue.of samples respectively. Sixty nine percent (69% were found positive for dengue NS1 antigen, 65% were Ig

  16. Conservation of myeloid surface antigens on primate granulocytes.

    Science.gov (United States)

    Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

    1983-02-01

    Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

  17. Protection against Escherichia coli K1 infection in newborn rats by antibody to K1 capsular polysaccharide antigen.

    OpenAIRE

    Bortolussi, R; Ferrier, P

    1980-01-01

    The protective value of antibody to the K1 capsular polysaccharide antigen of Escherichia coli was investigated in a newborn rat model of E. coli K1 infection. Pregnant rats were immunized intravenously with E. coli, and the agglutinating titer to meningococcal group B polysaccharide, which is identical to K1 polysaccharide, was measured in the serum of rats and their offspring. Convalescent serum from rat mothers showed an increased antibody titer in animals injected twice but not once with ...

  18. A monoclonal antibody against SV40 large T antigen (PAb416) does not label Merkel cell carcinoma.

    Science.gov (United States)

    Pelletier, Daniel J; Czeczok, Thomas W; Bellizzi, Andrew M

    2018-07-01

    Merkel cell carcinoma represents poorly differentiated neuroendocrine carcinoma of cutaneous origin. In most studies, the vast majority of Merkel cell carcinomas are Merkel cell polyomavirus (MCPyV)-associated. SV40 polyomavirus immunohistochemistry is typically used in the diagnosis of other polyomavirus-associated diseases, including tubulointerstitial nephritis and progressive multifocal leukoencephalopathy, given cross-reactivity with BK and JC polyomaviruses. MCPyV-specific immunohistochemistry is commercially available, but, if antibodies against SV40 also cross-reacted with MCPyV, that would be advantageous from a resource-utilisation perspective. Tissue microarrays were constructed from 39 Merkel cell carcinomas, 24 small-cell lung carcinomas, and 18 extrapulmonary visceral small-cell carcinomas. SV40 large T antigen immunohistochemistry (clone PAb416) was performed; MCPyV large T antigen immunohistochemistry (clone CM2B4) had been previously performed. UniProt was used to compare the amino acid sequences of the SV40, BK, JC and MCPyV large T antigens, focusing on areas recognised by the PAb416 and CM2B4 clones. SV40 immunohistochemistry was negative in all tumours; MCPyV immunohistochemistry was positive in 38% of Merkel cell carcinomas and in 0% of non-cutaneous poorly differentiated neuroendocrine carcinomas. UniProt analysis revealed a high degree of similarity between SV40, BK, and JC viruses in the region recognised by PAb416. There was less homology between SV40 and MCPyV in this region, which was also interrupted by two long stretches of amino acids unique to MCPyV. The CM2B4 clone recognises a unique epitope in one of these stretches. The PAb416 antibody against the SV40 large T antigen does not cross-react with MCPyV large T antigen, and thus does not label Merkel cell carcinoma. © 2018 John Wiley & Sons Ltd.

  19. Cross-reactivity among antigens of different air-borne fungi detected by ELISA using five monoclonal antibodies against Penicillium notatum.

    Science.gov (United States)

    Shen, H D; Lin, W L; Chen, R J; Han, S H

    1990-10-01

    Cross-reactivity among antigens of 12 genera of air-borne fungi, 13 species of Penicillium, and 5 species of Aspergillus was studied by ELISA using five monoclonal antibodies (MoAbs) against Penicillium notatum. Epitopes recognized by all the five MoAbs were susceptible to treatment of mild periodate oxidation and may therefore be associated with carbohydrates. Furthermore, our results showed that there is cross-reactivity among antigens of Penicillium, Aspergillus, and Eurotium species. By using these MoAbs, cross reactivity was not detected between antigens of Penicillium notatum and antigens of Fusarium solani, Alternaria porri, Cladosporium cladosporoides, Curvularia species, Nigrospora species, Aureobasidium pullulans, Wallemia species, Rhizopus arrhizus, and Candida albicans. Cross-reactivity among antigens of 11 species of Penicillium and 5 species of Aspergillus could be detected by ELISA using one of the five MoAbs (MoAb P15). The fact that there may be cross-reactivity among antigens of closely related fungi species should be considered in the diagnosis and treatment of mold allergic diseases.

  20. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens.

    Directory of Open Access Journals (Sweden)

    Christoph B Geier

    Full Text Available Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

  1. Antibody profiling sensitivity through increased reporter antibody layering

    Science.gov (United States)

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  2. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2017-03-28

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  3. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  4. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  5. Purification of immunoreactive radiolabeled moniclonal antibodies with anti-iodiotypic moniclonal antibodies

    International Nuclear Information System (INIS)

    Temponi, M.; Pupa, S.; Ferrone, S.

    1990-01-01

    A method is described to purify immunoreactive moniclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified an elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yeald was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies. (author). 12 refs.; 4 figs.; 1 tab

  6. [Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

    Science.gov (United States)

    Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

    2012-06-01

    To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

  7. Immunoglobulin variable region sequences of two human monoclonal antibodies directed to an onco-developmental carbohydrate antigen, lactotetraosylceramide (LcOse4Cer).

    Science.gov (United States)

    Yago, K; Zenita, K; Ohwaki, I; Harada, R; Nozawa, S; Tsukazaki, K; Iwamori, M; Endo, N; Yasuda, N; Okuma, M

    1993-11-01

    A human monoclonal antibody, 11-50, was generated and was shown to recognize an onco-developmental carbohydrate antigen, LcOse4Cer. The isotype of this antibody was IgM, lambda, similar to the previously known human anti-LcOse4 antibodies, such as IgMWOO and HMST-1. We raised a murine anti-idiotypic antibody G3 (IgG1, kappa) against 11-50, and tested its reactivity towards the affinity purified human polyclonal anti-LcOse4 antibodies prepared from pooled human sera using a Gal beta 1-->3GlcNAc beta-immobilized column. The results indicated that at least a part of the human polyclonal anti-LcOse4 antibodies shared the G3 idiotype with 11-50. We further analyzed the sequence of variable regions of the two anti-LcOse4 antibodies, 11-50 and HMST-1. Sequence analysis of the heavy chain variable regions indicated that the VH regions of these two antibodies were highly homologous to each other (93.5% at the nucleic acid level), and these antibodies utilized the germline genes VH1.9III and hv3005f3 as the VH segments, which are closely related germline genes of the VHIII family. It was noted that these germline VH genes are frequently utilized in fetal B cells. The JH region of both antibodies was encoded by the JH4 gene. For the light chain, the V lambda segments of the two antibodies were 96.3% homologous to each other at the nucleic acid level. The V lambda segments of both antibodies showed the highest homology to the rearranged V lambda gene called V lambda II.DS among reported V lambda genes, while the exact germline V lambda genes encoding the two antibodies were not yet registered in available sequence databanks. The amino acid sequences of the J lambda segments of both antibodies were identical. These results indicate that the two human antibodies recognizing the onco-developmental carbohydrate antigen Lc4 are encoded by the same or very homologous germline genes.

  8. Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti-liver-kidney microsome type-1 antibodies (LKM1).

    Science.gov (United States)

    Yamamoto, A M; Cresteil, D; Boniface, O; Clerc, F F; Alvarez, F

    1993-05-01

    Anti-liver-kidney microsome type-1 antibodies (LKM1), present in sera from a group of patients with autoimmune hepatitis, are directed against P450IID6. Previous work, using cDNA constructions spanning most of the P450IID6 protein defined the main immunogenic site between the amino acids (aa), 254-271 and predicted the presence of other putative immunogenic sites in the molecule. Fusion proteins from new cDNA constructions, spanning so-far-untested regions between aa 1-125 and 431-522, were not recognized by LKM1-positive sera. Synthetic peptides, representing sequences from putative immunogenic regions or previously untested regions, allowed a precise definition of four antigenic sites located between peptides 257-269, 321-351, 373-389 and 410-429, which were recognized, respectively, by 14, 8, 1 and 2 out of 15 LKM1-positive sera tested. The minimal sequence of the main antigenic site (peptide 257-269) recognized by the autoantibody was established to be WDPAQPPRD (peptide 262-270). In addition, deletion and replacement experiments showed that aa 263 (Asp) was essential for the binding of the autoantibody to peptide 262-270. Analysis of the second most frequently recognized peptide between aa 321-351, was performed using peptides 321-339 and 340-351 in competitive inhibition studies. Complete elimination of antibody binding to peptide 321-351 obtained by absorption of both shorter peptides indicated that peptide 321-351 is a discontinuous antigenic site. LKM1-positive sera reacting against peptide 321-351 recognized either both the shorter peptides or just one of them preferentially. Results of the present study suggest that the production of LKM1 antibodies is an antigen-driven, poly- or oligoclonal B cell response. The identification of antigenic sites will allow: (i) the development of specific diagnostic tests and (ii) further studies on the pathogenic value of LKM1 antibodies in autoimmune hepatitis.

  9. Use of JH4 joining segment gene by an anti-arsonate antibody that bears the major A-strain cross-reactive idiotype but displays diminished antigen binding.

    Science.gov (United States)

    Slaughter, C A; Jeske, D J; Kuziel, W A; Milner, E C; Capra, J D

    1984-06-01

    One of the antibody families utilized by the A/J mouse in its response to p-azophenylarsonate (Ars) is characterized by the expression of the major anti-arsonate cross-reactive idiotype (CRI) of the A strain. This family has been termed the Ars-A family. A hybridoma antibody (HP 101F11 ) obtained after immunization of an A/J mouse with Ars was identified initially as displaying the CRI, but was subsequently found to bind antigen at a level much lower than most members of the Ars-A family. The results of binding studies suggested that HP 101F11 possesses reduced avidity for antigen. When isolated light and heavy chains were allowed to recombine with the heavy and light chains of a strongly antigen-binding, strongly CRI-positive antibody of the Ars-A family (HP 93G7 ), the low level of antigen binding by HP 101F11 was found to be due to a structurally variant heavy chain. Whereas antibodies of the Ars-A family with normal avidity for antigen had been shown to use the JH2 joining segment gene, amino acid sequence analysis of HP 101F11 revealed that this antibody has a JH segment with a sequence identical to that encoded by a portion of a different JH gene, JH4 . The implication that 101F11 uses the JH4 gene instead of JH2 was supported by the observation that the productively rearranged gene is associated with an Eco R1 restriction fragment 0.95 Kb smaller than the corresponding fragments of Ars-A hybridomas with normal avidity for antigen. The size difference of 0.95 Kb corresponds exactly to the known distance between the JH2 and JH4 genes in BALB/c germline DNA. In addition to the structural differences immediately attributable to the use of JH4 , HP 101F11 has shown an amino acid interchange in the DH segment, and a single amino acid deletion at the DH-JH boundary. These results show that variation among members of the Ars-A family in the DH and/or JH segments provides alternative structural forms of Ars-A antibodies upon which selective processes can operate

  10. RosettaAntibodyDesign (RAbD): A general framework for computational antibody design

    Science.gov (United States)

    Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R.

    2018-01-01

    A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228–256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody–antigen complexes, using two design strategies—optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the

  11. Prevalence of Diego blood group antigen and the antibody in three ethnic population groups in Klang valley of Malaysia

    OpenAIRE

    Cheong Tar Wei; Faisal Muti Al-Hassan; Norris Naim; Aishah Knight; Sanmukh R Joshi

    2013-01-01

    Background: Diego blood group antigen, Di(a), is very rare among Caucasians and Blacks, but relatively common among the South American Indians and Asians of Mongolian origin. The antibody to Di(a) is clinically significant to cause hemolytic disease in a new-born or hemolytic transfusion reaction. Objectives: This study was designed to determine the prevalence of Di(a) antigen among the blood donors from the three major ethnic groups in Klang Valley of Malaysia as well as to find an incidence...

  12. Antigenic determinants of prostate-specific antigen (PSA) and development of assays specific for different forms of PSA.

    OpenAIRE

    Nilsson, O.; Peter, A.; Andersson, I.; Nilsson, K.; Grundstr?m, B.; Karlsson, B.

    1997-01-01

    Monoclonal antibodies were raised against prostate-specific antigen (PSA) by immunization with purified free PSA, i.e. not in complex with any protease inhibitor (F-PSA) and PSA in complex with alpha1-anti-chymotrypsin (PSA-ACT). Epitope mapping of PSA using the established monoclonal antibody revealed a complex pattern of independent and partly overlapping antigenic domains in the PSA molecule. Four independent antigenic domains and at least three partly overlapping domains were exposed both...

  13. Anti-D Antibodies in Pregnant D Variant Antigen Carriers Initially Typed as RhD.

    Science.gov (United States)

    Lukacevic Krstic, Jelena; Dajak, Slavica; Bingulac-Popovic, Jasna; Dogic, Vesna; Mratinovic-Mikulandra, Jela

    2016-11-01

    To evaluate the incidence, the consequences, and the prevention strategy of anti-D alloimmunizations of D variant carriers in the obstetric population of Split-Dalmatia County, Croatia. RhD immunization events were evaluated retrospectively for the period between 1993 and 2012. Women were tested for RhD antigen and irregular antibodies. Those with anti-D antibody who were not serologically D- were genotyped for RHD. They were evaluated for their obstetric and transfusion history and their titer of anti-D. The neonates were evaluated for RhD status, direct antiglobulin test (DAT), hemoglobin and bilirubin levels, transfusion therapy as well as phototherapy and outcome. Out of 104,884 live births 102,982 women were tested for RhD antigen. Anti-D immunization occurred in 184 women which accounts for 0.9% of individuals at risk of anti-D formation. 181 cases occurred in women serologically typed as D-. Three women were partial D carriers (DVa n = 2, DNB n = 1), initially typed RhD+, and recognized as D variant carriers after the immunization occurred. Anti-D titer varied from 1:1 to 1:16. Six children were RhD+, four had positive DAT, and two underwent phototherapy. Anti-D immunization occurred in pregnant partial D carriers (DVa, DNB). RhD+ children had serologic markers of hemolytic disease of the fetus and newborn (HDFN), with no cases of severe HDFN.

  14. Thyroid Antibodies

    Science.gov (United States)

    ... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

  15. Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

    Science.gov (United States)

    Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

    1996-03-01

    A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

  16. Evaluation of the recombinant antigens Wb14 and WbT for the capture antibody diagnosis of lymphatic filariasis

    Directory of Open Access Journals (Sweden)

    André Filipe Pastor

    2018-03-01

    Full Text Available BACKGROUND Lymphatic filariasis (LF is a parasitic disease caused mainly by the Wuchereria bancrofti worm and that affects up to 120 million people worldwide. LF is the second cause of chronic global deformity, responsible for 15 million people with lymphedema (elephantiasis and 25 million men with scrotal hydrocele. Its diagnosis is still associated with numerous difficulties, such as the sample collection periods (microfilaria nocturnal periodicity and limited diagnostic kits. OBJECTIVES The aim of this work was to evaluate two recombinant antigens (Wb14 and WbT as part of an enzyme-linked immunosorbent assay (ELISA based antibody capture tests for LF. METHODS The recombinant antigens rWb14 and rWbT were expressed in Escherichia coli BL21 and an antibody capture ELISA was performed. For this, sera were used from microfilaremic individuals with W. bancrofti (MF, chronic pathology (CP, individuals infected with Strongyloides (SP and healthy controls from endemic (EN and non-endemic (NE areas. FINDINGS Both tests showed similar results, with 90% sensitivity and 96.6% specificity. In comparison with the BM14 ELISA commercial test, the Wb14 and WbT antigens performed with identical sensitivity but greater specificity. Reduced positivity with the CP suggested a potential to monitor cure. This was not confirmed, however, when sera from individuals up to seven years after treatment were assayed. MAIN CONCLUSIONS The Wb14 and WbT ELISAs were considered efficient and promising diagnostic tests. Due to the importance of antibody capture analysis to evaluate the Global Program to Eliminate Lymphatic Filariasis (GPELF, the tests proposed here appear as great alternatives to the available commercial system.

  17. Factors associated with anti-human leukocyte antigen antibodies in patients supported with continuous-flow devices and effect on probability of transplant and post-transplant outcomes

    DEFF Research Database (Denmark)

    Alba, Ana C; Tinckam, Kathryn; Foroutan, Farid

    2015-01-01

    BACKGROUND: One major disadvantage of ventricular assist device (VAD) therapy is the development of human-leukocyte antigen (HLA) antibodies. We aimed to identify factors associated with HLA antibodies during continuous flow (CF)-VAD support and assess the effect on transplant probability...

  18. Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision

    Directory of Open Access Journals (Sweden)

    Patrick R. Adair

    2017-09-01

    Full Text Available Human regulatory CD4+ T cells (Tregs are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs, and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

  19. A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

    Science.gov (United States)

    Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

    2010-10-01

    Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

  20. Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

    Directory of Open Access Journals (Sweden)

    Gema Álvarez García

    2006-08-01

    Full Text Available A Neospora caninum 17 kDa protein fraction (p17 has been described as an immunodominant antigen (IDA under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17 was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86.

  1. The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer

    NARCIS (Netherlands)

    Somovilla, Víctor J; Bermejo, Iris A.; Albuquerque, Inês S; Martínez-Sáez, Nuria; Castro-López, Jorge; García-Martín, Fayna; Compañón, Ismael; Hinou, Hiroshi; Nishimura, Shin-Ichiro; Jiménez-Barbero, Jesús; Asensio, Juan L.; Avenoza, Alberto; Busto, Jesús H.; Hurtado-Guerrero, Ramón; Peregrina, Jesús M.; Bernardes, Gonçalo J L; Corzana, Francisco

    2017-01-01

    A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic

  2. NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination.

    Directory of Open Access Journals (Sweden)

    Yoshie Kametani

    Full Text Available Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD, which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg. After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2 cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP or keyhole limpet hemocyanin (KLH successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

  3. The molecular determinants of antibody recognition and antigenic drift in the H3 hemagglutinin of swine influenza A virus

    Science.gov (United States)

    Influenza A virus (IAV) of the H3 subtype is an important pathogen that affects both humans and swine. The main intervention strategy for preventing infection is vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA). However, due to antigenic drift, vaccin...

  4. Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.

    Science.gov (United States)

    Lee, Kwang Hoon; Chung, Hae-Shin; Kim, Hyoung Sup; Oh, Sang-Ho; Ha, Moon-Kyung; Baik, Ja-Hyun; Lee, Sungnack; Bang, Dongsik

    2003-07-01

    To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

  5. Bispecific antibodies targeting human CD73

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen.......The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen....

  6. Possible role of specific immunoglobulin M antibodies to Plasmodium falciparum antigens in immunoprotection of humans living in a hyperendemic area, Burkina Faso

    DEFF Research Database (Denmark)

    Boudin, C; Chumpitazi, B; Dziegiel, M

    1993-01-01

    of antibodies to crude extracts of Plasmodium falciparum (IgG or IgM antisomatic and IgG antiexoantigens) were tested by IFI or enzyme-linked immunosorbent assay and were followed up according to the fluctuations of the parasite densities. Specific IgG antibodies had the same evolution as parasite densities....... Group 3 was composed of immunoprotected adults. Specific IgM and IgG antibodies to crude extracts or a recombinant antigen (glutamate-rich protein) of P. falciparum were tested. Specific IgM antibodies were lower in group 1 (nonimmune) than in groups 2 (semiimmune) and 3 (immunoprotected). Furthermore...

  7. Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.

    Science.gov (United States)

    Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

    2015-02-01

    Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. © 2014 British Society for Immunology.

  8. Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

    Science.gov (United States)

    Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

    2015-01-01

    Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. PMID:25255895

  9. The Effectiveness of Implementing an E-Book: Antigen and Antibody Reaction for Diagnosis of Diseases in Microbiology Learning

    Science.gov (United States)

    Samrejrongroj, Phakakrong; Boonsiri, Tanit; Thunyaharn, Sudaluck; Sangarun, Preeyapan

    2014-01-01

    Currently very few Thai Immunology e-Books are available online. The authors created an online e-Book titled, "Antigen and Antibody Reaction for Diagnosis of Diseases" and used a quasi experimental research design to assess the effectiveness of its implementation in terms of knowledge gained, written exam scores and student satisfaction.…

  10. Ontogeny of adaptive antibody response to a model antigen in captive altricial zebra finches.

    Directory of Open Access Journals (Sweden)

    Tess L Killpack

    Full Text Available Based on studies from the poultry literature, all birds are hypothesized to require at least 4 weeks to develop circulating mature B-cell lineages that express functionally different immunoglobulin specificities. However, many altricial passerines fledge at adult size less than four weeks after the start of embryonic development, and therefore may experience a period of susceptibility during the nestling and post-fledging periods. We present the first study, to our knowledge, to detail the age-related changes in adaptive antibody response in an altricial passerine. Using repeated vaccinations with non-infectious keyhole limpet hemocyanin (KLH antigen, we studied the ontogeny of specific adaptive immune response in altricial zebra finches Taeniopygia guttata. Nestling zebra finches were first injected at 7 days (7d, 14 days (14d, or 21 days post-hatch (21d with KLH-adjuvant emulsions, and boosted 7 days later. Adults were vaccinated in the same manner. Induced KLH-specific IgY antibodies were measured using ELISA. Comparisons within age groups revealed no significant increase in KLH-specific antibody levels between vaccination and boost in 7d birds, yet significant increases between vaccination and boost were observed in 14d, 21d, and adult groups. There was no significant difference among age groups in KLH antibody response to priming vaccination, yet KLH antibody response post-boost significantly increased with age among groups. Post-boost antibody response in all nestling age groups was significantly lower than in adults, indicating that mature adult secondary antibody response level was not achieved in zebra finches prior to fledging (21 days post-hatch in zebra finches. Findings from this study contribute fundamental knowledge to the fields of developmental immunology and ecological immunology and strengthen the utility of zebra finches as a model organism for future studies of immune ontogeny.

  11. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    Directory of Open Access Journals (Sweden)

    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  12. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    International Nuclear Information System (INIS)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M.

    1982-01-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys. (Auth.)

  13. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  14. Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

    Science.gov (United States)

    Galanti, L M; Cornu, C; Masson, P L; Robert, A R; Becheanu, D; Lamy, M E; Cambiaso, C L

    1991-05-01

    An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

  15. Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells. Immunological study using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Hareyama, Masato

    1988-12-01

    We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-..gamma... In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G/sub 2//M phases of the cell cycle than in G/sub 0//G/sub 1/. The expression of YH206 antigen was enhanced in the S and G/sub 2//M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens.

  16. Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

    Science.gov (United States)

    Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B

    2016-01-01

    Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. © 2015 British Society for Immunology.

  17. Enhancement by gamma-interferon of in vivo tumor radiolocalization by a monoclonal antibody against HLA-DR antigen

    International Nuclear Information System (INIS)

    Rowlinson, G.; Balkwill, F.; Snook, D.; Hooker, G.; Epenetos, A.A.

    1986-01-01

    Athymic nu/nu (nude) mice bearing s.c. human breast tumors were treated systemically with recombinant human gamma-interferon. These tumors were phenotypically negative for HLA-DR prior to therapy, but after 4 days of treatment, 80% of the cells expressed this antigen in vivo as assessed by immunoperoxidase (F. R. Balkwill et al., Eur. J. Cancer Clin. Oncol., in press, 1986). A radioiodine-labeled murine monoclonal antibody (TAL-1B5) against HLA-DR specifically localized to the tumors in recombinant human gamma-interferon-treated but not in control mice. An isotype-identical murine monoclonal antibody that did not react with control or recombinant human gamma-interferon-treated tumors did not show any specific localization. These results demonstrate that specific localization to tumors of radio-labeled monoclonal antibodies to HLA-DR can be facilitated by systemic therapy with gamma-interferon

  18. Solid phase radioimmunoassays using labelled antibodies: a conceptual framework for designing assays

    International Nuclear Information System (INIS)

    Kalmakoff, J.; Parkinson, A.J.; Crawford, A.M.; Williams, B.R.G.

    1977-01-01

    A simple theoretical model for the antigen-antibody reaction is presented and used to evaluate the optimum conditions for designing solid phase radioimmunoassays (RIA) using labelled antibodies. Both theoretical and experimental data are presented, using a wide variety of antigens and their corresponding antibodies. The types of RIA described include the direct, the indirect, the direct sandwich assays for detecting either antigen or antibody. The experimental results confirm in a semiquantitative manner that the greatest sensitivity of the RIA is achieved when the smallest amount of labelled antibody is used, that whenever possible the antigen/antibody ratio should be greater than unity(>1), and that the formation of the antigen-antibody complex is dependent on the mass action effect

  19. The Development of Bispecific Hexavalent Antibodies as a Novel Class of DOCK-AND-LOCKTM (DNLTM Complexes

    Directory of Open Access Journals (Sweden)

    Chien-Hsing Chang

    2013-05-01

    Full Text Available The DOCK-AND-LOCKTM (DNLTM method provides a modular approach to develop multivalent, multifunctional complexes of defined structures, of which bispecific hexavalent antibodies (bsHexAbs are prominent examples with potential applications in targeted therapy for malignant, autoimmune, and infectious diseases. Currently, bsHexAbs are constructed by derivatizing a divalent IgG, at the carboxyl termini of either the heavy chain (the CH3-format or the light chain (the Ck-format, to contain two stabilized dimers of Fab having a different specificity from the IgG. In this review, we briefly outline the features of the DNLTM method and describe key aspects of bsHexAbs examined with diverse preclinical studies, which include binding affinity to target cells, induction of signaling pathways, effector functions, serum stability, pharmacokinetics, and antitumor activity in human tumor xenograft models. Our findings favor the selection of the CK- over the CH3-format for further exploration of bsHexAbs in clinical trials.

  20. Monoclonal antibody

    International Nuclear Information System (INIS)

    Oyamada, Hiyoshimaru

    1987-01-01

    Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

  1. A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells

    Directory of Open Access Journals (Sweden)

    Alba Marina Gimenez

    2016-11-01

    Full Text Available Abstract Background Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides. Results The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4+ T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c. Conclusions These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.

  2. Anti-soluble liver antigen/liver-pancreas (SLA/LP) antibodies in pediatric patients with autoimmune hepatitis.

    Science.gov (United States)

    Vitozzi, Susana; Djilali-Saiah, Idriss; Lapierre, Pascal; Alvarez, Fernando

    2002-12-01

    Antibodies against soluble liver antigen/liver-pancreas (SLA/LP) have been associated with severe autoimmune hepatitis (AIH) and poor outcome, but most of these reports have focused on adult patients. The aim of this study was to assess the prevalence and clinical significance of anti-SLA/LP antibodies in a pediatric population with AIH. We developed a quantitative enzyme-linked immunoassay (ELISA), a Western blot (WB) and an immunoprecipitation assay (IPA) based on recombinant cDNA from activated Jurkat cells. The specificity of these tests was validated by testing 200 serum samples from healthy subjects, and from patients with liver and non-liver diseases. Anti-SLA/LP antibodies were found in patients with type 1 and type 2 AIH. The prevalence of these antibodies in patients with type 1 AIH was: 42% when tested by ELISA, 15% by WB and 50% by IPA. In patients with type 2 AIH, the prevalence rates were 42% by ELISA, 18% by WB and 44% by IPA. The mean titer values for anti-SLA/LP antibodies was significantly higher in type 2 AIH (1:1,300 +/- 339) than in type 1 AIH (1:600 +/- 71; p LKM1) and anti-liver cytosol type 1 (LC1) antibodies in sera. The presence of anti-SLA/LP showed a significant female preponderance in type 1 and 2 AIH patients (p = 0.0003 and p = 0.003, respectively), and was significantly correlated with a lower age at diagnosis (p = 0.05) in type 1 AIH patients. In conclusion, anti-SLA/LP antibodies in pediatric patients are associated with both type 1 and 2 AIH.

  3. 19-DEJ-1, a hemidesmosome-anchoring filament complex-associated monoclonal antibody. Definition of a new skin basement membrane antigenic defect in junctional and dystrophic epidermolysis bullosa

    DEFF Research Database (Denmark)

    Fine, J D; Horiguchi, Y; Couchman, J R

    1989-01-01

    A murine monoclonal antibody (19-DEJ-1) was recently produced that recognizes a unique antigenic epitope of human skin basement membrane localized to the midlamina lucida exclusively in those areas bordered by overlying hemidesmosomes. To determine whether the antigen defined by 19-DEJ-1 is norma...

  4. Sensitivity of hepatitis C virus core antigen and antibody combination assays in a global panel of window period samples

    Science.gov (United States)

    Laperche, Syria; Nubling, C. Micha; Stramer, Susan L.; Brojer, Ewa; Grabarczyk, Piotr; Yoshizawa, Hiroshi; Kalibatas, Vytenis; El Elkyabi, Magdy; Moftah, Faten; Girault, Annie; van Drimmelen, Harry; Busch, Michael P.; Lelie, Nico

    2016-01-01

    BACKGROUND Hepatitis C virus (HCV) antigen and antibody combination assays have been launched as a cost-effective alternative to nucleic acid testing (NAT) for reducing the antibody-negative window period (WP). Later, a HCV antigen chemiluminescence immunoassay (CLIA) became available. STUDY DESIGN AND METHODS A panel composed of 337 HCV NAT–yield samples that were characterized for viral load (VL) and genotype was used to compare the sensitivity of two combination enzyme-linked immunosorbent assays (Monolisa, Bio-Rad; and Murex, formerly Abbott) and a HCV antigen CLIA (Abbott). Analytic sensitivity was compared with HCV RNA detection using Ultrio (Grifols) by testing serial dilutions of 10 genotype (gt)1 to gt4 samples. RESULTS HCV antigen CLIA detected 92.4% of samples, whereas Monolisa and Murex detected 38.3 and 47.5%, respectively. In the HCV RNA VL range of 105 to 107 IU/mL, Monolisa and Murex detected 38% to 56% of gt1, 85% to 78% of gt2, and 21% to 37% of gt3. The overall geometric mean 50% limit of detection (range) of Ultrio on gt1 to gt4 dilution series was 3.5 (1.2–7.7) copies/mL, compared to 3.3 × 106 (4.4 × 105-2.7 × 107), 3.4 × 106 (2.2 × 105–4.2 × 107), and 2728 (415–7243) copies/mL for Monolisa, Murex, and HCV antigen CLIA, respectively. CONCLUSION Analytical sensitivity of NAT was on average 1 million- and 780-fold higher than combination assays and HCV antigen CLIA, respectively. Relative sensitivities of combination assays differed for genotypes with Murex being more sensitive for gt1 and gt3 and Monolisa more sensitive for gt2. Although being less sensitive than NAT, combination assays could be considered in resource-limited settings since they detect 38% to 47% of seronegative WP donations. PMID:26013970

  5. Monoclonal antibodies: an overview of their advantages and limitations in nuclear medicine

    International Nuclear Information System (INIS)

    Revillard, J.P.; Cohen, J.

    1982-01-01

    The following topics were reviewed: antigen recognition by the immune system; development of immunoassays for antigenic components of biological fluids; monoclonal antibodies against infectious agents; monochonal antibodies against tumor and differentiation antigens; human monoclonal antibodies

  6. Reduced antibody responses against Plasmodium falciparum vaccine candidate antigens in the presence of Trichuris trichiura

    DEFF Research Database (Denmark)

    Esen, Meral; Mordmüller, Benjamin; de Salazar, Pablo Martinez

    2012-01-01

    BACKGROUND: Helminth infections are highly prevalent in the tropics and may have an effect on immune responses to vaccines due to their immunomodulatory effect. The prevalence of helminth infections in young children, the target group for malaria and most other vaccines, is high. Therefore we...... assessed the influence of helminth infection on vaccine-induced immune responses in a phase I clinical trial of the malaria vaccine candidate GMZ2. METHODS: Twenty Gabonese preschool-age children were vaccinated with GMZ2, a blood stage malaria vaccine candidate. Humoral immune response against the vaccine...... antigens and parasitological status were assessed. Vaccine-specific antibody concentrations and memory B-cell numbers were compared in worm infected and non-infected participants. RESULTS: Antibody response to GMZ2 was 3.4-fold (95% confidence interval: 1.6, 7.4) higher in Trichuris trichiura negative...

  7. Effect of antibody charge and concentration on deposition of antibody to glomerular basement membrane

    International Nuclear Information System (INIS)

    Madaio, M.P.; Salant, D.J.; Adler, S.; Darby, C.; Couser, W.G.

    1984-01-01

    Fixed anionic sites within the glomerular capillary wall influence the permeation of serum proteins, the localization of various antigens, and the deposition of antibody in the subepithelial space. In anti-GBM nephritis antibody deposition occurs very rapidly to antigenic sites located relatively proximal in the glomerular capillary wall. The authors examined the influence of the glomerular charge barrier on anti-GBM antibody deposition by comparing the rate of deposition of antibodies with cationic and anionic isoelectric points. Purified sheep anti-rat GBM IgG was isolated from acid eluates of kidneys obtained 24 hr after rats were injected with sheep antiserum to rat GBM. Anti-GBM IgG was separated into cationic (pI 6.4-8.5) and anionic (pI 4.2-6.8) fractions, which were radiolabelled with 131 I and 125 I, respectively, shown to have equal antibody contents measured by in vitro binding to normal glomeruli, mixed in equal amounts, and injected in incremental doses to ten rats. At 1 hr the glomerular antibody binding of each fraction was directly related to the blood level (r . 0.95, r . 0.97) and delivery of antibody (r . 0.98, r . 0.98). Glomerular binding of cationic antibody was four times greater than anionic antibody over the entire range of deliveries studied (P less than 0.001). The authors conclude that glomerular deposition of anti-GBM antibody is directly related to blood concentration and delivery of antibody. Furthermore, the deposition of cationic antibodies to GBM antigens was significantly greater than the deposition of anionic antibodies

  8. Improved porous silicon (P-Si) microarray based PSA (prostate specific antigen) immunoassay by optimized surface density of the capture antibody

    Science.gov (United States)

    Lee, SangWook; Kim, Soyoun; Malm, Johan; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas

    2014-01-01

    Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5ngmL−1, 80pgmL−1, and 800fgmL−1 when arraying the PSA antibody, H117 at the concentration 15µgmL−1, 35µgmL−1 and 154µgmL−1, respectively. We further investigated PSA spiked into human female serum in the range of 800fgmL−1 to 500ngmL−1. The microarray showed a LOD of 800fgmL−1 and a dynamic range of 800 fgmL−1 to 80ngmL−1 in serum spiked samples. PMID:24016590

  9. Radioimmunoassay method for detection of gonorrhea antibodies

    International Nuclear Information System (INIS)

    1975-01-01

    A novel radioimmunoassay for the detection of gonorrhea antibodies in serum is described. A radionuclide is bound to gonorrhea antigens produced by a growth culture. In the presence of gonorrhea antibodies in the serum, an antigen-antibody conjugate is formed, the concentration of which can be measured with conventional radiometric methods. The radioimmunoassay is highly specific

  10. Radioimmunodetection of tumor with Ga-67 labeled antibodies

    International Nuclear Information System (INIS)

    Furukawa, Takako; Endo, Keigo; Ohmomo, Yoshiro

    1986-01-01

    Antibodies against tumor associated antigen; anti-AFP polyclonal antibody, anti-thyroglobulin monoclonal antibody and anti-hCG monoclonal antibody, were labeled with Ga-67, using deferoxamine (DF) as a bifunctional chelating agent. The immunoreactivity and in vivo stability of the Ga-67 labeled antibodies were examined. The effect of DF conjugation to antibodies on the antigen-binding activity was evaluated by RIA and Scatchard analysis or tanned sheep red blood cell hemagglutination technique. When DF was conjugated to antibody at the molar ratio of 1 : 1, the antibody activity of the DF-conjugated antibodies was fully retained. Whereas, in heavily conjugated antibodies, the maximum antigen binding capacity was reduced. Biodistribution study in normal mice demonstrated the high in vivo stability of Ga-67 labeled antibodies. The labeling of DF-antibody conjugated with Ga-67 was performed easily and quickly, with a high labeling efficiency, requiring no further purification. Thus, this labeling method, providing in vivo stability of Ga-67 labeled antibody and full retention of immunoreactivity, would be useful for the radioimmunodetection of various cancers. (author)

  11. Identification of Eimeria acervulina conoid antigen using chicken monoclonal antibody.

    Science.gov (United States)

    Matsubayashi, Makoto; Minoura, Chisa; Kimura, Shintaro; Tani, Hiroyuki; Furuya, Masaru; Lillehoj, Hyun S; Matsuda, Haruo; Takenaka, Shigeo; Hatta, Takeshi; Tsuji, Naotoshi; Sasai, Kazumi

    2016-11-01

    In the poultry industry, Eimeria spp. is one of the important pathogens which cause significant economic losses. We have previously generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina and with cross-reactive among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium spp. Furthermore, the protein of Cryptosporidium parvum recognized by the 6D-12-G10 has been identified as elongation factor-1α (EF-1α). In the present study, to identify the target molecule of E. acervulina by the mAb, we performed two-dimensional Western blotting analysis. Finally, we found two positive molecules which are identified as EF-1α and a related protein. Our previous finding using C. parvum and the results in this study suggest that EF-1α could be associated with the invasion facilitated by the cytoskeleton at the apical region of zoites.

  12. Isocyanate test antigens

    International Nuclear Information System (INIS)

    Karol, M.H.; Alarie, Y.C.

    1980-01-01

    A test antigen for detecting antibodies to a diisocyanate comprises the reaction product of a protein and a monoisocyanate derived from the same radical as the diisocyanate. The diisocyanates most usually encountered and therefore calling for antibody detection are those of toluene, hexamethylene, methylene, isophorone and naphthylene. The preferred protein is human serum albumin. (author)

  13. Antibodies to AB blood group antigens mimic anti-salivary duct autoantibodies in patients with limited sicca symptoms.

    Science.gov (United States)

    Goldblatt, F; Beroukas, D; Gillis, D; Cavill, D; Bradwell, A; Rischmueller, M; Gordon, T P

    2000-10-01

    We evaluated the clinical relevance and pathogenic significance of anti-salivary duct autoantibodies (ASDA) in Sjögren's syndrome (SS) and rheumatoid arthritis (RA) by examining (1) their frequency in healthy controls, patients with sicca symptoms, and patients with various autoimmune and infective disorders; (2) their localization by confocal microscopy; and (3) their tissue distribution and cross reactivity with blood group antigens. Indirect immunofluorescence (IF) was performed on commercial cryostat sections of monkey parotid salivary gland. Sections were examined by fluorescence and confocal laser scanning microscopy. Sera giving positive staining on the ducts were tested by IF on a range of monkey tissues and salivary glands from several mammalian species. Blocking experiments were performed with human erythrocytes of different ABO blood groups and AB antigens. We identified 2 distinct ductal staining patterns. The first resembled ASDA described in earlier studies and showed patchy bright staining of the apical (luminal) surfaces of the ducts and staining of apical cytoplasmic vesicles. The other was only observed with anti-mitochondrial antibody positive sera and stained the mitochondrial-rich ductal epithelium in a distinctive punctate pattern. Antibodies staining the apical surface of ducts were detected rarely in patients with antiRo/La autoantibody-positive primary SS (1/76) and RA (1/36) and were found in only 1115 with RA and secondary SS. ASDA were detected in sera from 13/51 (25.5%) of patients referred to our clinic with limited sicca symptoms who were anti-Ro/La antibody-negative and had no typical clinical or laboratory features of classical primary SS. The apical ductal staining pattern was not observed with sera from 63 healthy controls without sicca symptoms or in patients with autoimmune and infective disorders. Twelve of the 13 patients whose sera gave ASDA-like staining were blood group O and one group A. Ductal staining was abolished in

  14. Molecular docking.

    Science.gov (United States)

    Morris, Garrett M; Lim-Wilby, Marguerita

    2008-01-01

    Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. The goal of ligand-protein docking is to predict the predominant binding mode(s) of a ligand with a protein of known three-dimensional structure. Successful docking methods search high-dimensional spaces effectively and use a scoring function that correctly ranks candidate dockings. Docking can be used to perform virtual screening on large libraries of compounds, rank the results, and propose structural hypotheses of how the ligands inhibit the target, which is invaluable in lead optimization. The setting up of the input structures for the docking is just as important as the docking itself, and analyzing the results of stochastic search methods can sometimes be unclear. This chapter discusses the background and theory of molecular docking software, and covers the usage of some of the most-cited docking software.

  15. Distance between two binding sites of the same antibody molecule

    International Nuclear Information System (INIS)

    Cser, L.; Gladkikh, I.A.; Ostanevich, Y.M.; Franek, F.; Novotny, J.; Nezlin, R.S.

    1978-01-01

    Neutron small-angle scattering experiments are reported, aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. Although the distance values could be determined only with a large statistical error, the data allowed the conclusion that the geometrical parameters of the complexes formed with the early (i.e., precipitating) antibody are significantly different from those of the complexes formed with the late (i.e, non-precipitating) antibody. The data suggest that the precipitating antibody complexed with a high molecular weight antigen assumes an extended shape with an antigen to antigen distance of 35.8 +- 1.3 nm. (Auth.)

  16. Seroprevalence of antibodies and antigens against hepatitis A-E viruses in refugees and asylum seekers in Germany in 2015.

    Science.gov (United States)

    Jablonka, Alexandra; Solbach, Philipp; Wöbse, Michael; Manns, Michael P; Schmidt, Reinhold E; Wedemeyer, Heiner; Cornberg, Markus; Behrens, Georg M N; Hardtke, Svenja

    2017-08-01

    Migration because of miscellaneous political crises in countries in the Middle East and Africa is a global challenge for whole Europe from an economic, social, and public health view. There is an urgent need to generate comprehensive, evidence-based data to expedite further screening and vaccination strategies. A total of 604 individuals ranging in age from 2 to 68 years who enrolled at a single reception center were tested for the prevalence of serologic markers for hepatitis virus types A, B, C, D, and E (HAV, HBV, HCV, HDV, HEV), respectively. Anti-HAV antibody prevalence was 91.2 and 70.3% in children younger than 18 years of age. The prevalence of anti-HEV antibodies was 20.1% among the individuals. 3.0% were positive for hepatitis B surface antigen, whereas 15.2% tested positive for anti-hepatitis B core antigen. None of the refugees tested positive for anti-HDV. 14.1% of refugees were vaccinated against hepatitis B and had a protective anti-hepatitis B surface level of at least 10 mIU/ml. Significant differences in vaccination status were found between the regions (Eastern Mediterranean Region with 77/482 (16.0%; 95% confidence interval=12.7-19.3%) versus African Region with 1/55 (1.8%; 95% confidence interval=0-5.0%). The prevalence of anti-HCV antibodies was 1.2% (n=7), with 0.7% HCV RNA positivity; 16.7% of hepatitis B surface antigen-positive individuals were HCV coinfected (n=3). The prevalence of refugees with previous exposure to hepatitis viruses was higher than that in the general German population, but lower than in other migrant populations in Germany. The vaccination status against hepatitis B was poor.

  17. Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); N. Juntti; B. Klingeborn; J. Groen (Jan); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractAn enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of

  18. The preparation and use of radiolabelled specific helminth antibodies

    International Nuclear Information System (INIS)

    Movsesijan, M.; Jovanovic, B.; Borojevic, D.; Petrovic, M.

    1983-01-01

    Specific antibodies from the serum of sheep infected with Haemonchus contortus were isolated by combination with a ''solid phase antigen'' (soluble antigen coupled to an activated crystalline cellulose). The antibodies were labelled with 125 I while bound to the solid phase then eluted and their potential demonstrated: (1) to determine amounts of specific antibody in unknown sera; (2) to determine amounts of soluble antigen in unknown preparations. (author)

  19. Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency

    NARCIS (Netherlands)

    van der Zee, J. S.; van Swieten, P.; Aalberse, R. C.

    1986-01-01

    Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophagoïdes pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound

  20. Characteristics Studies of 125I- and total PSA antibody's Binding with prostate specific antigen (PSA) in Human Uterus Tumors

    International Nuclear Information System (INIS)

    Al-Mudaffar, S.; Al-Salihi, J.

    2005-01-01

    Two groups of uterus tumors (benign and malignant) postmenopausal patients were used to investigate the presence of prostate specific antigen (PSA). Preliminary experiments were performed to follow the binding of '1 25 I-anti total PSA antibody with PSA in uterus tissues homogenates of the two groups with their corresponding antigen and found to be (8.8,7.1%) for benign and malignant tumors, respectively. An Immuno Radio Metric Assay (IRMA) procedure was developed for measuring PSA in benign and malignant uterus tumors homogenates. The optimum conditions of the binding of 125 I-anti total PSA antibody with PSA were as follows: PSA concentration (150,200 μg protein),tracer antibody concentration (125,250 μg protein), p H (7.6,7.2), temp (15,25?C) and time (1.5 hrs) for postmenopausal benign and malignant uterus tumors tissue homogenates, respectively. The use of different concentrations of Na + and Mg 2+ ions were shown to cause an increase in the binding at concentration of (125,75 mΜ) of Na 1+ ions (75,225 mΜ) of Mg 2+ ions for benign and malignant uterus tumors homogenates, respectively, while the use of different concentrations of urea and polyethylene glycol (PEG) Caused a decrease in the binding with the increase in the concentration of each of urea and PEG in the both cases

  1. A competitive-inhibiton radioimmunoassay for influenza virus envelope antigens

    International Nuclear Information System (INIS)

    Russ, G.; Styk, B.; Vareckova, E.; Polakova, K.

    1976-01-01

    A double-antibody competitive-inhibition radioimmunoassay for influenza virus envelope antigens is described. A viral antigen preparation from influenza A virus recombinant MRC11 [antigenically identical to A/Port Chalmers/1/73 (H3N2)] consisting of haemagglutinin and neuraminidase was labelled with radioiodine. Rabbit antisera were allowed to react with the labelled antigen and the resultant antigen-antibody complexes were precipitated with the appropriate antiglobulin. The competitive-inhibition radioimmunoassay very sensitively elucidated differences even among closely related influenza virus strains. Attempts have been made to eliminate neuraminidase from radioimmunoprecipitation to obtain a competitive-inhibition radioimmunoassay system for haemagglutinin alone. (author)

  2. Combined radioimmunoassay of HBs-antigen and anti-HBs using the Biotest combRIA-Au kit

    International Nuclear Information System (INIS)

    Kselikova, M.; Novak, J.; Urbankova, J.

    1979-01-01

    Hepatitis B antigen and antibody were determined simultaneously by radioimmunoassay using the Biotest combRIA-Au kit. The results in determining antigens and antibodies in sera with a known content of these substances by this kit nearly equal those of isolated determinations of antigen by the Abbott AUSRIA II-125 kit, and of antibody by the Abbott AUSAB kit, resp., except for a negligible number of sera with a very low content of either antigen or antibody. (author)

  3. Exact docking flight controller for autonomous aerial refueling with back-stepping based high order sliding mode

    Science.gov (United States)

    Su, Zikang; Wang, Honglun; Li, Na; Yu, Yue; Wu, Jianfa

    2018-02-01

    Autonomous aerial refueling (AAR) exact docking control has always been an intractable problem due to the strong nonlinearity, the tight coupling of the 6 DOF aircraft model and the complex disturbances of the multiple environment flows. In this paper, the strongly coupled nonlinear 6 DOF model of the receiver aircraft which considers the multiple flow disturbances is established in the affine nonlinear form to facilitate the nonlinear controller design. The items reflecting the influence of the unknown flow disturbances in the receiver dynamics are taken as the components of the "lumped disturbances" together with the items which have no linear correlation with the virtual control variables. These unmeasurable lumped disturbances are estimated and compensated by a specially designed high order sliding mode observer (HOSMO) with excellent estimation property. With the compensation of the estimated lumped disturbances, a back-stepping high order sliding mode based exact docking flight controller is proposed for AAR in the presence of multiple flow disturbances. Extensive simulation results demonstrate the feasibility and superiority of the proposed docking controller.

  4. Imaging of non-small cell lung cancers with a monoclonal antibody, KC-4G3, which recognizes a human milk fat globule antigen

    International Nuclear Information System (INIS)

    Dienhart, D.G.; Schmelter, R.F.; Lear, J.L.; Miller, G.J.; Glenn, S.D.; Bloedow, D.C.; Kasliwal, R.; Moran, P.; Seligman, P.; Murphy, J.R.

    1990-01-01

    To determine the role of lung cancer tumor imaging with monoclonal antibodies directed against high molecular weight human milk fat globule antigens, we administered i.v. 111In-KC-4G3 to 24 patients with advanced non-small cell lung cancer. One mg of 111In-KC-4G3 was mixed with 0, 9, 49, 99, or 499 mg of unlabeled KC-4G3 and infused i.v. over 1 to 5 h. The mean 111In-KC-4G3 radiochemical purity was greater than 97% and the resultant immunoreactivity averaged 62%. Successful imaging of cancer sites was accomplished in 92% of 24 patients, and 57% of 91 total lesions were visualized. Successful localization of tumor sites related to size (P less than 0.001), with 81% of lesions greater than 3.0 cm in diameter, 50% of lesions 1.5 to 3 cm, and 6% of lesions less than 1.5 cm successfully imaging, and to location (P less than 0.05), with 69% of pulmonary lesions, 80% of soft tissue lesions, and only 32% of bone metastases being visualized. Nonspecific reticulo-endothelial uptake of radioactivity was a major problem. Approximately 35% of 111In was chelated to serum transferrin by 24 and 48 h after infusion. The mean t 1/2 beta for plasma radioisotope and immunoreactive KC-4G3 was 29 and 27 h, respectively. There was no correlation between total infused antibody dose and imaging success or between total dose and effect on 111In and KC-4G3 kinetics. Circulating free KC-4 antigen was measurable in all but one patient before study. Tumor biopsy following infusion could demonstrate antibody presence but not saturable antigen binding. We conclude that (a) 111In-KC-4G3 demonstrates successful tumor localization in non-small cell lung cancers bearing generally high expression of its antigen and (b) further investigations to diminish nonspecific radioactivity for imaging and utilization of high dose radiolabeled antibody for therapeutic intent are warranted

  5. Immunodiagnosis of paracoccidioidomycosis due to Paracoccidioides brasiliensis using a latex test: detection of specific antibody anti-gp43 and specific antigen gp43.

    Directory of Open Access Journals (Sweden)

    Priscila Oliveira Dos Santos

    2015-02-01

    Full Text Available Paracoccidioidomycosis (PCM is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations.A latex immunoassay using sensitized latex particles (SLPs with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43 was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF, and bronchoalveolar lavage (BAL from patients with PCM due to P. brasiliensis. The gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7-100.0, specificity (93.94%, 95% CI 87.3-97.7, and positive (91.4% and negative (98.9% predictive values. In addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3-99.6, specificity (88.89%, 95% CI 81.0-94.3, and positive (85.1% and negative (97.8% predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924 and mAb17c-SLPs (k = 0.850, which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. In addition, the latex test was useful for monitoring PCM patients receiving therapy.The high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but mainly in remote locations with limited laboratory infrastructure

  6. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

    Science.gov (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

    2000-03-01

    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  7. Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

    Science.gov (United States)

    Hoelzle, Ludwig E; Hoelzle, Katharina; Wittenbrink, Max M

    2004-10-05

    Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.

  8. Cloning, bacterial expression and crystallization of Fv antibody fragments

    Science.gov (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  9. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen...

  10. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  11. Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

    DEFF Research Database (Denmark)

    Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang

    1988-01-01

    Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....

  12. Commercial bacterins did not induce detectable levels of antibodies in mice against Mycoplasma hyopneumoniae antigens strongly recognized by swine immune system

    Directory of Open Access Journals (Sweden)

    Andressa Fisch

    2016-01-01

    Full Text Available Enzootic Pneumonia (EP caused by Mycoplasma hyopneumoniae results in major economic losses to the swine industry. Hence, the identification of factors that provide protection against EP could help to develop effective vaccines. One such factor that provides partial protection are bacterins. Therefore, the aim of this study was to verify the induction of antibodies against fifteen M. hyopneumoniae antigens, strongly recognized by the swine immune system during natural infection, in mice vaccinated with six commercial bacterins. Each group of mice was inoculated with one bacterin, and seroconversion was assessed by indirect ELISA using recombinant antigens and M. hyopneumoniae 7448 whole cell extract. Sera from one inoculated group recognized antigen MHP_0067, and sera from four inoculated groups recognized antigens MHP_0513 and MHP_0580. None of the bacterins was able to induce seroconversion against the twelve remaining antigens. This absence of a serological response could be attributed to the lack of antigen expression in M. hyopneumoniae strains used in bacterin production. Additionally the partial protection provided by these vaccines could be due to low expression or misfolding of antigens during vaccine preparation. Therefore, the supplementation of bacterins with these recombinant antigens could be a potential alternative in the development of more effective vaccines.

  13. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    Science.gov (United States)

    Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

    2015-01-01

    Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

  14. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    Directory of Open Access Journals (Sweden)

    Sindy Liao-Chan

    Full Text Available Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

  15. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery....... To better understand the underlying mechanisms of antibody-antigen interaction here we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class....

  16. Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

    Science.gov (United States)

    Williams, L A; McLellan, A D; Summers, K L; Sorg, R V; Fearnley, D B; Hart, D N

    1996-01-01

    Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro. PMID:8911149

  17. An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

    Science.gov (United States)

    Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

    2017-10-01

    The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Use of a solid-phase radioimmunoassay and formalin-fixed whole bacterial antigen in the detection of antigen-specific immunoglobulin in prostatic fluid.

    OpenAIRE

    Shortliffe, L M; Wehner, N; Stamey, T A

    1981-01-01

    The prostatic fluid of two patients with Escherichia coli bacterial prostatitis was analyzed for evidence of a local immune response to bacterial infection. A solid-phase radioimmunoassay was modified to measure the immunoglobulin (Ig)A and IgG antigen-specific antibody responses to infecting bacteria in serum and prostatic fluid from patient. Formalin-fixed whole E. coli were used as antigen. In one patient with acute E. coli prostatic infection, measurements of antigen-specific antibody con...

  19. Understanding original antigenic sin in influenza with a dynamical system.

    Science.gov (United States)

    Pan, Keyao

    2011-01-01

    Original antigenic sin is the phenomenon in which prior exposure to an antigen leads to a subsequent suboptimal immune response to a related antigen. Immune memory normally allows for an improved and rapid response to antigens previously seen and is the mechanism by which vaccination works. I here develop a dynamical system model of the mechanism of original antigenic sin in influenza, clarifying and explaining the detailed spin-glass treatment of original antigenic sin. The dynamical system describes the viral load, the quantities of healthy and infected epithelial cells, the concentrations of naïve and memory antibodies, and the affinities of naïve and memory antibodies. I give explicit correspondences between the microscopic variables of the spin-glass model and those of the present dynamical system model. The dynamical system model reproduces the phenomenon of original antigenic sin and describes how a competition between different types of B cells compromises the overall effect of immune response. I illustrate the competition between the naïve and the memory antibodies as a function of the antigenic distance between the initial and subsequent antigens. The suboptimal immune response caused by original antigenic sin is observed when the host is exposed to an antigen which has intermediate antigenic distance to a second antigen previously recognized by the host's immune system.

  20. Antibody responses to a major Pneumocystis carinii antigen in human immunodeficiency virus-infected patients with and without P. carinii pneumonia

    DEFF Research Database (Denmark)

    Lundgren, Bettina; Lundgren, Jens Dilling; Nielsen, T

    1992-01-01

    of pulmonary symptoms. Significantly more patients with P. carinii pneumonia (PCP) had detectable antibodies compared with HIV-infected patients without PCP and with HIV-negative controls (50 [66%] of 76 vs. 18 [34%] of 53 and 7 [35%] of 20, respectively; P less than .001), and the level of antibody response......Antibody responses to a major purified human Pneumocystis carinii surface antigen (gp95) were determined by ELISA in human immunodeficiency virus (HIV)-infected patients. Serum IgG directed against gp95 was measured in 129 consecutive HIV-infected patients who underwent bronchoscopy for evaluation...... response, compared with only 1 (3%) of 31 patients without PCP (P less than .001). This patient had PCP on the basis of clinical criteria, including response to therapy. Thus, despite severe immunosuppression, a proportion of HIV-infected patients with PCP can mount a specific IgG-mediated antibody...

  1. Radioimmunoassay in the detection of the hepatitis Be antigen/antibody system in asymptomatic carriers of hepatitis B surface antigen

    International Nuclear Information System (INIS)

    Pastore, G.; Dentico, P.; Angarano, G.; Schiraldi, O.; Zanetti, A.R.; Ferroni, P.

    1980-01-01

    A radioimmunoassay for hepatitis e antigen (HBeAg) and antibody to e (anti-HBe) was developed and sera of 71 asymptomatic chronic carriers of hepatitis B surface antigen (HBsAg), in 44 of whom liver biopsy was obtained, were tested. In addition, testing for Dane particle associated DNA polymerase activity was performed in all sera. HBeAg was detected in 14 subjects (19.7%) and anti-HBe in 46 (64.8%). The highest proportion of HBeAg positivity (40%) was found among carriers with histological evidence of chronic hepatitis, whereas anti-HBe was present in 80% of carriers with normal liver histology, in 58% of carriers with non-specific reactive hepatitis and in 60% of carriers with chronic liver lesions. DNA polymerase activity was present in 92.8% of sera positive for HBeAg, in 13% of sera positive for anti HBe, and in 9% of sera negative for both markers. Our results demonstrate that not all HBsAg carriers reactive to HBeAg show evidence of chronic hepatitis nor, conversely, that anti-HBe is invariably associated with the healthy carrier state of HBsAg. Finally, circulating Dane particles, as revealed by the presence of serum specific DNA polymerase activity, may also be present in anti-HBe positive sera other than those of some HBsAg carriers lacking both HBeAg and anti-HBe. (orig.) [de

  2. Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

    Science.gov (United States)

    Schmitz, H

    1981-01-01

    An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

  3. Detection of human leukocyte antigen compatibility and antibodies in liver transplantation in China

    Institute of Scientific and Technical Information of China (English)

    Xue-Qin Meng; Xuan Zhang; Jun Fan; Lin Zhou; Bing Hao; Xiao-Ming Chen; Wei-Hang Ma; Shu-Sen Zheng

    2009-01-01

    BACKGROUND: The exact roles of human leukocyte antigen (HLA) compatibility, HLA antibodies and underlying diseases in acute rejection of liver transplants are not clear. Moreover, cytomegalovirus (CMV) infection, one of the most common infections after transplantation, is related to HLA genotype and the incidence of acute rejection. METHODS: Since there are controversial reports, we analyzed the impact of HLA matching, HLA antibodies and underlying diseases in 38 liver transplant recipients in China, and assessed the association of CMV infection and HLA compatibility. RESULTS: The frequency of no HLA compatibility was high in patients without antigenemia (P=0.019). All 17 patients with HLA-A matching developed antigenemia (P0.05). In patients with acute rejection, no differences were found in the incidence of acute rejection in transplants for hepatitis B, tumors, or combined hepatitis B and tumors (P>0.05).CONCLUSIONS: There are fewer acute rejections in transplants with more HLA compatibilities. Speciifc investigations of underlying diseases and HLA typing may be necessary in liver transplantation. The mechanisms of CMV infection and HLA matching should be further studied. HLA before transplantation should be examined for the prevention of acute rejection and CMV infection.

  4. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

    Science.gov (United States)

    Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

    2010-03-01

    The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

  5. Imaging of colorectal carcinoma with radiolabeled antibodies.

    Science.gov (United States)

    Goldenberg, D M; Goldenberg, H; Sharkey, R M; Lee, R E; Higgenbotham-Ford, E; Horowitz, J A; Hall, T C; Pinsky, C M; Hansen, H J

    1989-10-01

    Colorectal cancer has been the tumor type most frequently studied with radiolabeled antibodies. Among the various antibodies, a majority of patients with colorectal cancer have received xenogeneic polyclonal or monoclonal antibodies against carcino-embryonic antigen. This review summarizes the current status of colorectal cancer imaging with radiolabeled antibodies, ie, radioimmunodetection (RAID), and examines the published studies involving carcinoembryonic antigen (CEA) antibodies and 17-1A, 19-9, and B72.3, and other monoclonal antibodies. In order to better address the issue of the current and future clinical usefulness of this emerging technology, particular attention is given to the protocols, methods, and results of the published studies. Despite differences in study parameters, antibodies and forms, labels, administration routes and doses, and scanning instruments and methods, it has been found that (1) almost no adverse reactions have been evident; (2) antibody fragments are preferred over whole immunoglobulin G reagents because they achieve higher tumor-to-background ratios earlier, thus reducing or precluding the need for dual-isotope subtraction methods or long delays before imaging; (3) use of antibody fragments, including the monovalent Fab' form, permits imaging with short-lived radionuclides of excellent photon properties, such as 123I and 99mTc; (4) circulating antigens against which the imaging antibody is directed can complex with the injected antibody, but such complexes have not prevented successful RAID; (5) patients with high serum titers of the appropriate antigen target usually have higher rates of positive RAID; (6) patients who are seronegative for the tumor antigen being studied can have positive RAID findings, which can represent the detection of occult lesions; (7) single photon emission computed tomography appears to provide better image resolution than planar scanning; (8) regardless of the sensitivity reported in any particular

  6. The fate of heterologous antigen (131I-HSA) in the organs of chickens exposed to total-body X-irradiation before a secondary antigenic stimulus

    International Nuclear Information System (INIS)

    Prohazka, Z.; Hampl, J.; Krejci, J.

    1975-01-01

    A study was made on the effect of ionizing radiation on the rate of elimination of 131 I-labelled human serum albumin from the blood and its organ deposition in chickens exposed to 1200 R (LD 50 ) at various intervals before secondary antigen injection. In unirradiated control chickens, the elimination of antigen after its secondary injection followed the typical three-phase pattern, characterized by an early onset and a rapid progress of the third phase. The elimination curve from irradiated birds paralleled rather closely that from the controls during the first and second phases while the phase of immune elimination was hardly perceptible. No major differences were found between the individual irrradiated groups. The irradiated birds also showed less formation of antibodies and antigen-antibody complexes and a lower antigen content of the organs than the unirradiated controls. From the results it appears that the specific antigen uptake from the blood of chickens during the first and second phases of elimination of a secondary dose of antigen is radioresistant; the temporal relation between X-irradiation and secondary antigen injection does not play a substantial role in impairment of the secondary antibody response to soluble antigens in chickens

  7. Serum IgG antibodies from healthy subjects up to 100 years old react to JC polyomavirus.

    Science.gov (United States)

    Bononi, Ilaria; Mazzoni, Elisa; Pietrobon, Silvia; Manfrini, Marco; Torreggiani, Elena; Rossini, Marika; Lotito, Francesca; Guerra, Giovanni; Rizzo, Paola; Martini, Fernanda; Tognon, Mauro

    2018-08-01

    JC polyomavirus (JCPyV) was identified in 1971 in the brain tissue of a patient (J.C.) affected by the progressive multifocal leukoencephalopathy (PML). JCPyV encodes for the oncoproteins large T antigen (Tag) and small t-antigen (tag). These oncoproteins are responsible of the cell transformation and tumorigenesis in experimental animals. JCPyV is ubiquitous in human populations. After the primary infection, which is usually asymptomatic, JCPyV remains lifelong in the host in a latent phase. Its reactivation may occur in heathy subjects and immunocompromised patients. Upon reactivation, JCPyV could reach (i) the CNS inducing the PML, (ii) the kidney of transplant patients causing the organ rejection. Association between JCPyV, which is a small DNA tumor virus, and gliomas and colorectal carcinomas has been published. In the present investigation, we report on a new indirect ELISA with two specific synthetic peptides mimicking JCPyV VP1 immunogenic epitopes to detect specific serum IgG antibodies against JCPyV. Serum samples of healthy subjects (n = 355) ranging 2-100 years old, were analyzed by this new indirect ELISA. The linear peptides VP1 K and VP1 N resemble the natural JCPyV VP1 capsidic epitopes constituting a docking site for serum antibodies. Data from this innovative immunologic assay indicate that the overall prevalence of JCPyV-VP1 antibodies in healthy subjects is at 39%. The innovative indirect ELISA with JCPyV VP1 mimotopes seems to be a useful method to detect specific IgG antibodies against this virus, without cross-reactivity with the closely related SV40 and BKPyV polyomaviruses. © 2018 Wiley Periodicals, Inc.

  8. Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays.

    Science.gov (United States)

    Principato, MaryAnn; Njoroge, Joyce M; Perlloni, Andrei; O' Donnell, Michael; Boyle, Thomas; Jones, Robert L

    2010-10-01

    There is a critical need for qualitative and quantitative methodologies that provide the rapid and accurate detection of food contaminants in complex food matrices. However, the sensitivity of the assay can be affected when antigen-capture is applied to certain foods or beverages that are extremely acidic. This study was undertaken to assess the effects of orange juice and popular carbonated soft drink upon the fidelity of antibody-based antigen-capture assays and to develop simple approaches that could rescue assay performance without the introduction of additional or extensive extraction procedures. We examined the effects of orange juice and a variety of popular carbonated soft drink beverages upon a quantitative Interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) assay system and a lateral flow device (LFD) adapted for the detection of staphylococcal enterotoxin B (SEB) in foods. Alterations in the performance and sensitivity of the assay were directly attributable to the food matrix, and alterations in pH were especially critical. The results demonstrate that approaches such as an alteration of pH and the use of milk as a blocking agent, either singly or in combination, will partially rescue ELISA performance. The same approaches permit lateral flow to efficiently detect antigen. Practical Application: The authors present ways to rescue an ELISA assay compromised by acidity in beverages and show that either the alteration of pH, or the use of milk as a blocking agent are not always capable of restoring the assay to its intended efficiency. However, the same methods, when employed with lateral flow technology, are rapid and extremely successful.

  9. 9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Erysipelothrix Rhusiopathiae Antibody... REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae Antibody is a specific antibody product containing antibodies directed against one or more somatic antigens...

  10. A small antigenic determinant of the Chikungunya virus E2 protein is sufficient to induce neutralizing antibodies which are partially protective in mice.

    Directory of Open Access Journals (Sweden)

    Christopher Weber

    2015-04-01

    Full Text Available The mosquito-borne Chikungunya virus (CHIKV causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses.E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L and surface-exposed parts of the E2 domain A (sA alone did not induce neutralizing antibodies, a construct containing domain B and a part of the β-ribbon (called B+ was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+ induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA, MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice.The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine.

  11. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http://www.biocomputing.it/tabhu CONTACT: anna.tramontano@uniroma1.it, pierpaolo.olimpieri@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  12. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

    Toledo e Souza, I.T. de; Okada, H.

    1990-05-01

    Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

  13. Timing of the human prenatal antibody response to Plasmodium falciparum antigens.

    Directory of Open Access Journals (Sweden)

    Samuel Tassi Yunga

    Full Text Available Plasmodium falciparum (Pf-specific T- and B-cell responses may be present at birth; however, when during fetal development antibodies are produced is unknown. Accordingly, cord blood samples from 232 preterm (20-37 weeks of gestation and 450 term (≥37 weeks babies were screened for IgM to Pf blood-stage antigens MSP1, MSP2, AMA1, EBA175 and RESA. Overall, 25% [95% CI = 22-28%] of the 682 newborns were positive for IgM to ≥1 Pf antigens with the earliest response occurring at 22 weeks. Interestingly, the odds of being positive for cord blood Pf IgM decreased with gestational age (adjusted OR [95% CI] at 20-31 weeks = 2.55 [1.14-5.85] and at 32-36 weeks = 1.97 [0.92-4.29], with ≥37 weeks as reference; however, preterm and term newborns had similar levels of Pf IgM and recognized a comparable breadth of antigens. Having cord blood Pf IgM was associated with placental malaria (adjusted OR [95% CI] = 2.37 [1.25-4.54]. To determine if in utero exposure occurred via transplacental transfer of Pf-IgG immune complexes (IC, IC containing MSP1 and MSP2 were measured in plasma of 242 mother-newborn pairs. Among newborns of IC-positive mothers (77/242, the proportion of cord samples with Pf IC increased with gestational age but was not associated with Pf IgM, suggesting that fetal B cells early in gestation had not been primed by IC. Finally, when cord mononuclear cells from 64 term newborns were cultured in vitro, only 11% (7/64 of supernatants had Pf IgM; whereas, 95% (61/64 contained secreted Pf IgG. These data suggest fetal B cells are capable of making Pf-specific IgM from early in the second trimester and undergo isotype switching to IgG towards term.

  14. Evaluation of an Antigen-Antibody

    African Journals Online (AJOL)

    GB

    replication would lead to the production of various antigens. Today with BMT history of over 30 years, infection ... Study design: The study involved both retrospective and prospective laboratory-based analysis of ..... core protein of a molecular mass 19 x 103 Da, one picogram (pg) of virus core corresponds to 1.3 x. 105 HCV ...

  15. Evaluation of Serum Specific Antibody against Recombinant ESAT-6 Antigen in Patients with Tuberculosis and Comparing to Normal Controls

    Directory of Open Access Journals (Sweden)

    Homeira Izadi

    2017-02-01

    Full Text Available Background & Objective: Tuberculosis (TB is a zoonotic disease which is caused by Mycobacterium tuberculosis. Because of common structural and secretory antigens between pathogen and nonpathogenic mycobacterium, the specific diagnosis of TB is difficult. Therefore, it is very important to find a new method with high specificity and sensitivity for accurate and rapid diagnosis of tuberculosis. In this study, the serodiagnostic potential of Mycobacterium tuberculosis recombinant ESAT-6 in TB infected patients was evaluated by Enzyme Linked Immunosorbent Assay (ELISA. Materials & Methods: 55 TB patients with active disease and 28 healthy controls have been collected and evaluated in different dilutions in ELISA methods for the presence of specific anti-ESAT-6 antibody. The specificity and the sensitivity of this method was compared with the culture test. Results: TB patients have high levels of specific antibody against ESAT-6 antigens. The specificity and the sensitivity of this method was calculated as 80.90% and 85.45%, respectively. Conclusion: These findings provide useful information on the importance of ESAT-6 protein and suggested this serologic test as a good alternative method for rapid and prefect diagnosis of tuberculosis.

  16. Monoclonal antibodies to Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Halpern, J L; Lundgren, B

    1989-01-01

    To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii r...

  17. Prevalence of Bovine Viral Diarrhoea Virus antibodies and antigen among the aborted cows in industrial dairy cattle herds in Mashhad area of Iran

    Directory of Open Access Journals (Sweden)

    Naseri, Z.

    2011-06-01

    Full Text Available The measurement of antibody responses of animals exposed to BVDV either through a natural exposure or an immunization protocol is still a standard procedure. For BVDV, the test formats have been largely limited to ELISA which is a valuable diagnostic test to measure the level of BVDV specific antibodies as well as antigen in blood samples. In the present study, 120 blood samples were collected from the cows with the history of abortion in different period of pregnancy from different industrial dairy cattle herds of Mashhad area of Iran. Also 30 samples were collected from the cows with no history of abortion as control. The presence of antibody against BVDV from the 120 serum samples was investigated by indirect ELISA. From 120 serum samples which were collected from aborted cows, 89 samples were positive (%74.16. From these positive samples, 12(13.48%, 54 (60.68% and 23 (25.84% samples belong to the first, second and third trimester of pregnancy, respectively. From 89 positive samples, 12 (13.48% samples were related to stillbirth and 8 (8.99% samples were belongs to the mummified fetus. From 89 positive samples, 71 (79.78% were related to cattle between 2-5 years old and 18 (20.22% were associated to cattle more than 5 years old. In control group, 20 samples (66.66% were antibody positive. Also the presence of BVDV antigen in serum samples was investigated by Ag-capture ELISA. From 120 serum samples, 2 samples were positive (1.67%, which belongs to the second period of pregnancy. In control group, none of the samples were antigen positive. The results of this study showed that the prevalence of BVDV infection is high among the aborted cows of Mashhad area. Although this prevalence is higher than the control group, the observed difference is not significant.

  18. Lea blood group antigen on human platelets

    International Nuclear Information System (INIS)

    Dunstan, R.A.; Simpson, M.B.; Rosse, W.F.

    1985-01-01

    One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125 I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125 I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125 I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined

  19. Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.

    Science.gov (United States)

    Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

    2011-04-01

    The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Intra-uterine exposure of horses to Sarcocystis spp. antigens

    Directory of Open Access Journals (Sweden)

    A.M. Antonello

    2016-04-01

    Full Text Available The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT and immunoblot analysis. In 84.1% (159/189 of the pregnant mares and in 7.4% (14/189 of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.

  1. Hepatitis A virus antibody

    International Nuclear Information System (INIS)

    Novak, J.; Kselikova, M.; Urbankova, J.

    1980-01-01

    A description is presented of a radioimmunoassay designed to prove the presence of the antibody against the hepatitis A virus (HA Ab, anti-Ha) using an Abbott HAVAB set. This proof as well as the proof of the antibody against the nucleus of the hepatitis B virus is based on competition between a normal antibody against hepatitis A virus and a 125 I-labelled antibody for the binding sites of a specific antigen spread all over the surface of a tiny ball; this is then indirect proof of the antibody under investigation. The method is described of reading the results from the number of impulses per 60 seconds: the higher the titre of the antibody against the hepatitis A virus in the serum examined, the lower the activity of the specimen concerned. The rate is reported of incidence of the antibody against the hepatitis A virus in a total of 68 convalescents after hepatitis A; the antibody was found in 94.1%. The immunoglobulin made from the convalescents' plasma showed the presence of antibodies in dilutions as high as 1:250 000 while the comparable ratio for normal immunoglobulin Norga was only 1:2500. Differences are discussed in the time incidence of the antibodies against the hepatitis A virus, the antibodies against the surface antigen of hepatitis B, and the antibody against the nucleus of the hepatitis V virus. (author)

  2. The effect of parenteral immunisation on antibody production in the pig colon.

    Science.gov (United States)

    Rees, A S; Lysons, R J; Stokes, C R; Bourne, F J

    1989-11-30

    Local and systemic antibody production was studied in pigs to compare responses to live and killed bacterial antigen and purified protein antigen, with and without prior mucosal stimulation. Recovery from challenge with live bacteria and intramuscular injection with killed bacteria gave rise to similar high levels of serum IgG antibody, but the ratio of specific IgA to IgG in the colon was significantly higher after infection than following vaccination with killed bacteria. Vaccination with a protein antigen gave rise to serum and local antibody production. Prior feeding of the antigen had a tolerising effect on the serum antibody response, but production of IgG and IgA antibody by the colon was not suppressed.

  3. Assessing drivers of the IgG4 antibody reactivity to recombinant antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa

    DEFF Research Database (Denmark)

    Hansen, Johanne Damgaard; Meyrowitsch, Dan W.; Rwegoshora, Rwehumbiza T.

    2016-01-01

    to the recombinant filarial antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa. Sera collected during previous studies from 395 well characterized individuals with regard to age, sex, mf, CFA, household vector biting and household exposure to infective filarial larvae were tested for IgG4......A high proportion of the human population in lymphatic filariasis (LF) endemic areas is positive for filarial specific IgG4 antibodies, including many individuals without microfilariae (mf; circulating larvae in the human blood) or circulating filarial antigens (CFA; marker of adult worm infection...

  4. Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

    Directory of Open Access Journals (Sweden)

    MORALES Olga Lucía

    2002-01-01

    Full Text Available Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB assay was performed using excretory/secretory antigens (E/S of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

  5. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination

    NARCIS (Netherlands)

    Mahan, Alison E.; Jennewein, Madeleine F.; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W.; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D.; Baden, Lindsey; Barouch, Dan H.; Alter, Galit

    2016-01-01

    Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain

  6. HLA-DP antigens and HTLV-1 antibody status among Japanese with multiple sclerosis: evidence for an increased frequency of HLA-DPw4

    DEFF Research Database (Denmark)

    Ødum, Niels; Saida, T; Ohta, M

    1989-01-01

    , personal communication). Sera from all 34 patients and 38 controls (both from the HTLV-1 nonendemic, Kyoto region) were examined for the presence of HTLV-1 reacting antibodies by a highly sensitive radioimmuno assay (RIA) using two sources of HTLV-1 antigens, namely total crude protein preparations from...... disrupted HTLV-1 virions and affinity purified p24 HTLV-1 core proteins. The frequency of DPw4 was significantly increased to 35.3% in Japanese MS patients compared to 16.5% in controls (Relative Risk, RR = 2.8, p = 1.9 x 10(-2)). 41.6% of the MS patients gave clear typing responses with a PLT reagent which...... recognized a Dw2+ related specificity, which is higher than the frequency of Dw2 (6.8%) in Japanese. Fourteen of the 34 patient sera contrasting to none of the sera from 38 controls contained antibodies of IgG and/or IgM subclasses reacting with the HTLV-1 derived antigens. This difference is highly...

  7. Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats.

    Science.gov (United States)

    Kalaiyarasu, Semmannan; Mishra, Niranjan; Rajukumar, Katherukamem; Nema, Ram Kumar; Behera, Sthita Pragnya

    2015-01-01

    The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

  8. AnchorDock: Blind and Flexible Anchor-Driven Peptide Docking.

    Science.gov (United States)

    Ben-Shimon, Avraham; Niv, Masha Y

    2015-05-05

    The huge conformational space stemming from the inherent flexibility of peptides is among the main obstacles to successful and efficient computational modeling of protein-peptide interactions. Current peptide docking methods typically overcome this challenge using prior knowledge from the structure of the complex. Here we introduce AnchorDock, a peptide docking approach, which automatically targets the docking search to the most relevant parts of the conformational space. This is done by precomputing the free peptide's structure and by computationally identifying anchoring spots on the protein surface. Next, a free peptide conformation undergoes anchor-driven simulated annealing molecular dynamics simulations around the predicted anchoring spots. In the challenging task of a completely blind docking test, AnchorDock produced exceptionally good results (backbone root-mean-square deviation ≤ 2.2Å, rank ≤15) for 10 of 13 unbound cases tested. The impressive performance of AnchorDock supports a molecular recognition pathway that is driven via pre-existing local structural elements. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Antigen antibody interactions

    CERN Document Server

    DeLisi, Charles

    1976-01-01

    1. 1 Organization of the Immune System One of the most important survival mechanisms of vertebrates is their ability to recognize and respond to the onslaught of pathogenic microbes to which they are conti- ously exposed. The collection of host cells and molecules involved in this recognition­ 12 response function constitutes its immune system. In man, it comprises about 10 cells 20 (lymphocytes) and 10 molecules (immunoglobulins). Its ontogenic development is c- strained by the requirement that it be capable of responding to an almost limitless variety of molecular configurations on foreign substances, while simultaneously remaining inert to those on self components. It has thus evolved to discriminate, with exquisite precision, between molecular patterns. The foreign substances which induce a response, called antigens, are typically large molecules such as proteins and polysaccharides. The portions of these with which immunoglobulins interact are called epitopes or determinants. A typical protein epitope m...

  10. Utility of HLA Antibody Testing in Kidney Transplantation

    Science.gov (United States)

    Konvalinka, Ana

    2015-01-01

    HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

  11. IGHV1-69 B cell chronic lymphocytic leukemia antibodies cross-react with HIV-1 and hepatitis C virus antigens as well as intestinal commensal bacteria.

    Directory of Open Access Journals (Sweden)

    Kwan-Ki Hwang

    Full Text Available B-cell chronic lymphocytic leukemia (B-CLL patients expressing unmutated immunoglobulin heavy variable regions (IGHVs use the IGHV1-69 B cell receptor (BCR in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s (≥21 aa. IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54 of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54 allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.

  12. Primary and secondary antibody reaction to acute radiation syndrome of calves after parenteral and oral immunization with Salmonella antigen

    International Nuclear Information System (INIS)

    Koch, F.; Mehlhorn, G.; Johannsen, U.; Panndorf, H.

    1984-01-01

    After active immunization against Salmonella dublin 25 calves (2.5 to 4 weeks old) were whole-body irradiated with sublethal to medium lethal X-ray doses, with 5 sham-irradiated control animals in each group. Sublethal and medium lethal doses failed in affecting the antibody titers in general, though short-time effects were observed temporarily. These depressive effects correlated with clinical responses, especially with reduced food intake probably caused by nutritive disorders. Higher antibody levels in the recovery period following sublethal and medium lethal doses indicate an antigenic stimulation released by the radiation syndrome. The depressive action of medium lethal doses on the booster response on the 30th postirradiation day refers to damage of the memory cell pool

  13. Monoclonal antibodies in oncology

    International Nuclear Information System (INIS)

    Chan, S.Y.T.; Sikora, K.

    1986-01-01

    Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. (Auth.)

  14. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    International Nuclear Information System (INIS)

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. The authors have studied the binding of 125 I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind =Ka[Ag total]/1 + Ka[Ag total]. Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10 8 M -1 are not likely to be useful for drug targeting or tumor imaging

  15. Discovery of a Prefusion Respiratory Syncytial Virus F-Specific Monoclonal Antibody That Provides Greater In Vivo Protection than the Murine Precursor of Palivizumab.

    Science.gov (United States)

    Zhao, Min; Zheng, Zi-Zheng; Chen, Man; Modjarrad, Kayvon; Zhang, Wei; Zhan, Lu-Ting; Cao, Jian-Li; Sun, Yong-Peng; McLellan, Jason S; Graham, Barney S; Xia, Ning-Shao

    2017-08-01

    persistent infection is limited because of cost, determined from the high doses needed to compensate for its relatively low neutralizing potency. Prior efforts to improve the in vitro potency of site II-specific antibodies did not translate to significant in vivo dose sparing. We isolated a pre-F protein-specific, high-potency neutralizing antibody (5C4) that recognizes antigenic site Ø and compared its efficacy to that of the murine precursor of palivizumab (antibody 1129) matched for isotype and pre-F protein binding affinities. Our findings demonstrate that epitope specificity is an important determinant of antibody neutralizing potency, and defining the mechanisms of neutralization has the potential to identify improved products for the prevention and treatment of RSV infection. Copyright © 2017 American Society for Microbiology.

  16. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  17. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

    DEFF Research Database (Denmark)

    de Jong, R. N.; Beurskens, F. J.; Verploegen, S.

    2016-01-01

    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology......) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce...... conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD...

  18. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity...... to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  19. Antigenic characterisation of lyssaviruses in South Africa

    Directory of Open Access Journals (Sweden)

    Ernest Ngoepe

    2014-09-01

    Full Text Available There are at least six Lyssavirus species that have been isolated in Africa, which include classical rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus. In this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in South Africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. A total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. The lyssaviruses were differentiated into two species, namely rabies virus (99.5% and Mokola virus (0.5%. Furthermore, rabies virus was further delineated into two common rabies biotypes in South Africa: canid and mongoose. Initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. This difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. Mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. Although Mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. These data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones.

  20. Evaluation of a novel enzyme-linked immunosorbent assay for detection of antibodies against Salmonella, employing a stable coating of lipopolysaccharide-derived antigens covalently attached to polystyrene microwells

    DEFF Research Database (Denmark)

    Wiuff, C.; Jauho, E. S.; Stryhn, H.

    2000-01-01

    to microtiter polystyrene plates by ultraviolet irradiation. Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate. The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from......Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled...

  1. Proteomic Identification of Non-Gal Antibody Targets After Pig-to-Primate Cardiac Xenotransplantation

    Science.gov (United States)

    Byrne, Guerard W.; Stalboerger, Paul G.; Davila, Eduardo; Heppelmann, Carrie J.; Gazi, Mozammel H.; McGregor, Hugh C. J.; LaBreche, Peter T.; Davies, William R.; Rao, Vinay P.; Oi, Keiji; Tazelaar, Henry D.; Logan, John S.; McGregor, Christopher G. A.

    2008-01-01

    Background Experience with non-antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non-Gal endothelial cell (EC) antigens. Methods To gauge the diversity of the non-Gal antibody response we used antibody derived from CD46 transgenic heterotopic cardiac xenografts performed without T-cell immunosuppression, Group A (n = 4) and Gal knockout (GT-KO) heart transplants under tacrolimus and sirolimus immunosuppression, Group B (n = 8). Non-Gal antibody was measured by flow cytometry and by Western blots using GT-KO EC membrane antigens. A nanoLC/MS/MS analysis of proteins recovered from 2D gels was used to identify target antigens. Results Group A recipients exhibited a mixed cellular and humoral rejection. Group B recipients mainly exhibited classical DXR. Western blot analysis showed a non-Gal antibody response induced by GT+ and GT-KO hearts to an overlapping set of pig aortic EC membrane antigens. Proteomic analysis identified 14 potential target antigens but failed to define several immunodominant targets. Conclusions These experiments indicate that the non-Gal antibody response is directed to a number of stress response and inflammation related pig EC antigens and a few undefined targets. Further analysis of these antibody specificities using alternative methods is required to more fully define the repertoire of non-Gal antibody responses. PMID:18957049

  2. Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

    Science.gov (United States)

    Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

    1991-01-01

    The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

  3. Hepatitis B and A virus antibodies in alcoholic steatosis and cirrhosis

    DEFF Research Database (Denmark)

    Gluud, C; Aldershvile, J; Henriksen, J

    1982-01-01

    Sera from 74 alcoholics with cirrhosis and 63 alcoholics with steatosis were tested for antibody to hepatitis B surface antigen, to hepatitis B core antigen, and to hepatitis A virus by radioimmunoassay or enzyme-linked immunosorbent assay. No significant difference between the two groups...... of alcoholics could be found concerning the prevalence of these antibodies. The total group of patients had antibody to hepatitis B surface antigen or hepatitis B core antigen, or both, significantly (p less than 0.001) more often (26%) than sex- and age-matched controls (4%). No significant difference...... was found between patients and controls concerning the prevalence of antibody to hepatitis A virus (46% v 40%). In patients with cirrhosis, no correlation between wedged hepatic vein pressure or wedged-to-free hepatic vein pressure and any of the viral antibodies could be established. The present results...

  4. Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection.

    Science.gov (United States)

    Lenzi, M; Manotti, P; Muratori, L; Cataleta, M; Ballardini, G; Cassani, F; Bianchi, F B

    1995-01-01

    Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7797126

  5. Localization of tumors by radiolabelled antibodies

    International Nuclear Information System (INIS)

    Hansen, H.J.; Primus, F.J.

    1975-01-01

    A method of utilizing radiolabelled antibodies to carcinoembryonic antigens for determining the site of tumors which produce or are associated with carcinoembryonic antigen is disclosed. 3 claims, no drawings

  6. Anti-cytotoxic T lymphocyte antigen-4 antibodies in melanoma

    Directory of Open Access Journals (Sweden)

    Tosti G

    2013-10-01

    Full Text Available Giulio Tosti, Emilia Cocorocchio, Elisabetta PennacchioliDivisione Melanomi e Sarcomi, Istituto Europeo di Oncologia, Milano, ItalyAbstract: Approaches aimed at enhancement of the tumor specific response have provided proof for the rationale of immunotherapy in cancer, both in animal models and in humans. Ipilimumab, an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4 antibody, is a new generation immunotherapeutic agent that has shown activity in terms of disease free and overall survival in metastatic melanoma patients. Its use was approved by the US Food and Drug Administration in March 2011 to treat patients with late stage melanoma that has spread or that cannot be removed by surgery. The mechanism of action of CTLA-4 antibodies in the activation of an antitumor immune response and selected clinical studies of ipilimumab in advanced melanoma patients are discussed. Ipilimumab treatment has been associated with immune related adverse events due to T-cell activation and proliferation. Most of these serious adverse effects are associated with the gastrointestinal tract and include severe diarrhea and colitis. The relationship between immune related adverse events and antitumor activity associated with ipilimumab was explored in clinical studies. Potential biomarkers predictive for clinical response and survival in patients treated with anti-CTLA-4 therapy are presently under investigation. Besides the conventional patterns of response and stable disease as defined by standard Response Evaluation Criteria in Solid Tumors criteria, in subsets of patients, ipilimumab has shown patterns of delayed clinical activity which were associated with an improved overall survival. For this reason a new set of response criteria for tumor immunotherapy has been proposed, which was termed immune related response criteria. These new criteria are presently used to better analyze clinical activity of immunotherapeutic regimens. Ipilimumab is currently under

  7. A python-based docking program utilizing a receptor bound ligand shape: PythDock.

    Science.gov (United States)

    Chung, Jae Yoon; Cho, Seung Joo; Hah, Jung-Mi

    2011-09-01

    PythDock is a heuristic docking program that uses Python programming language with a simple scoring function and a population based search engine. The scoring function considers electrostatic and dispersion/repulsion terms. The search engine utilizes a particle swarm optimization algorithm. A grid potential map is generated using the shape information of a bound ligand within the active site. Therefore, the searching area is more relevant to the ligand binding. To evaluate the docking performance of PythDock, two well-known docking programs (AutoDock and DOCK) were also used with the same data. The accuracy of docked results were measured by the difference of the ligand structure between x-ray structure, and docked pose, i.e., average root mean squared deviation values of the bound ligand were compared for fourteen protein-ligand complexes. Since the number of ligands' rotational flexibility is an important factor affecting the accuracy of a docking, the data set was chosen to have various degrees of flexibility. Although PythDock has a scoring function simpler than those of other programs (AutoDock and DOCK), our results showed that PythDock predicted more accurate poses than both AutoDock4.2 and DOCK6.2. This indicates that PythDock could be a useful tool to study ligand-receptor interactions and could also be beneficial in structure based drug design.

  8. DockingShop: A Tool for Interactive Molecular Docking

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Ting-Cheng; Max, Nelson L.; Ding, Jinhui; Bethel, E. Wes; Crivelli, Silvia N.

    2005-04-24

    Given two independently determined molecular structures, the molecular docking problem predicts the bound association, or best fit between them, while allowing for conformational changes of the individual molecules during construction of a molecular complex. Docking Shop is an integrated environment that permits interactive molecular docking by navigating a ligand or protein to an estimated binding site of a receptor with real-time graphical feedback of scoring factors as visual guides. Our program can be used to create initial configurations for a protein docking prediction process. Its output--the structure of aprotein-ligand or protein-protein complex--may serve as an input for aprotein docking algorithm, or an optimization process. This tool provides molecular graphics interfaces for structure modeling, interactive manipulation, navigation, optimization, and dynamic visualization to aid users steer the prediction process using their biological knowledge.

  9. DockQ: A Quality Measure for Protein-Protein Docking Models.

    Directory of Open Access Journals (Sweden)

    Sankar Basu

    Full Text Available The state-of-the-art to assess the structural quality of docking models is currently based on three related yet independent quality measures: Fnat, LRMS, and iRMS as proposed and standardized by CAPRI. These quality measures quantify different aspects of the quality of a particular docking model and need to be viewed together to reveal the true quality, e.g. a model with relatively poor LRMS (>10Å might still qualify as 'acceptable' with a descent Fnat (>0.50 and iRMS (<3.0Å. This is also the reason why the so called CAPRI criteria for assessing the quality of docking models is defined by applying various ad-hoc cutoffs on these measures to classify a docking model into the four classes: Incorrect, Acceptable, Medium, or High quality. This classification has been useful in CAPRI, but since models are grouped in only four bins it is also rather limiting, making it difficult to rank models, correlate with scoring functions or use it as target function in machine learning algorithms. Here, we present DockQ, a continuous protein-protein docking model quality measure derived by combining Fnat, LRMS, and iRMS to a single score in the range [0, 1] that can be used to assess the quality of protein docking models. By using DockQ on CAPRI models it is possible to almost completely reproduce the original CAPRI classification into Incorrect, Acceptable, Medium and High quality. An average PPV of 94% at 90% Recall demonstrating that there is no need to apply predefined ad-hoc cutoffs to classify docking models. Since DockQ recapitulates the CAPRI classification almost perfectly, it can be viewed as a higher resolution version of the CAPRI classification, making it possible to estimate model quality in a more quantitative way using Z-scores or sum of top ranked models, which has been so valuable for the CASP community. The possibility to directly correlate a quality measure to a scoring function has been crucial for the development of scoring functions for

  10. Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells

    DEFF Research Database (Denmark)

    Blixt, Klas Ola; Lavrova, Olga I; Mazurov, Dmitriy V

    2012-01-01

    antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which...... are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal Gal...

  11. Application of fluorescent monocytes for probing immune complexes on antigen microarrays.

    Directory of Open Access Journals (Sweden)

    Zoltán Szittner

    Full Text Available Microarrayed antigens are used for identifying serum antibodies with given specificities and for generating binding profiles. Antibodies bind to these arrayed antigens forming immune complexes and are conventionally identified by secondary labelled antibodies.In the body immune complexes are identified by bone marrow derived phagocytic cells, such as monocytes. In our work we were looking into the possibility of replacing secondary antibodies with monocytoid cells for the generation of antibody profiles. Using the human monocytoid cell line U937, which expresses cell surface receptors for immune complex components, we show that cell adhesion is completely dependent on the interaction of IgG heavy chains and Fcγ receptors, and this recognition is susceptible to differences between heavy chain structures and their glycosylation. We also report data on a possible application of this system in autoimmune diagnostics.Compared to secondary antibodies, fluorescent monocytesas biosensors are superior in reflecting biological functions of microarray-bound antibodies and represent an easy and robust alternative for profiling interactions between serum proteins and antigens.

  12. New tests to detect antiphospholipid antibodies: antiprothrombin (aPT) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies.

    Science.gov (United States)

    Sciascia, Savino; Khamashta, Munther A; Bertolaccini, Maria Laura

    2014-05-01

    Antiprothrombin antibodies have been proposed as potential new biomarkers for thrombosis and/or pregnancy morbidity in the setting of the antiphospholipid syndrome (APS). Antiprothrombin antibodies are commonly detected by ELISA, using prothrombin coated onto irradiated plates (aPT), or prothrombin in complex with phosphatidylserine (aPS/PT), as antigen. Although these antibodies can co-exist in the same patient, aPT and aPS/PT seem to belong to different populations of autoantibodies. Early research explored the role of antibodies to prothrombin as potential antigenic targets for the lupus anticoagulant (LA). To date their clinical significance is being investigated and their potential role in identifying patients at higher risk of developing thrombotic events or pregnancy morbidity is being probed.

  13. Antibody proteases: induction of catalytic response.

    Science.gov (United States)

    Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D

    2002-10-01

    Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

  14. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    Science.gov (United States)

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

  15. Antibody-radioisotope conjugates for tumor localization and treatment

    International Nuclear Information System (INIS)

    Larson, S.M.; Carrasquillo, J.A.

    1985-01-01

    In principle, anti-tumor antibodies can be used to carry radioactivity to tumors for in-vivo diagnosis and treatment of cancer. First, for diagnostic purposes, an antibody that targets a specific antigen (for example, the p97 antigen of human melanoma tumor), is labeled with a tracer amount of radioactivity. When this antibody-radioisotope conjugate is injected into the blood stream, the antibody carries the radioactivity throughout the body and in time, percolates through all the tissues of the body. Because the tumor has specific antigens to which the antibody can bind, the antibody conjugate progressively accumulates in the tumor. Using conventional nuclear medicine imaging equipment, the body of the patient is scanned for radioactivity content, and a map of the distribution of the radioactivity is displayed on photographic film. The tumor shows up as a dense area of radio-activity. These same antibody-radioisotope conjugates may be used for therapy of tumors, except that in this case large amounts of radioactivity are loaded on the antibody. After localization of the conjugate there is sufficient radiation deposited in the tumor of radiotherapy. The success of this approach in the clinic is determined in large measure by the concentration gradient that can be achieved between tissue antibody conjugate in tumor versus normal tissue

  16. Intramuscular Immunisation with Chlamydial Proteins Induces Chlamydia trachomatis Specific Ocular Antibodies.

    Directory of Open Access Journals (Sweden)

    Alexander Badamchi-Zadeh

    Full Text Available Ocular infection with Chlamydia trachomatis can cause trachoma, which is the leading cause of blindness due to infection worldwide. Despite the large-scale implementation of trachoma control programmes in the majority of countries where trachoma is endemic, there remains a need for a vaccine. Since C. trachomatis infects the conjunctival epithelium and stimulates an immune response in the associated lymphoid tissue, vaccine regimens that enhance local antibody responses could be advantageous. In experimental infections of non-human primates (NHPs, antibody specificity to C. trachomatis antigens was found to change over the course of ocular infection. The appearance of major outer membrane protein (MOMP specific antibodies correlated with a reduction in ocular chlamydial burden, while subsequent generation of antibodies specific for PmpD and Pgp3 correlated with C. trachomatis eradication.We used a range of heterologous prime-boost vaccinations with DNA, Adenovirus, modified vaccinia Ankara (MVA and protein vaccines based on the major outer membrane protein (MOMP as an antigen, and investigated the effect of vaccine route, antigen and regimen on the induction of anti-chlamydial antibodies detectable in the ocular lavage fluid of mice.Three intramuscular vaccinations with recombinant protein adjuvanted with MF59 induced significantly greater levels of anti-MOMP ocular antibodies than the other regimens tested. Intranasal delivery of vaccines induced less IgG antibody in the eye than intramuscular delivery. The inclusion of the antigens PmpD and Pgp3, singly or in combination, induced ocular antigen-specific IgG antibodies, although the anti-PmpD antibody response was consistently lower and attenuated by combination with other antigens.If translatable to NHPs and/or humans, this investigation of the murine C. trachomatis specific ocular antibody response following vaccination provides a potential mouse model for the rapid and high throughput

  17. Uses of monoclonal antibody 8H9

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Nai-Kong V.

    2018-04-10

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  18. Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route.

    Science.gov (United States)

    Carey, John B; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V S; Draper, Simon J; Moore, Anne C

    2014-08-21

    Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP1₄₂, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP1₄₂ also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP1₄₂ using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies.

  19. Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route

    Science.gov (United States)

    Carey, John B.; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V. S.; Draper, Simon J.; Moore, Anne C.

    2014-01-01

    Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies. PMID:25142082

  20. Electron spectroscopic imaging of antigens by reaction with boronated antibodies.

    Science.gov (United States)

    Qualmann, B; Kessels, M M; Klobasa, F; Jungblut, P W; Sierralta, W D

    1996-07-01

    Two small homogeneous markers for electron spectroscopic imaging (ESI) containing eight dodecaborane cages linked to a poly-alpha, epsilon-L-lysine dendrimer were synthesized; one of these was made water soluble by the attachment of a polyether. The markers were coupled to the sulfhydryl group of (monovalent) antibody fragments (Fab') by a homobifunctional cross-linker. While the coupling ratios of the poorly water-soluble compound did not exceed 20%, the polyether-containing variant reacted quantitatively. Its suitability for immunolabelling was tested in a study of the mechanism of the transcellular transport of an administered heterologous protein (bovine serum albumin, BSA) through ileal enterocytes of newborn piglets by endocytotic vesicles in comparison to conventional immunogold reagents. The post-embedding technique was employed. The boronated Fab' gave rise to considerably higher tagging frequencies than seen with immunogold, as could be expected from its form- and size-related physical advantages and the dense packing of BSA in the vesicles. The new probe, carrying the antigen-combining cleft at one end and the boron clusters at the opposite end of the oval-shaped conjugate, add to the potential of ESI-based immunocytochemistry.

  1. Antibody isotype analysis of malaria-nematode co-infection: problems and solutions associated with cross-reactivity

    Directory of Open Access Journals (Sweden)

    Graham Andrea L

    2010-02-01

    Full Text Available Abstract Background Antibody isotype responses can be useful as indicators of immune bias during infection. In studies of parasite co-infection however, interpretation of immune bias is complicated by the occurrence of cross-reactive antibodies. To confidently attribute shifts in immune bias to the presence of a co-infecting parasite, we suggest practical approaches to account for antibody cross-reactivity. The potential for cross-reactive antibodies to influence disease outcome is also discussed. Results Utilising two murine models of malaria-helminth co-infection we analysed antibody responses of mice singly- or co-infected with Plasmodium chabaudi chabaudi and Nippostrongylus brasiliensis or Litomosoides sigmodontis. We observed cross-reactive antibody responses that recognised antigens from both pathogens irrespective of whether crude parasite antigen preparations or purified recombinant proteins were used in ELISA. These responses were not apparent in control mice. The relative strength of cross-reactive versus antigen-specific responses was determined by calculating antibody titre. In addition, we analysed antibody binding to periodate-treated antigens, to distinguish responses targeted to protein versus carbohydrate moieties. Periodate treatment affected both antigen-specific and cross-reactive responses. For example, malaria-induced cross-reactive IgG1 responses were found to target the carbohydrate component of the helminth antigen, as they were not detected following periodate treatment. Interestingly, periodate treatment of recombinant malaria antigen Merozoite Surface Protein-119 (MSP-119 resulted in increased detection of antigen-specific IgG2a responses in malaria-infected mice. This suggests that glycosylation may have been masking protein epitopes and that periodate-treated MSP-119 may more closely reflect the natural non-glycosylated antigen seen during infection. Conclusions In order to utilize antibody isotypes as a measure of

  2. Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.; Suzuki, M.; Adachi, S.

    1983-01-01

    Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

  3. Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system

    International Nuclear Information System (INIS)

    Kuroki, Masahide; Arakawa, Fumiko; Matsunaga, Akira; Okamoto, Naomi; Takakura, Kyoko; Matsuoka, Yuji; Higuchi, Hiroshi.

    1987-01-01

    A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radio-immunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels. (author)

  4. Characterization of fully functional spray-on antibody thin films

    Energy Technology Data Exchange (ETDEWEB)

    Figueroa, Jhon [Department of Chemistry, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620-5250 (United States); Magaña, Sonia; Lim, Daniel V. [Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620-7115 (United States); Schlaf, Rudy, E-mail: schlaf@eng.usf.edu [Department of Electrical Engineering, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620-5101 (United States)

    2014-02-15

    The authors recently demonstrated that fully functional Escherichia coli O157:H7 antibody thin films can be prepared using a simple pneumatic nebulizer on glass surface [1]. This paper focuses on the investigation of the morphology and physical properties of these films with the aim to better understand their performance. A series of E. coli O157:H7 antibody spray-on thin films were investigated by ellipsometry, X-ray photoelectron spectroscopy (XPS), immunoassays, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), fluorescence microscopy, atomic force microscope (AFM) and contact angle analysis. These data were compared to measurements on films prepared with the biotin–avidin covalent bonding scheme. The investigation showed that films created by a 2 min pneumatic spray deposition time can capture antigens similar as the avidin–biotin wet-chemical method. The results also suggests that an influential factor for the comparable capture cell ability between sprayed and covalent films is an increased antibody surface coverage for the sprayed films (non-equilibrium technique), which compensates for the lack of its antibody orientation. There was no significant antibody denaturation detected on any of the sprayed films. Both techniques led to the formation of cluster-aggregates, a factor that seems unavoidable due to the natural tendency of protein to cluster. The avidin–biotin bridge films generally had a higher roughness, which manifested itself in a higher wettability compared to the sprayed films.

  5. Tumor immunolocalization using 124I-iodine-labeled JAA-F11 antibody to Thomsen-Friedenreich alpha-linked antigen

    International Nuclear Information System (INIS)

    Chaturvedi, Richa; Heimburg, Jamie; Yan, Jun; Koury, Stephen; Sajjad, Munawwar; Abdel-Nabi, Hani H.; Rittenhouse-Olson, Kate

    2008-01-01

    Clinical immunolocalization has been attempted by others with an anti-Thomsen-Friedenreich antigen (TF-Ag) mAb that bound both alpha- and beta-linked TF-Ag. In this report, 124 I-labeled mAb JAA-F11 specific for alpha-linked TF-Ag showed higher tumor specificity in in vivo micro-positron emission tomography (micro-PET) of the mouse mammary adenocarcinoma line, 4T1, showing no preferential uptake by the kidney. Labeled product remained localized in the tumor for at least 20 days. Glycan array analysis showed structural specificity of the antibody

  6. Characterization of crystals of an antibody-recognition fragment of the cancer differentiation antigen mesothelin in complex with the therapeutic antibody MORAb-009

    International Nuclear Information System (INIS)

    Ma, Jichun; Tang, Wai Kwan; Esser, Lothar; Pastan, Ira; Xia, Di

    2012-01-01

    The therapeutic antibody MORAb-009 disrupts the interaction of mesothelin and the ovarian cancer antigen CA-125. Crystals have been grown of the Fab fragment derived from MORAb-009 and of its complex with an N-terminal fragment of mesothelin. The mesothelin-specific monoclonal antibody MORAb-009 is capable of blocking the binding of mesothelin to CA-125 and displays promising anticancer potential. It is currently undergoing clinical trials. In order to understand the basis of the interaction between MORAb-009 and mesothelin at atomic resolution, both the Fab fragment of MORAb-009 and the complex between the Fab and an N-terminal fragment of mesothelin (residues 7–64) were crystallized. The crystals of the Fab diffracted X-rays to 1.75 Å resolution and had the symmetry of space group P4 1 2 1 2, with unit-cell parameters a = b = 140.6, c = 282.0 Å. The crystals of the mesothelin–Fab complex diffracted to 2.6 Å resolution and belonged to the hexagonal space group P6 4 , with unit-cell parameters a = b = 146.2, c = 80.9 Å. Structural analyses of these molecules are in progress

  7. DockQ: A Quality Measure for Protein-Protein Docking Models

    Science.gov (United States)

    Basu, Sankar

    2016-01-01

    The state-of-the-art to assess the structural quality of docking models is currently based on three related yet independent quality measures: Fnat, LRMS, and iRMS as proposed and standardized by CAPRI. These quality measures quantify different aspects of the quality of a particular docking model and need to be viewed together to reveal the true quality, e.g. a model with relatively poor LRMS (>10Å) might still qualify as 'acceptable' with a descent Fnat (>0.50) and iRMS (iRMS to a single score in the range [0, 1] that can be used to assess the quality of protein docking models. By using DockQ on CAPRI models it is possible to almost completely reproduce the original CAPRI classification into Incorrect, Acceptable, Medium and High quality. An average PPV of 94% at 90% Recall demonstrating that there is no need to apply predefined ad-hoc cutoffs to classify docking models. Since DockQ recapitulates the CAPRI classification almost perfectly, it can be viewed as a higher resolution version of the CAPRI classification, making it possible to estimate model quality in a more quantitative way using Z-scores or sum of top ranked models, which has been so valuable for the CASP community. The possibility to directly correlate a quality measure to a scoring function has been crucial for the development of scoring functions for protein structure prediction, and DockQ should be useful in a similar development in the protein docking field. DockQ is available at http://github.com/bjornwallner/DockQ/ PMID:27560519

  8. Cross-reactive Legionella antigens and the antibody response during infection

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Shand, G; Pearlman, E

    1991-01-01

    In order to define cross-reactive Legionella antigens suitable for diagnostic purposes, we investigated sonicate antigens from two Legionella species, including two serogroups of L. pneumophila. The antigens were reacted with heterologous and homologous rabbit antisera in Western blot. Sera from ...

  9. TriFabs—Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery

    Directory of Open Access Journals (Sweden)

    Klaus Mayer

    2015-11-01

    Full Text Available TriFabs are IgG-shaped bispecific antibodies (bsAbs composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH fused to CH3 with “knob”-mutations, and the variable region of the light chain (VL fused to CH3 with matching “holes”. The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent “in between” these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic payloads.

  10. Immunity to rhabdoviruses in rainbow trout: the antibody response

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lapatra, S.E.

    1999-01-01

    to their occasional detrimental effect on rainbow trout farming. Research efforts have been focused on understanding the mechanisms involved in protective immunity. Several specific and nonspecific cellular and humoral parameters are believed to be involved, but only the antibody response has been characterised......, have demonstrated that rainbow trout can produce specific and highly functional antibodies that are able to neutralise virus pathogenicity in vitro as well as in vivo. The apparently more restricted antibody response to IHNV and VHSV antigens in fish compared to mammals could possibly be explained...... aspects of antibody response and antibody reactivity with IHNV and VHSV antigens....

  11. Uses of monoclonial antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  12. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

    NARCIS (Netherlands)

    Brakenhoff, R H; Gerretsen, M; Knippels, E M; van Dijk, M.; van Essen, H; Weghuis, D O; Sinke, R J; Snow, G B; van Dongen, G A

    The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody-based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose

  13. TSOL18 Vaccine Antigen of Taenia solium: Development of Monoclonal Antibodies and Field Testing of the Vaccine in Cameroon

    Directory of Open Access Journals (Sweden)

    Assana, E.

    2010-01-01

    Full Text Available Chapter 1 reviews the literature about the immunological aspects of taeniid cestode infections and the existing vaccines against Taenia solium cysticercosis in pigs. One of the most promising vaccines is TSOL18, a protein that has been identified in the oncosphere of Taenia solium and expressed as a recombinant molecule in E. coli. Repeated experimental trials have shown that this vaccine is able to protect up to 100% of the immunised pigs against a challenge infection with T. solium. Antibodies raised by the vaccine are capable of killing the parasite in in vitro cultures and it is believed that antibody and complement mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. The identification of the villages with a high risk of T. solium infection, which could subsequently be used in the vaccine trial, is reported in chapter 2. A survey was conducted in 150 households owning 1756 pigs in the rural areas of Mayo-Danay division in the far north region of Cameroon. A questionnaire survey was carried out to collect information on the pig farming system and to identify potential risk factors for T. solium cysticercosis infection in pigs. Blood samples were collected from 398 pigs with the aim of estimating the sero-prevalence of Taenia solium cysticercosis. The results showed that 90.7% of the pigs were free roaming during the dry season and that 42.7% of households keeping pigs in the rural areas had no latrine facility. Seventy six percent of the interviewed pig owners affirmed that the members of the household used open field defecation. ELISA for antigen and antibody detection showed an apparent prevalence of porcine cysticercosis of 24.6% and 32.2%, respectively. A Bayesian approach using the conditional dependence between the two diagnostic tests indicated that the true sero-prevalence of cysticercosis in Mayo-Danay was 26.6%. Binary logistic regression analysis indicated that the

  14. Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1991-01-01

    textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these

  15. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    Science.gov (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  16. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

  17. Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

    Science.gov (United States)

    Spanier, Justin A; Frederick, Daniel R; Taylor, Justin J; Heffernan, James R; Kotov, Dmitri I; Martinov, Tijana; Osum, Kevin C; Ruggiero, Jenna L; Rust, Blake J; Landry, Samuel J; Jenkins, Marc K; McLachlan, James B; Fife, Brian T

    2016-06-13

    Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes.

  18. SAMPL4 & DOCK3.7: lessons for automated docking procedures

    Science.gov (United States)

    Coleman, Ryan G.; Sterling, Teague; Weiss, Dahlia R.

    2014-03-01

    The SAMPL4 challenges were used to test current automated methods for solvation energy, virtual screening, pose and affinity prediction of the molecular docking pipeline DOCK 3.7. Additionally, first-order models of binding affinity were proposed as milestones for any method predicting binding affinity. Several important discoveries about the molecular docking software were made during the challenge: (1) Solvation energies of ligands were five-fold worse than any other method used in SAMPL4, including methods that were similarly fast, (2) HIV Integrase is a challenging target, but automated docking on the correct allosteric site performed well in terms of virtual screening and pose prediction (compared to other methods) but affinity prediction, as expected, was very poor, (3) Molecular docking grid sizes can be very important, serious errors were discovered with default settings that have been adjusted for all future work. Overall, lessons from SAMPL4 suggest many changes to molecular docking tools, not just DOCK 3.7, that could improve the state of the art. Future difficulties and projects will be discussed.

  19. Application of murine monoclonal antibodies to the serodiagnosis of tuberculosis

    International Nuclear Information System (INIS)

    Ivanyl, J.; Coates, A.R.M.; Krambovitis, E.

    1982-01-01

    The immune response during infectious diseases leads to a rise in antibody titre to the various different antigenic determinants of the causative organism. The response is further complicated by the fact that it is relatively unusual for one individual to respond to all antigenic components of an organism. Demonstration of the specific immune response of an infected host by serological tests is often hampered by the broad cross-reactivity between several bacterial antigens. The authors report on a serodiagnostic application of murine monoclonal antibodies (MAB), specific for a human pathogen, M. tuberculosis by a technique which is applicable in principle to the serodiagnosis of many other infectious diseases. The serum diagnostic test is based on the competitive inhibition by human sera of the binding of 125 I-labelled murine monoclonal antibodies to M. tuberculosis-coated polyvinyl plates. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of antibodies from patients' sera. When compared with healthy controls, increased titres of inhibitory antibodies were found in about 70% of patients with active tuberculosis. The diagnostic value of the individual monoclonal antibodies as well as the benefit from the use of multiple specificity probes has been qualified

  20. A generalized quantitative antibody homeostasis model: maintenance of global antibody equilibrium by effector functions.

    Science.gov (United States)

    Prechl, József

    2017-11-01

    The homeostasis of antibodies can be characterized as a balanced production, target-binding and receptor-mediated elimination regulated by an interaction network, which controls B-cell development and selection. Recently, we proposed a quantitative model to describe how the concentration and affinity of interacting partners generates a network. Here we argue that this physical, quantitative approach can be extended for the interpretation of effector functions of antibodies. We define global antibody equilibrium as the zone of molar equivalence of free antibody, free antigen and immune complex concentrations and of dissociation constant of apparent affinity: [Ab]=[Ag]=[AbAg]= K D . This zone corresponds to the biologically relevant K D range of reversible interactions. We show that thermodynamic and kinetic properties of antibody-antigen interactions correlate with immunological functions. The formation of stable, long-lived immune complexes correspond to a decrease of entropy and is a prerequisite for the generation of higher-order complexes. As the energy of formation of complexes increases, we observe a gradual shift from silent clearance to inflammatory reactions. These rules can also be applied to complement activation-related immune effector processes, linking the physicochemical principles of innate and adaptive humoral responses. Affinity of the receptors mediating effector functions shows a wide range of affinities, allowing the continuous sampling of antibody-bound antigen over the complete range of concentrations. The generation of multivalent, multicomponent complexes triggers effector functions by crosslinking these receptors on effector cells with increasing enzymatic degradation potential. Thus, antibody homeostasis is a thermodynamic system with complex network properties, nested into the host organism by proper immunoregulatory and effector pathways. Maintenance of global antibody equilibrium is achieved by innate qualitative signals modulating a

  1. Composition and method for detecting cancer with technetium labeled antibody fragments

    International Nuclear Information System (INIS)

    Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.

    1984-01-01

    F(ab') 2 or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab') 2 or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab') 2 or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab') 2 or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab') 2 or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased

  2. Antigen-Dissociation from Antibody-Modified Nanotransistor Sensor Arrays as a Direct Biomarker Detection Method in Unprocessed Biosamples.

    Science.gov (United States)

    Krivitsky, Vadim; Zverzhinetsky, Marina; Patolsky, Fernando

    2016-10-12

    The detection of biomolecules is critical for a wide spectrum of applications in life sciences and medical diagnosis. Nonetheless, biosamples are highly complex solutions, which contain an enormous variety of biomolecules, cells, and chemical species. Consequently, the intrinsic chemical complexity of biosamples results in a significant analytical background noise and poses an immense challenge to any analytical measurement, especially when applied without prior efficient separation and purification steps. Here, we demonstrate the application of antigen-dissociation regime, from antibody-modified Si-nanowire sensors, as a simple and effective direct sensing mechanism of biomarkers of interest in complex biosamples, such as serum and untreated blood, which does not require ex situ time-consuming biosample manipulation steps, such as centrifugation, filtering, preconcentration, and desalting, thus overcoming the detrimental Debye screening limitation of nanowire-based biosensors. We found that two key parameters control the capability to perform quantitative biomarkers analysis in biosamples: (i) the affinity strength (k off rate) of the antibody-antigen recognition pair, which dictates the time length of the high-affinity slow dissociation subregime, and (ii) the "flow rate" applied during the solution exchange dissociation step, which controls the time width of the low-affinity fast-dissociation subregime. Undoubtedly, this is the simplest and most convenient approach for the SiNW FET-based detection of antigens in complex untreated biosamples. The lack of ex situ biosample manipulation time-consuming processes enhances the portability of the sensing platform and reduces to minimum the required volume of tested sample, as it allows the direct detection of untreated biosamples (5-10 μL blood or serum), while readily reducing the detection cycle duration to less than 5 min, factors of great importance in near-future point-of-care medical applications. We believe

  3. Microbials for the production of monoclonal antibodies and antibody fragments.

    Science.gov (United States)

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Studies on antigenic cross-reactivity of Trichuris ovis with host mucosal antigens in goat

    Directory of Open Access Journals (Sweden)

    Gautam Patra

    2015-12-01

    Full Text Available Objective: To ascertain whether immunodominant antigens of Trichuris ovis might share and cross react with host molecule. Methods: Two crude protein preparations from anterior and posterior parts of Trichuris ovis were characterized along with host mucosal antigen by double immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting technique. Conventional scanning electron microscopy was performed as per standard procedure. Results: Sharp and distinct bands of three antigens have been found in double immunodiffusion using hyperimmune serum raised in rabbit indicating the presence of specific antibody against each antigen. All three antigens have shown major and minor bands with molecular weight ranging from 15 to 110 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Conclusions: The antigenic cross-reactivity was thought to result from shared antigens. The existence of paracloacal papillae found in the anterior part of the male was not a unique feature for species differentiation.

  5. Non-HLA antibodies post-transplantation: clinical relevance and treatment in solid organ transplantation.

    Science.gov (United States)

    Dragun, Duska; Hegner, Bjorn

    2009-01-01

    Antibodies and B cells are increasingly recognized as major modulators of allograft function and survival. Improved immunohistochemical and serologic diagnostic procedures have been developed to monitor antibody responses against HLA antigens during the last decade. Acute and chronic allograft rejection can occur in HLA-identical sibling transplants implicating the importance of immune response against non-HLA targets. Non-HLA anti-bodies may occur as alloantiboides, yet they seem to be predominantly autoantibodies. Antigenic targets of non-HLA antibodies described thus far include various minor histocompatibility antigens, vascular receptors, adhesion molecules, and intermediate filaments. Non-HLA antibodies may function as complement- and non-complement-fixing antibodies and they may induce a wide variety of allograft injuries, reflecting the complexity of their acute and chronic actions. Refined approaches considering the subtle mechanistic differences in the individual antibody responses directed against non-HLA antigens may help to define patients at particular risk for irreversible acute or chronic allograft injuries and improve over-all outcomes. We attempted to summarize the current state of research, development in diagnostic and therapeutic strategies, and to address some emerging problems in the area of humoral response against non-HLA antigens beyond ABO blood group and MHC class I chain-related gene A and B (MICA and MICB) antigens in solid organ transplantation. Copyright (c) 2009 S. Karger AG, Basel.

  6. Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

    Science.gov (United States)

    Sorice, M; Pittoni, V; Griggi, T; Losardo, A; Leri, O; Magno, M S; Misasi, R; Valesini, G

    2000-01-01

    The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-β2-glycoprotein I (β2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to ‘pure’ phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific ‘pure’ anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein–Barr virus infection. However, anti-β2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins. PMID:10792380

  7. Detection of different Leishmania spp. and Trypanosoma cruzi antibodies in cats from the Yucatan Peninsula (Mexico) using an iron superoxide dismutase excreted as antigen.

    Science.gov (United States)

    Longoni, Silvia S; López-Cespedes, Angeles; Sánchez-Moreno, Manuel; Bolio-Gonzalez, Manuel E; Sauri-Arceo, Carlos H; Rodríguez-Vivas, Roger I; Marín, Clotilde

    2012-09-01

    Although human leishmaniasis has been reported in 20 states in Mexico, no case of leishmaniasis has been reported in cats to date. In the Yucatan Peninsula, it has been found that dogs may act as reservoirs for at least three Leishmania species (Leishmania mexicana, Leishmania braziliensis, and Leishmania panamensis). In this study we identified specific antibodies against these three Leishmania spp. and Trypanosoma cruzi in the sera from 95 cats from two States on the Yucatan Peninsula, namely Quintana Roo and Yucatan, by ELISA and Western blot techniques using whole extract and an iron superoxide dismutase excreted by the parasites as antigens. As well as demonstrating the presence of trypanosomatid antibodies in the feline population on the Yucatan Peninsula, we were also able to confirm the high sensitivity and specificity of the iron superoxide dismutase antigen secreted by them, which may prove to be very useful in epidemiological studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Antigenicity and diagnostic potential of vaccine candidates in human Chagas disease.

    Directory of Open Access Journals (Sweden)

    Shivali Gupta

    Full Text Available Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease.Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd-phase for antibody response to the recombinant antigens (individually or mixed by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175. The IgG antibodies to TcG1, TcG2, and TcG4 (individually and TcG(mix were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%, TcG2- (96%, TcG4- (94.6% and TcG(mix- (98% based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8% in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects.Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.

  9. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  10. The antigen presenting cells instruct plasma cell differentiation.

    Science.gov (United States)

    Xu, Wei; Banchereau, Jacques

    2014-01-06

    The professional antigen presenting cells (APCs), including many subsets of dendritic cells and macrophages, not only mediate prompt but non-specific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells (PCs), which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only "signal 1" (the antigen), but also "signal 2" to directly instruct the differentiation process of PCs in a T-cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching, and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  11. Deteksi Antigen pada Kriptokokosis

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    Robiatul Adawiyah

    2014-12-01

    Full Text Available AbstrakKriptokokosis merupakan infeksi sistemik yang disebabkan Cryptococcus sp. Predileksi jamur tersebut adalah susunan saraf pusat dan selaput otak. Terdapat 5 spesies Cryptococcus sp. yang menyebabkan penyakit pada manusia; yang paling banyak adalah Cr. neoformans dan Cr. gattii. Diagnosis kriptokokosis ditegakkan berdasarkan gejala klinis, pemeriksaan laboratoris serta radiologis. Pemeriksaan laboratoris dilakukan dengan identifikasi morfologi, serologi danPCR. Pemeriksaan secara morfologi dengan tinta India positif  bila jumlah sel jamur 10  sel/ml spesimen. Kultur dilakukan di media sabouraud dextrose agar (SDA dan niger sheed agar (NSA, jamur tumbuh setelah 5-7 hari. Deteksi antigen dan antibodi dilakukan pada cairan tubuh dan tidak membutuhkan waktu lama. Deteksi antibodi Cr.neoformans memiliki kelemahan yaitu tidak menunjukkan hasil positif pada infeksi akut, IgA masih positif setelah 1-2 tahun fase penyembuhan, IgG dapat persisten, pada individu imunokompromis menunjukkan hasil yang sangat kompleks dan dalam menentukan diagnosis sering tidak konsisten. Polisakarida adalah komponen paling berperan dalam virulensi Cr. neoformans. Komponen polisakarida terutama glucuronoxylomannan merupakan petanda penting dalam diagnosis kriptokokosis secara serologis. Deteksi antigen Cr. neoformans memiliki kelebihan yaitu menunjukkan hasil positif pada infeksi akut/kronis, sensitivitas dan spesifisitas tinggi, dapat mendeteksi polisakarida hingga 10 ng/ml sehingga dengan kadarantigen yang minimal tetap dapat mendiagnosis kriptokokosis.Kata kunci: Cr. neoformans, glucuronoxylomannan, antigenAbstractCryptococcosis is systemic infection that caused by Cryptococcus sp. Predilection of this fungi is the central nervous system and brain membrane. There are 5 species of Cryptococcus sp. that cause cryptococcosis in human; but the majority are caused by Cr. neoformans and Cr. gattii. The diagnosis of cryptococcosis is made based on clinical symptoms

  12. Involvement of lymphocyte function-associated antigen-1 (LFA-1) in HIV infection: inhibition by monoclonal antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Mathiesen, Lars Reinhardt

    1991-01-01

    Monoclonal antibodies (MAbs) against the alpha- and beta-chain of lymphocyte-associated antigen-1 (LFA-1) were examined for inhibition of HIV-1 infection in vitro. Infection of the T cell line MT4 and the monocytic cell line U937 by isolates HTLVIIIB and SSI-002, respectively was inhibited...... in a concentration dependent manner by MAb against the beta-chain but not against the alpha-chain. No cross-reactivity was found between MAb against LFA-1 and against the CD4 receptor (MAb Leu3a). MAbs against the beta-chain and the CD4 receptor were found to act synergistically in inhibiting HIV infection...

  13. Use of Recombinant Antigens for the Diagnosis of Invasive Candidiasis

    Directory of Open Access Journals (Sweden)

    Ana Laín

    2008-01-01

    Full Text Available Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.

  14. Immunohistochemical detection of VHS virus in paraffin-embedded specimens of rainbow trout (Oncorhynchus mykiss); The influence of primary antibody, fixative, and antigen unmasking on method sensitivity

    DEFF Research Database (Denmark)

    Evensen, O.; Olesen, Niels Jørgen

    1997-01-01

    performed on parallel specimens, and the virus titer (TCID50/ml) was determined. Purified nucleocapsid protein (N-protein) of the virus was incorporated in an artificial antigen substrate (polymerized bovine serum albumin), fixed as described above, and embedded in paraffin wax. Microwave unmasking...... was performed an formalin-, PLP-, and Bouin's fluid-fixed specimens. The presence of virus peptides in situ or N-protein in the artificial antigen substrates was Visualized using an immunohistochemical method based on alkaline phosphatase or peroxidase and one polyclonal and five monoclonal polypeptide......-specific antibodies. VHS virus was identified in situ in specimens with high virus titers (10(7-8) TCID50/ml) regardless of the fixative and without the need of an unmasking procedure. A pronounced masking effect was observed for the cross-linking formalin and PLP fixatives. Regardless of the primary antibodies used...

  15. Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

    Science.gov (United States)

    Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

    2014-01-25

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Excess antibody immunoassays for rat glandular kallikreins. Measurement of kallikrein from different organs in the presence of cross-reacting antigens

    International Nuclear Information System (INIS)

    Johansen, L.; Oerstavik, T.B.; Holck, M.; Nustad, K.

    1983-01-01

    An immunoradiometric assay has previously been developed for measurement of rat glandular kallikrein. In the present paper, further studies on the specificity and sensitivity of the method are described. Problems of interference of immunologically cross-reacting antigens were overcome by proper preabsorption of the antibody. A method was thus established in which enzymatic activity of the immunoreactive kallikrein could be measured even in the presence of enzymes sharing immunological determinants and substrate specificity with kallikrein. Two variants of the immunoradiometric assay have been evaluated. A simplified version with simultaneous addition of all reagents gave results equal to those obtained in the original assay. A further modification with delayed addition of the solid-phase antibody, gave considerable improvement in assay sensitivity. (Auth.)

  17. Evaluation of monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) for antigen detection of foot-and-mouth disease virus using clinical samples.

    Science.gov (United States)

    Morioka, Kazuki; Fukai, Katsuhiko; Sakamoto, Kenichi; Yoshida, Kazuo; Kanno, Toru

    2014-01-01

    A monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) method was previously developed for foot-and-mouth disease (FMD) viral antigen detection. Here we evaluated the sensitivity and specificity of two FMD viral antigen detection MSD-ELISAs and compared them with conventional indirect sandwich (IS)-ELISA. The MSD-ELISAs were able to detect the antigen in saliva samples of experimentally-infected pigs for a longer term compared to the IS-ELISA. We also used 178 RT-PCR-positive field samples from cattle and pigs affected by the 2010 type-O FMD outbreak in Japan, and we found that the sensitivities of both MSD-ELISAs were about 7 times higher than that of the IS-ELISA against each sample (P<0.01). In terms of the FMD-positive farm detection rate, the sensitivities of the MSD-ELISAs were about 6 times higher than that of the IS-ELISA against each farm (P<0.01). Although it is necessary to conduct further validation study using the other virus strains, MSD-ELISAs could be appropriate as a method to replace IS-ELISA for FMD antigen detection.

  18. Immunotherapy with GD2 specific monoclonal antibodies

    International Nuclear Information System (INIS)

    Cheung, N.K.V.; Medof, E.M.; Munn, D.

    1988-01-01

    Targeted immunotherapy focuses anti-tumor activity of antibodies and effector cells, which are actively developed by the host or adoptively transferred, onto tumor cells and into tumor sites. Such tumor selective therapy can be more specific and efficient. The value of such an approach is evident in the classical interaction of antibodies. This paper reports that the ganglioside G D2 is an ideal antigen for specific tumor targeting because of its relative lack of heterogeneity among human neuroblastoma, its high density on tumor cells, its lack of antigen modulation upon binding to antibody, and its restricted distribution in normal tissues

  19. Baseline Antibody Titre against Salmonella enterica in Healthy Population of Mumbai, Maharashtra, India.

    Science.gov (United States)

    Patki, Rucha; Lilani, Sunil; Lanjewar, Dhaneshwar

    2017-01-01

    The aim of this study was to establish a baseline titre for the population of Mumbai, Maharashtra, India. Four hundred healthy blood donors, attending blood donation camps, were screened using a survey questionnaire. Widal tube agglutination test was performed on the diluted sera (with 0.9% normal saline) of blood donors, with final dilution ranging from 1 : 40 to 1 : 320. Out of 400 individuals providing samples, 78 (19.5%) individuals showed antibody titres ≥ 1 : 40 for at least one antigen and 322 (80.5%) showed no agglutination. The baseline antibody titres against O antigen and H antigen of Salmonella enterica serotype Typhi were found to be 1 : 40 and 1 : 80, respectively. Similarly, the baseline antibody titres for the H antigen of Salmonella enterica serotypes Paratyphi A and Paratyphi B were found to be 1 : 40 and 1 : 80, respectively. Thus, it was noted that the diagnostically significant cutoff of antibody titre from acute phase sample was ≥ 1 : 80 for S. Typhi O antigen and titre of ≥ 1 : 160 for both S. Typhi H antigen and S. Paratyphi BH antigen. Antibody titre of ≥ 1 : 80 can be considered significant for S. Paratyphi AH antigen.

  20. Baseline Antibody Titre against Salmonella enterica in Healthy Population of Mumbai, Maharashtra, India

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    Rucha Patki

    2017-01-01

    Full Text Available Objective. The aim of this study was to establish a baseline titre for the population of Mumbai, Maharashtra, India. Method. Four hundred healthy blood donors, attending blood donation camps, were screened using a survey questionnaire. Widal tube agglutination test was performed on the diluted sera (with 0.9% normal saline of blood donors, with final dilution ranging from 1 : 40 to 1 : 320. Results. Out of 400 individuals providing samples, 78 (19.5% individuals showed antibody titres ≥ 1 : 40 for at least one antigen and 322 (80.5% showed no agglutination. The baseline antibody titres against O antigen and H antigen of Salmonella enterica serotype Typhi were found to be 1 : 40 and 1 : 80, respectively. Similarly, the baseline antibody titres for the H antigen of Salmonella enterica serotypes Paratyphi A and Paratyphi B were found to be 1 : 40 and 1 : 80, respectively. Conclusion. Thus, it was noted that the diagnostically significant cutoff of antibody titre from acute phase sample was ≥ 1 : 80 for S. Typhi O antigen and titre of ≥ 1 : 160 for both S. Typhi H antigen and S. Paratyphi BH antigen. Antibody titre of ≥ 1 : 80 can be considered significant for S. Paratyphi AH antigen.