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Sample records for shapes specific antibody

  1. In-depth analysis of subclass-specific conformational preferences of IgG antibodies

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    Xinsheng Tian

    2015-01-01

    Full Text Available IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering.

  2. Staphylococcal enterotoxin-specific IgE antibodies in atopic dermatitis.

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    Ide, Fumihito; Matsubara, Tomoyo; Kaneko, Miho; Ichiyama, Takashi; Mukouyama, Tokuko; Furukawa, Susumu

    2004-06-01

    The authors clarified the clinical significance of the measurement of serum concentrations of specific IgE antibodies to staphylococcal enterotoxin (SE) A- and SEB in atopic dermatitis (AD). The serum concentrations of SEA- and SEB-specific IgE antibodies in 140 pediatric patients with AD were measured with an immuno CAP -radioallergosorbent test system (RAST). To check the cross-reaction of specific IgE antibodies to SEA/SEB and other allergens, the CAP RAST fluorescent enzyme immunoassay inhibition test was performed. Forty-seven patients (33.6%) tested positive for either SEA- or SEB-specific IgE antibodies. School children showed higher positive rates of SEA/SEB-specific IgE antibodies than infants or young children. The patients with severe AD and those with exacerbation of symptoms in summer, had higher positive rates of SEA/SEB-specific IgE antibodies than patients with mild AD or those with exacerbation in winter. In addition, the positive rates of specific IgE antibodies to both dog-dander and cat-dander were higher in patients with positive SEA/SEB-specific IgE antibodies than in patients with negative ones. No cross-reactions occurred among specific IgE antibodies to SEA/SEB and dog/cat dander with one patient's serum, which had positive IgE-specific antibodies against cat/dog dander and SEA/SEB. The positive rate of SEA/SEB-specific IgE antibodies in the patients with dogs and/or cats as pets was 48.4%, which was higher than in those with no pets. Atopic dermatitis patients who exhibit high positive rates of SEA/SEB-specific IgE antibodies were found to be school children, severe cases, cases with high serum concentrations of total IgE, cases with exacerbation in summer, and cases with dogs and/or cats as pets. The measurement of serum concentrations of specific IgE antibodies to SEA and SEB, thus has some value for evaluating AD patients.

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

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    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.

  4. Radioimmunoassay of class-specific antibodies (RIACA): chicken antibodies to DNP

    International Nuclear Information System (INIS)

    Viljanen, M.K.; Granfors, K.; Toivanen, P.

    1977-01-01

    A radioimmunological method for the quantitation of class-specific antibodies has been developed. The method allows the quantitation of nanogram per ml concentrations of IgG and IgM-anti-DNP antibodies without any physical or chemical pretreatment of the sample. DNP was coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies were allowed to react with the coupled DNP and then quantitated by their capacity to bind 125 I-labelled anti-chicken-μ or anti-chicken-γ. The inter-assay variation coefficients ranged from 8.1 to 14.7% and the mean standard deviations of duplicate determinations were about 11%. The combination of this method with the exact immunoradiometric quantitation of the total serum IgM and IgG, and with an immunoabsorption technique, makes it possible to quantitate class-specific antibodies on weight units

  5. Production of Monoclonal Antibodies specific for Progesterone

    OpenAIRE

    YÜCEL, Fatıma

    2014-01-01

    Progesterone levels in milk and serum are indicators of pregnancy in cattle. The progesterone level reaches a peak on the 21 st and 22 nd days of pregnancy. Monoclonal antibodies specific to progesterone could be used for the immunodetection of milk and serum progesterone levels. We report here the development of hybrid cells prdoducing monoclonal antibodies specific for progesterone using hybridoma technology. Hybridoma cells secreting monoclonal antibodies against progesterone (MAM 2H1...

  6. 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

  7. Dissection of antibody specificities induced by yellow fever vaccination.

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    Oksana Vratskikh

    Full Text Available The live attenuated yellow fever (YF vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific

  8. Development of an EGFRvIII specific recombinant antibody

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    Li Gordon

    2010-10-01

    Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

  9. Timothy-specific IgG antibody levels vary with the pollen seasons.

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    Nordvall, S L; Larsson, P H; Johansson, S G

    1986-11-01

    Serum samples were collected from eight grass pollen hypersensitive children during a 4-year period. The sera were assayed for contents of timothy-specific IgE antibodies by RAST. Timothy-specific IgG and IgA antibodies were quantified by a refined ELISA in which covalent binding of the antigen to the polystyrene solid phase had been performed. IgG antibodies were also assayed by a Sepharose-protein-A technique with radiolabelled timothy allergens as the antigen. It was possible to register clearcut seasonal variations with postseasonally boosted antibody levels not only of timothy-specific IgE but also of IgG antibody. Both IgG1 and IgG4 antibodies specific for timothy showed seasonal variations of a similar degree. It was not possible to register seasonal variations of the same magnitude of timothy-specific IgA antibodies.

  10. Characterization of a monoclonal antibody with specificity for holo-transcobalamin

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    Fedosov Sergey N

    2006-01-01

    Full Text Available Abstract Background Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12, which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin. Methods The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. Results An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA. Conclusion The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.

  11. Antibody specific epitope prediction-emergence of a new paradigm.

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    Sela-Culang, Inbal; Ofran, Yanay; Peters, Bjoern

    2015-04-01

    The development of accurate tools for predicting B-cell epitopes is important but difficult. Traditional methods have examined which regions in an antigen are likely binding sites of an antibody. However, it is becoming increasingly clear that most antigen surface residues will be able to bind one or more of the myriad of possible antibodies. In recent years, new approaches have emerged for predicting an epitope for a specific antibody, utilizing information encoded in antibody sequence or structure. Applying such antibody-specific predictions to groups of antibodies in combination with easily obtainable experimental data improves the performance of epitope predictions. We expect that further advances of such tools will be possible with the integration of immunoglobulin repertoire sequencing data. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. 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)

  13. Monoclonal antibody PAL-E specific for endothelium

    NARCIS (Netherlands)

    Schlingemann, R. O.; Dingjan, G. M.; Emeis, J. J.; Blok, J.; Warnaar, S. O.; Ruiter, D. J.

    1985-01-01

    A monoclonal antibody, PAL-E, is described that is specific for endothelial cells. The monoclonal antibody, an IgG2a, markedly stains endothelium of capillaries, medium-sized and small veins, and venules in frozen sections of human and some animal tissues tested. It reacts not at all or only weakly

  14. Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

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    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

  15. The future of antibody therapeutics: ADCs bi-specifics and RIT

    International Nuclear Information System (INIS)

    Reichert, J.

    2015-01-01

    Full text of publication follows. Antibodies are widely accepted as remarkably versatile therapeutic agents. As evidence of this, the ∼ 30 antibody products marketed worldwide had total global sales of more than 50 billion dollars in 2012, and the commercial clinical pipeline currently comprises over 350 antibody-based product candidates. In a testament to scientific ingenuity, the investigational molecules (clinical and preclinical) are notably diverse in their composition of matter and include antibodies conjugated to a variety of agents (drugs, radioisotopes), bi-specific antibodies, and fragments or domains of antibodies. The concepts that form the basis of these agents were established decades ago, but advances in technology are now allowing new opportunities for their development. In this presentation, future directions in antibody therapeutics development will be discussed, with a focus on antibody-drug conjugates, bi-specific antibodies and radioimmunotherapy. (author)

  16. A Recombinant Antibody with the Antigen-Specific, Major Histocompatibility Complex-Restricted Specificity of T Cells

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    Andersen, Peter S.; Stryhn, Anette; Hansen, Bjarke E.; Fugger, Lars; Engberg, Jan; Buus, Soren

    1996-03-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

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

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    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.

  18. Anti-citrullinated heat shock protein 90 antibodies identified in bronchoalveolar lavage fluid are a marker of lung-specific immune responses.

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    Harlow, Lisa; Gochuico, Bernadette R; Rosas, Ivan O; Doyle, Tracy J; Osorio, Juan C; Travers, Timothy S; Camacho, Carlos C; Oddis, Chester V; Ascherman, Dana P

    2014-11-01

    Previous work has demonstrated a correlation between serum anti-citrullinated HSP90 antibodies and rheumatoid arthritis-associated interstitial lung disease (RA-ILD). To further investigate this potential pathogenic relationship, we used ELISA-based techniques to assess anti-citrullinated HSP90 antibody profiles in bronchoalveolar lavage fluid (BALF) of patients with different stages of RA-ILD. 9/21 RA-derived BALF specimens demonstrated IgG and/or IgA antibodies targeting citrullinated HSP90 proteins/peptides, highlighting disease specific responses (with a predilection for RA-ILD) that did not occur in IPF patients (0/5) or healthy control subjects (0/5). Comparison of antibody profiles between BALF and matching serum specimens revealed various recognition patterns favoring predominant production of anti-citrullinated HSP90 antibodies within the lung microenvironment-further supporting the connection between this antibody specificity and parenchymal lung disease. Equally important, qualitative as well as quantitative differences in anti-citrullinated HSP90 profiles between BALF and serum indicate that the lung plays a direct role in shaping the immune repertoire of RA/RA-ILD. Published by Elsevier Inc.

  19. Micro solid-phase radioimmunoassay for detection of herpesvirus type-specific antibody: specificity and sensitivity

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    Adler-Storthz, K.; Matson, D.O.; Adam, E.; Dreesman, G.R. (Baylor Univ., Houston, TX (USA). Coll. of Medicine)

    1983-02-01

    The specificity and sensitivity of a micro solid-phase radioimmunoassay (micro-SPRIA) that detects type-specific IgG antibody to herpes simplex virus types 1 and 2 (HSV1 and HSV2) were evaluated. Glycoproteins VP123 (molecular weight, 123,000) of HSV1 and VP119 (molecular weight, 119,000) of HSV2 were found to display the greatest degree of antigenic type-specificity of several HSV antigens tested with the micro-SPRIA technique. When testing a group of sera, negative for anti-HSV antibodies by microneutralization, in the micro-SPRIA, a range of negative reactivities was noted, suggesting that cut-points should be determined for each antigen preparation. The micro-SPRIA detected appropriate antibody activity in patients with recurrent infection and a marked agreement was noted in comparison to detection of anti-HSV antibodies measured with the microneutralization test. The type-specificity of the micro-SPRIA was substantiated by the independence of test results using VP119 and VP123 antigens for a random group of positive sera. The assay is rapid, specific, and sensitive and allows the testing of multiple serum samples with a standardized set of reagents.

  20. 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...

  1. Immunoradiometric assay for cytomegalovirus-specific IgG antibodies

    International Nuclear Information System (INIS)

    Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J.

    1990-01-01

    An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab

  2. C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

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    Fuss, Alexander; Hope, Christopher M; Deayton, Susan; Bennett, Greg Donald; Holdsworth, Rhonda; Carroll, Robert P; Coates, P Toby H

    2015-07-01

    Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers. © 2015 Asian Pacific Society of Nephrology.

  3. Intramuscular Immunisation with Chlamydial Proteins Induces Chlamydia trachomatis Specific Ocular Antibodies.

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    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

  4. Characterization of antibodies specific for UV-damaged DNA by ELISA

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    Eggset, G; Volden, G; Krokan, H

    1987-04-01

    The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was inhibited. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO/sub 4/. Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin.

  5. Characterization of antibodies specific for UV-damaged DNA by ELISA

    International Nuclear Information System (INIS)

    Eggset, G.; Volden, G.; Krokan, H.; Norsk Hydro Research Centre, Porsgrunn

    1987-01-01

    The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was inhibited. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO 4 . Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin. (author)

  6. Critical contribution of aromatic rings to specific recognition of polyether rings. The case of ciguatoxin CTX3C-ABC and its specific antibody 1C49.

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    Tsumoto, Kouhei; Yokota, Akiko; Tanaka, Yoshikazu; Ui, Mihoko; Tsumuraya, Takeshi; Fujii, Ikuo; Kumagai, Izumi; Nagumo, Yoko; Oguri, Hiroki; Inoue, Masayuki; Hirama, Masahiro

    2008-05-02

    To address how proteins recognize polyether toxin compounds, we focused on the interaction between the ABC ring compound of ciguatoxin 3C and its specific antibody, 1C49. Surface plasmon resonance analyses indicated that Escherichia coli-expressed variable domain fragments (Fv) of 1C49 had the high affinity constants and slow dissociation constants typical of antigen-antibody interactions. Linear van't Hoff analyses suggested that the interaction is enthalpy-driven. We resolved the crystal structure of 1C49 Fv bound to ABC ring compound of ciguatoxin 3C at a resolution of 1.7A. The binding pocket of the antibody had many aromatic rings and bound the antigen by shape complementarity typical of hapten-antibody interactions. Three hydrogen bonds and many van der Waals interactions were present. We mutated several residues of the antibody to Ala, and we used surface plasmon resonance to analyze the interactions between the mutated antibodies and the antigen. This analysis identified Tyr-91 and Trp-96 in the light chain as hot spots for the interaction, and other residues made incremental contributions by conferring enthalpic advantages and reducing the dissociation rate constant. Systematic mutation of Tyr-91 indicated that CH-pi and pi-pi interactions between the aromatic ring at this site and the antigen made substantial contributions to the association, and van der Waals interactions inhibited dissociation, suggesting that aromaticity and bulkiness are critical for the specific recognition of polyether compounds by proteins.

  7. Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control.

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    Margaret E Ackerman

    2016-01-01

    Full Text Available Elite controllers (ECs represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

  8. Application of Food-specific IgG Antibody Detection in Allergy Dermatosis

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    Yine Hu

    2015-01-01

    Full Text Available The application of food-specific IgG antibody detection in allergy dermatoses was explored. 181 patients with allergy dermatoses were diagnosed from January to September 2014 and 20 healthy subjects were selected. Fourteen kinds of food-specific IgG antibodies were detected by ELISA method among all the subjects. The positive rates of IgG antibody of the patient group and the healthy group were respectively 65.2% and 5.0%. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults (p0.05. Allergy dermatoses are closely related to food-specific IgG antibodies, and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in the serum of patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  9. Prion-Specific Antibodies Produced in Wild-Type Mice

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Bergström, Ann-Louise; Andersen, Heidi Gertz

    2015-01-01

    Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward...... method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well......-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely...

  10. Data on atherosclerosis specific antibody conjugation to nanoemulsions

    Directory of Open Access Journals (Sweden)

    Geoffrey Prévot

    2017-12-01

    Full Text Available This article present data related to the publication entitled “Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging” (Prévot et al., 2017 [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs using a heterobifunctional linker (DSPE-PEG-maleimide. Atheroma labelling was assayed by immunochemistry on arterial sections from rabbits.

  11. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    International Nuclear Information System (INIS)

    Quinn, T.P.

    2003-01-01

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with 99m Tc and 188 Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic nuclear

  12. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  13. Detection and specifity of class specific antibodies to whole bacteria cells using a solid phase radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Czerkinsky, C.; Rees, A.S.; Bergimeier, L.A.; Challacombe, S.J. (Guy' s Hospital Medical and Dental Schools, London (UK))

    1983-07-01

    A solid phase radioimmunoassay has been developed which can be used for the detection of isotype specific antibodies to whole bacteria and other particulate antigens, and is applicable to a variety of species. Bacteria are bound to the solid phase by the use either of antibodies, or of methyl glyoxal. Both methods result in a sensitive and reproducible assay, and bacteria do not appear to desorb from the solid phase. The specificity of antibodies to whole bacteria was examined by absorption of antisera with various species of bacteria and retesting, or by determining the binding of antisera to various bacteria bound to the solid phase. Both methods revealed specificity for the bacteria examined. Inhibition studies showed that antibodies to Streptococcus mutans whole cells could be inhibited by purified cell surface antigens glucosyltransferase and antigen I/II, but only minimally by lipoteichoic acid, c polysaccharide or dextran. In murine antisera antibodies of the IgG, IgM, and IgA classes could be detected at amounts of less than 1 ng/ml.

  14. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

    Science.gov (United States)

    Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

    Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

  15. 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.

  16. A double antibody radioimmunoassay specific for placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Dass, S.; Bagshawe, K.D.

    1984-01-01

    Placental alkaline phosphatase (PLAP) is normally found in enzymically measurable amounts in second and third trimester pregnancy serum. Its occurrence in sera and tumours from patients with malignant disease has led to the development of methods to specifically identify and quantitate the enzyme. Recently immunological techniques have been used, employing antibodies raised to purified PLAP; these include solid phase radioimmunoassays and enzyme-immunoassay. The development of a sensitive, specific, automated double-antibody radioimmunoassay for the measurement of PLAP in serum is reported. (Auth.)

  17. High-affinity uranyl-specific antibodies suitable for cellular imaging

    International Nuclear Information System (INIS)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C.

    2008-01-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO 2 2+ -DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO 2 2+ -DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K D = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO 2 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Ca 2+ ) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO 2 2+ equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  18. Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats

    NARCIS (Netherlands)

    Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

    2006-01-01

    The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and

  19. Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function.

    Science.gov (United States)

    Minkoff, Marjorie Sue

    A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the

  20. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

    DEFF Research Database (Denmark)

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders

    2014-01-01

    of more sensitive methods including real-time PCR (RT-PCR). Immunohistochemistry with mutation-specific antibodies might be a promising detection method. We evaluated 210 samples with NSCLC from an unselected Caucasian population. Extracted DNA was analyzed for EGFR mutations by RT-PCR (Therascreen EGFR......, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94...... was demonstrated. However, sensitivity was low, especially for exon19 deletions, and thus these antibodies cannot yet be used as screening method for EGFR mutations in NSCLC. Refinement of sensitivity for the mutation-specific antibodies is warranted to improve molecular diagnosis using EGFR immunohistochemistry....

  1. 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

  2. High-affinity uranyl-specific antibodies suitable for cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C. [CEA Valrho, DSV, IBEB, Serv Biochim et Toxicol Nucl, F-30207 Bagnols Sur Ceze (France)

    2008-07-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO{sub 2}{sup 2+}-DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO{sub 2}{sup 2+}-DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K{sub D} = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO{sub 2}{sup 2+}, Fe{sup 3+}, Zn{sup 2+}, Cu{sup 2+}, and Ca{sup 2+}) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO{sub 2}{sup 2+} equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  3. Mouse-specific antibody responses to a monoclonal antibody during repeated immunoscintigraphy investigations: Comparison of antibody titres and imaging studies in a rat model

    International Nuclear Information System (INIS)

    Pimm, M.V.; Gribben, S.J.; Markham, A.J.; Perkins, A.C.

    1990-01-01

    As a model for human mouse-specific antibody responses in patients undergoing immunoscintigraphy, we have investigated in rats the production of mouse-specific antibodies (MA) to the mouse monoclonal antibody 791T/36. At intervals of between 5 and 16 weeks the rats were given repeated cycles of intravenous (IV) injections of antibody with or without a simultaneous intradermal (ID) injection. The IV dose was 60 μg/kg, a dose similar to that used in many clinical immunoscintigraphy studies. The ID injection was 2 μg, which mimicks the skin test dose often given in clinical imaging protocols. The study was carried out with both 131 I-labelled antibody and with antibody labelled with 111 In by DTPA chelation. MA was measured with a passive haemagglutination assay using sheep red blood cells coated with the monoclonal antibody. Of rats given ID injections of unlabelled antibody at the same time as the IV imaging doses, 9/20 produced MA during 4 cycles of injections. In contrast, only 2/16 rats given only the IV dose produced MA. Both 131 I- and 111 In-labelled antibody appeared equally immunogenic with 5/18 and 6/18 overall responders, respectively. The production of MA was associated with a significant perturbation in the biodistribution of the IV dose of labelled antibody as seen by gamma-camera imaging of the rats given 111 In-labelled antibody. There was clearance of immune complexes to the liver, this organ accumulating up to 90% of the whole body count rate of radiolabel. MA titres of between 1/100 and 1/78000 caused equal perturbation of biodistribution, although below 1/100 the effect was more variable. (orig.)

  4. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  5. Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

    International Nuclear Information System (INIS)

    X. Li; A.M. Kriegel; T.C. Bishop; R.C. Blake; E. Figueiredo; H. Yu; D.A. Blake

    2005-01-01

    The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO 2 2+ was used as a source of RNA for the development of a recombinant (Fab) 2 fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire κ light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab) 2 protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab) 2 fragments that bound to the UO 2 2+ -DCP complex with an affinity indistinguishable from that of a (Fab) 2 fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the canonical structures method detailed by Morea

  6. The detection and specifity of class specific antibodies to whole bacteria cells using a solid phase radioimmunoassay

    International Nuclear Information System (INIS)

    Czerkinsky, C.; Rees, A.S.; Bergimeier, L.A.; Challacombe, S.J.

    1983-01-01

    A solid phase radioimmunoassay has been developed which can be used for the detection of isotype specific antibodies to whole bacteria and other particulate antigens, and is applicable to a variety of species. Bacteria are bound to the solid phase by the use either of antibodies, or of methyl glyoxal. Both methods result in a sensitive and reproducible assay, and bacteria do not appear to desorb from the solid phase. The specificity of antibodies to whole bacteria was examined by absorption of antisera with various species of bacteria and retesting, or by determining the binding of antisera to various bacteria bound to the solid phase. Both methods revealed specificity for the bacteria examined. Inhibition studies showed that antibodies to Streptococcus mutans whole cells could be inhibited by purified cell surface antigens glucosyltransferase and antigen I/II, but only minimally by lipoteichoic acid, c polysaccharide or dextran. In murine antisera antibodies of the IgG, IgM, and IgA classes could be detected at amounts of less than 1 ng/ml. (author)

  7. [Immunochemical Detection of Azospirilla in Soil with Genus-Specific Antibodies].

    Science.gov (United States)

    Shirokov, A A; Krasov, A I; Selivanov, N Yu; Burygin, G L; Shchegolev, S Yu; Matora, L Yu

    2015-01-01

    Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

  8. General shape and hapten-induced conformational changes of pig anti-dinitrophenyl antibody. A small-angle scattering study

    International Nuclear Information System (INIS)

    Cher, L.; Gladkikh, I.A.; Franek, F.; Kunchenko, A.B.; Ostanevich, Yu.M.

    1980-01-01

    Pig anti-dinitrophenyl antibodies were studied by neutron and X-ray small-angle scattering. Observed scattering curves show that the shapes of two antibody types, precipitating and non-precipitating, are similar, however the latter being more compact. A hapten binding induced conformational change of antibody is observed. This conformational change might be described as a contraction of the whole molecule via similarity transformation. The spatial models of pig antibody molecule based on the existing experimental data were designed. Most probable models have a cavity in the Fsub(c) part and the Fsub(ab) parts are either fully extended or slightly bent down to the Fsub(c) part

  9. A monoclonal antibody that specifically recognizes m6A nucleoside

    OpenAIRE

    Espuny, Ruth; Castro, Ana; Codony, Carles; Eritja Casadellà, Ramón; Bach-Elias, Montse

    1998-01-01

    A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.

  10. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    2008-08-01

    Full Text Available Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  11. Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

    Science.gov (United States)

    Smith, Kenneth; Muther, Jennifer J; Duke, Angie L; McKee, Emily; Zheng, Nai-Ying; Wilson, Patrick C; James, Judith A

    2013-05-01

    B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23. Copyright © 2012 Elsevier GmbH. All rights reserved.

  12. Potential for novel MUC1 glycopeptide-specific antibody in passive cancer immunotherapy

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Wandall, Hans H; Pedersen, Anders Elm

    2013-01-01

    MUC1 is an important target for antibodies in passive cancer immunotherapy. Antibodies against mucin glycans or mucin peptide backbone alone may give rise to cross reactivity with normal tissues. Therefore, attempts to identify antibodies against cancer-specific MUC1 glycopeptide epitopes havebeen...

  13. 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.

  14. Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

    Science.gov (United States)

    Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

    2006-05-01

    The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

  15. Titres of Specific Antibodies against Toxoplasma gondii in Goats and their Kids

    Directory of Open Access Journals (Sweden)

    Ľubica Mišurová

    2009-01-01

    Full Text Available The aim of our study was to perform repeated determination of specific antibody levels in mothers and their kids in order to assess indirectly the possibility of vertical transmission of toxoplasmosis in goats. Twenty-eight goats with their kids were included in the study. The following variables were assessed: number of born kids in relation to antibody titres of goats; levels of specific antibodies in the blood of goats and kids; and concentrations of immunoglobulins (Ig, total protein (TP and total globulins (G in order to define the end of colostral immunity and the start of active production of antibodies in kids under 69 days of age. Specific antibodies against Toxoplasma gondii in goats were detected by IFAT in titres ranging from 0 to 1 280. Out of a total of 28 animals, 5 goats were negative (17.9% and 23 goats were seropositive (82.1%. The goats delivered 42 kids. A total ratio of number of kids to number of mothers was 1.5. Partial evaluation of results in goats without positive titre against T. gondii before parturition and goats with positive titre showed that negative goats tended to have more kids (p p < 0.01 of monitored non-specific immunity indicators. During this period, we observed increased titres of specific antibodies against toxoplasmosis in 20 kids (5 kids 41 days old, 5 kids 55 days old, and 10 kids 69 days old and thus we could assume the possibility of vertical transmission of toxoplasmosis.

  16. Muscle-Specific Tyrosine Kinase and Myasthenia Gravis Owing to Other Antibodies.

    Science.gov (United States)

    Rivner, Michael H; Pasnoor, Mamatha; Dimachkie, Mazen M; Barohn, Richard J; Mei, Lin

    2018-05-01

    Around 20% of patients with myasthenia gravis are acetylcholine receptor antibody negative; muscle-specific tyrosine kinase antibodies (MuSK) were identified as the cause of myasthenia gravis in 30% to 40% of these cases. Anti MuSK myasthenia gravis is associated with specific clinical phenotypes. One is a bulbar form with fewer ocular symptoms. Others show an isolated head drop or symptoms indistinguishable from acetylcholine receptor-positive myasthenia gravis. These patients usually respond well to immunosuppressive therapy, but not as well to cholinesterase inhibitors. Other antibodies associated with myasthenia gravis, including low-density lipoprotein receptor-related protein 4, are discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. 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

  18. 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.

  19. Specific antibodies to detect Tamarillo leaf malformation virus (TALMV) in Tamarillo

    International Nuclear Information System (INIS)

    Gallo Garcia, Yuliana; Marin Montoya, Mauricio; Gutierrez, Pablo Andres

    2011-01-01

    In Colombia, yields of Tamarillo are seriously affected by a complex viral disease known as virosis. This pathology was first reported in 1991 in the north of Antioquia and currently affects all Tamarillo growing regions in the country. Recent works have demonstrated the association of two potyviruses (potyviridae) with this disease: potato virus y (PVY) and Tamarillo leaf malformation virus (TALMV, proposed species). Specific diagnostic tools are required for early asymptomatic detection of these viruses and Tamarillo certification programs. In this study, we report the obtention of TALMV specific antibodies using a 15 residues peptide mimicking the n-terminal coat protein. Specificity and sensitivity of the anti-TALMV antibodies was determined by Elisa and dot-blot using recombinant protein and synthetic peptides as controls. The usefulness of these antibodies was validated from a preliminary trial of TALMV detection in plant samples obtained from Tamarillo crops in eastern Antioquia and results were compared with a TALMV specific coat RT-PCR detection protocol.

  20. Specificity and polyreactivity of the antibody response during natural HIV-1 infection

    OpenAIRE

    Wang, Xin

    2006-01-01

    The specificity and polyreactivity of the antibody response in natural HIV-1 infection were studied. First, to investigate the overall antibody response, overlapping linear peptides were used to screen sera taken from HIV-1-infected individuals. The polyclonal antibody response was relatively stable during long-term infection, compared with acute infection, and mostly directed against immunodominant regions. Low level, transient antibody responses were detected against membrane proximal exter...

  1. Antibodies to dopamine: radioimmunological study of specificity in relation to immunocytochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Geffard, M.; Kah, O.; Onteniente, B.; Seguela, P.; Le Moal, M.; Delaage, M.

    1984-06-01

    Two classes of anti-3,4- dihydroxyphenylethylamine (dopamine) antibodies were raised in rabbits using dopamine conjugated to albumin either via formaldehyde or via glutaraldehyde. Each was usable for immunohistochemical detection of dopamine neurons provided that the tissue was fixed by the homologous cross-linking agent. However, anti-dopamine-glutaraldehyde antibodies turned out to be of more general use because of the better fixative properties of glutaraldehyde which fixed dopamine in rat and in teleost, whereas formaldehyde only worked in lower vertebrates (such as goldfish) and not in rat brain. The specificity of anti-dopamine-glutaraldehyde antibodies was firmly established by competition experiments in equilibrium dialysis, using an immunoreactive tritiated derivative synthesized by coupling dopamine to N-alpha-acetyl-L-lysine N-methylamide via glutaraldehyde. Specificity studies in vitro and immunohistological results demonstrating the specific staining of dopaminergic neurons were found to correlate well.

  2. Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

    Science.gov (United States)

    Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2015-05-27

    Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen

  3. Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

    Science.gov (United States)

    Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

    2013-04-01

    Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by

  4. A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.

    Science.gov (United States)

    Min, Junseon; Song, Eun Kyung; Kim, Hansol; Kim, Kyoung Taek; Park, Tae Joo; Kang, Sebyung

    2016-04-11

    We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.

  5. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  6. Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

    Science.gov (United States)

    Ranade, V V

    1989-10-01

    Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera

  7. Activity, specificity, and titer of naturally occurring canine anti-DEA 7 antibodies.

    Science.gov (United States)

    Spada, Eva; Proverbio, Daniela; Baggiani, Luciana; Canzi, Ilaria; Perego, Roberta

    2016-11-01

    The reported prevalence of naturally occurring anti-dog erythrocyte antigen (DEA) 7 antibodies in DEA 7-negative dogs is as high as 50%. Characterization of these antibodies may better define their importance in canine transfusion medicine. We determined in vitro activity, specificity, and titer of anti-DEA 7 antibodies in DEA 7-negative dogs. Plasma samples from 317 DEA 7-negative dogs were cross-matched with DEA 7-positive red blood cells (RBCs) using gel column technology. Agglutination occurred with DEA 7-positive RBCs but not with DEA 7-negative RBCs in 73 samples (23%), which were hence classified as containing anti-DEA 7 antibodies. These samples were evaluated for hemolytic and agglutinating activity, strength of agglutination, and antibody specificity and titers. All samples showed agglutination but none showed hemolysis. Gel agglutination was graded as 1+ for 20 samples (27%), 2+ for 49 samples (67%), 3+ for 4 samples (6%); no samples were graded 4+. The agglutination titer was DEA 7 antibodies were found in 23% of DEA 7-negative dogs. The presence of naturally occurring anti-DEA 7 antibodies suggests that cross-matching of canine blood recipients is advisable, even at first transfusion, to minimize delayed transfusion reactions. © 2016 The Author(s).

  8. Isolation of monoclonal antibodies with predetermined conformational epitope specificity.

    Directory of Open Access Journals (Sweden)

    Anton M Sholukh

    Full Text Available Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs. To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

  9. A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells

    DEFF Research Database (Denmark)

    Andersen, P S; Stryhn, A; Hansen, B E

    1996-01-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might ...

  10. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna

    2012-01-01

    -specific monoclonal antibodies (mAbs). METHODS: B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated......ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...

  11. 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....

  12. Estimation of antibodies specific for dextran

    International Nuclear Information System (INIS)

    Matsuuchi, L.; Morrison, S.L.

    1978-01-01

    Methods are described for the isolation and characterization of picogram quantities of anti-dextran antibodies. 14 C-dextrans produced by using the dextransucrases of Leuconostoc mesenteroides strains B1355 and B512 were used in a radioimmunoassay. The specificity of this assay was verified by using cell cytoplasmic lysates from mouse plasmacytomas, J558 (anti-α 1 → 3 dextran) and W3129 (anti-α 1 → 6 dextran). Dextran produced by strain B1355 and insolubilized with epichlorohydrin was used as an immunoabsorbent

  13. Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions.

    Directory of Open Access Journals (Sweden)

    Franck R Petry

    Full Text Available Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3, type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5, and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46. For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409 while others did not (pS199, pT205, pS396, pS404, pS422, A0024. With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i using secondary antibodies designed to bind only non-denatured Igs, ii preparation of a heat-stable fraction, iii clearing Igs from the homogenates, and iv using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes

  14. Incomplete separation of radioiodinated thyroid hormones in serum using specific antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Perrild, H; Skovsted, L; Korsgaard Christensen, L [Department of Internal Medicine and Endocrinology, Herlev Hospital, DK-2730 Herlev, Denmark

    1980-01-01

    Alkaline Sephadex G-25 columns were used to separate labelled 3,5,3',5'-thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine from the serum binding proteins followed by a quantitative elution of each hormone by coupling to its respective antibody. It is shown that although these antibodies (diluted 1:1500-1:100 000) in our radioimmunoassays are highly specific they show a high degree of non-specific binding when they are used in the concentrations necessary to get a maximal recovery of the hormones in column separating experiments.

  15. Monoclonal antibodies against pregnancy-specific β1-glycoprotein (SP1) in immunohistochemistry and radioimmunoassay

    International Nuclear Information System (INIS)

    Wahlstroem, T.; Heikinheimo, M.

    1983-01-01

    Monoclonal mouse antibodies against pregnancy-specific beta-1-glycoprotein (SP 1 ) have been studied for their suitability in immunoperoxidase staining and radioimmunoassay methodologies. These antibodies were useful in staining normal placentas, hydatidiform moles, invasive moles and choriocarcinomas. They showed good specificity, with minimal background staining, and will thus be superior to conventional polyclonal antisera in immunohistochemistry. However, the presently tested monoclonal anti-SP 1 antibodies were found not to be suitable for radioimmunoassay. (Auth.)

  16. Effects of long-term low dose radiation. Epstein-Barr virus-specific antibodies in radiological technologists

    Energy Technology Data Exchange (ETDEWEB)

    Kumagai, Etsuko; Higashida, Yoshiharu; Onomichi, Mitsukazu; Nakamura, Ikuo; Tanoue, Shozo; Tanaka, Ryuji; Kumagai, Takashi; Katsuki, Takato; Sawada, Shozo.

    1988-09-01

    To clarify the long-term effects of occupational exposure to low doses of radiation, Epstein-Barr virus (EBV)-specific antibody titers in sera from 104 radiological technologists (R.T.) and 118 controls in Kumamoto prefecture were measured by the immunofluorescence method. Antibody titers to viral capsid antigen (VCA)-IgG increased with the years of experience as R.T., and the prevalence of abnormal antibody titers to both VCA-IgG and early antigen (EA)-IgG were significantly higher in R.T. with over 15 years of experience or 30 rads of cumulative radiation dose than in the controls. However, there was no correlation between exposure and the frequency of abnormal EBV-associated nuclear antigen (EBNA) antibody titers. The EBV-specific antibody titers of 24 Hiroshima atomic-bomb survivors were also measured. They were similar to those of the R.T. with over 30 years of experience. The EBV-specific antibody titers of R.T. suggest that there may be an impairment of immunologic competence after continuous long-term exposure to low doses of radiation. Also, the correlation of EBV-specific antibody titers and frequency of cells with chromosome aberrations in 53 R.T. was studied. Some correlations were found between the antibody titers to both of the VCA-IgG and EBNA and the frequency of cells with chromosome aberrations.

  17. Agonistic effects of a monoclonal antibody specific for the interleukin-2 receptor

    International Nuclear Information System (INIS)

    Eardley, D.D.; Makrides, V.

    1986-01-01

    Interleukin-2 (IL-2) mediated immune responses can be blocked by monoclonal antibodies to the IL-2 receptor. The monoclonal antibody, M720, is defined as specific for the IL-2 receptor because it blocks 35 S-IL-2 binding to Con A blasts, reacts with lymphoblasts but not resting splenocytes, and inhibits IL-2 induced proliferation to mitogen, antigen, or allogeneic stimuli. Under appropriate culture conditions, the IL-2 receptor-specific antibody can act like IL-2 in that it will induce proliferation in T cells in the absence of additional antigen or mitogen. This agonistic effect is dependent on time, dose of antibody, and requires fetal calf serum (FCS) in the media. Because the FCS is not mitogenic by itself, the authors propose that the FCS components act as incomplete mitogen to induce appearance of IL-2 receptors but lack a factor which would push the majority of the cells into the S phase of the cell cycle. This factor is usually IL-2, but in the authors experiments, the IL-2 receptor-specific antibody can provide the same stimulus. These data indicate that factors like FCS can induce IL-2 receptors, but without additional IL-2 or receptor triggering, the cells will not proceed through the synthetic and proliferative phases of cell growth

  18. Potential Role of Specific Antibodies as Important Vaccine Induced Protective Mechanism against Aeromonas salmonicida in Rainbow Trout

    DEFF Research Database (Denmark)

    Rømer Villumsen, Kasper; Dalsgaard, Inger; Holten-Andersen, Lars

    2012-01-01

    of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred...

  19. Radioimmunoassay of serum antibodies with B-streptococcus specificity in pregnant women and infants

    International Nuclear Information System (INIS)

    Frey, C.W.

    1980-01-01

    In a specific competitive radioimmunoassay of purified rabbit antibodies, labeled with iodine 125 against group- and type-antigens of streptococcus agalactiae (streptococci type B), we investigated the amount of serum anti-bodies providing specificity of streptococci type B in not preselected pregnant women, newborn and babies with colonies of streptococci type B or with diseases due to streptococci type B and in some of their mothers. These antibodies could be detected in 26 of 45 pregnant women and in 3 of 7 children with colonies of streptococci type B. 5 of 18 newborn with the ''early-onset'' type of infection and 6 of 7 of their mothers provided antibodies with specificity of streptococci type B as did one of two newborn with the ''late onset'' type of infection. Contrary to the supposition of Baker and Kasper and in accordance with the findings of Wilkinson, the ''risk group'' cannot be determined only by detecting the antibodies against streptococci type B. The risk group comprises those persons in whom the colonisation of streptococci agalactiae leads to the frequently life-threatening infecton of neonatals with streptococci type B. (orig.) [de

  20. Radioimmunological demonstration of DNA specific antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Falck, P [Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung

    1976-01-01

    Using /sup 125/I chemically labelled denatured (d) and native (n) DNA, specifically binding antibodies were demonstrated in the sera of Lupus erythemathodes patients by means of the Farr technique. (NH/sub 4/)/sub 2/SO/sub 4/ was used to separate the immunologically bound /sup 125/I-d-DNA. For /sup 125/I-n-DNA the use of a secondary antiserum for the precipitation of the primary immune complex is advantageous. The influence of antigen concentration upon the binding rate was studied. Titre determinations can be made with the proposed method.

  1. Red blood cell antibodies in pregnancy and their clinical consequences: synergistic effects of multiple specificities.

    Science.gov (United States)

    Nordvall, Maria; Dziegiel, Morten; Hegaard, Hanne Kristine; Bidstrup, Mogens; Jonsbo, Finn; Christensen, Birgit; Hedegaard, Morten

    2009-10-01

    The objective was to determine clinical consequences of various specificities for the infant/fetus. The population was patients referred between 1998 and 2005 to the tertiary center because of detected red blood cell (RBC) alloimmunization. Altogether 455 infants were delivered by 390 alloimmunized women. This was a retrospective cohort study. Data were obtained from the blood bank register and the obstetric and neonatal database. As indicators of hemolytic activity of the antibodies, the frequency of the therapeutic interventions intrauterine transfusion, exchange transfusion, and simple transfusion was used. Anti-D was the most common antibody (46.6%), followed by anti-K (15.4%). A combination of antibodies was detected in 27%. All three types of therapeutic intervention were significantly more frequent in women with anti-D plus an additional antibody than in women with anti-D as the sole antibody. The anti-D titer closely paralleled the clinical importance of the antibody. One case of anti-s with a titer of 512 required all three types of transfusion. Anti-D was the single most frequent and harmful specificity closely followed by anti-K. Combinations of antibody specificities were more harmful than single specificities, and a potentially synergistic effect should be considered.

  2. General approach to standardization of the solid-phase radioimmunoassay for quantitation of class-specific antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Zollinger, W D; Boslego, J W [Walter Reed Army Inst. of Research, Washington, DC (USA)

    1981-10-30

    The feasibility of using an anti-human immunoglobulin/human immunoglobulin/(/sup 125/I)anti-human immunoglobulin 'sandwich' in a solid-phase radioimmunoassay to produce a standard curve which could be used to quantitate antigen-specific antibody of a particular immunoglobulin class was investigated. The amount of secondary antibody (SAb) bound was determined as a function of whether the primary antibody (PAb) was bound to its specific solid-phase antigen or by a solid-phase anti-human immunoglobulin. No significant difference between the two values was observed. Quantitation of anti-tetanus toxoid antibody by this method was in a good agreement with quantitative precipitin tests. Comparison of SAb binding as a function of the way the PAb is bound was extended to class-specific PAb by use of murine monoclonal antibodies to meningococcal antigens. In most cases somewhat greater binding of SAb occurred when PAb was bound to antigen, but in several cases where low avidity antibody and/or poor quality antigens were used, greater SAb binding occurred when PAb was bound by anti-mouse immunoglobulin. The results indicate that this approach may be useful as a general method for standardizing the SPRIA and other solid-phase immunoassays such as the ELISA to measure class-specific antibody.

  3. Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Miao Wang

    2017-05-01

    Full Text Available Dengue virus (DENV co-circulates as four serotypes (DENV1-4. Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS. Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR, a process known as antibody dependent enhancement (ADE. Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2 and DENV-2 prM monoclonal antibody (prM mAb could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6 were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10 and alaninea minotransferase (ALT in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to

  4. Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection.

    Science.gov (United States)

    Wang, Miao; Yang, Fan; Huang, Dana; Huang, Yalan; Zhang, Xiaomin; Wang, Chao; Zhang, Shaohua; Zhang, Renli

    2017-01-01

    Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo , interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo , suggested that anti-idiotypic antibodies might be a new choice to be considered to treat

  5. The impact of pretransplant donor-specific antibodies on graft outcome in renal transplantation: a six-year follow-up study

    Directory of Open Access Journals (Sweden)

    Elias David-Neto

    2012-01-01

    Full Text Available OBJECTIVE: The significance of pretransplant, donor-specific antibodies on long-term patient outcomes is a subject of debate. This study evaluated the impact and the presence or absence of donor-specific antibodies after kidney transplantation on short- and long-term graft outcomes. METHODS: We analyzed the frequency and dynamics of pretransplant donor-specific antibodies following renal transplantation from a randomized trial that was conducted from 2002 to 2004 and correlated these findings with patient outcomes through 2009. Transplants were performed against a complement-dependent T- and B-negative crossmatch. Pre- and posttransplant sera were available from 94 of the 118 patients (80%. Antibodies were detected using a solid-phase (LuminexH, single-bead assay, and all tests were performed simultaneously. RESULTS: Sixteen patients exhibited pretransplant donor-specific antibodies, but only 3 of these patients (19% developed antibody-mediated rejection and 2 of them experienced early graft losses. Excluding these 2 losses, 6 of 14 patients exhibited donor-specific antibodies at the final follow-up exam, whereas 8 of these patients (57% exhibited complete clearance of the donor-specific antibodies. Five other patients developed ''de novo'' posttransplant donor-specific antibodies. Death-censored graft survival was similar in patients with pretransplant donor-specific and non-donor-specific antibodies after a mean follow-up period of 70 months. CONCLUSION: Pretransplant donor-specific antibodies with a negative complement-dependent cytotoxicity crossmatch are associated with a risk for the development of antibody-mediated rejection, although survival rates are similar when patients transpose the first months after receiving the graft. Our data also suggest that early posttransplant donor-specific antibody monitoring should increase knowledge of antibody dynamics and their impact on long-term graft outcome.

  6. Determination of specific IgG antibody by crossed radioimmunoelectrophoresis

    International Nuclear Information System (INIS)

    Nordvall, S.L.; Uhlin, T.; Einarsson, R.

    1983-01-01

    A crossed radioimmunoelectrophoretic method was developed for detection of honey bee venom specific IgG antibodies in patient sera. At the serum concentration 1/200 the contrast between specific binding and backgroud was the most favourable. The detection limit was fairly low, approximately 30 kU/l(IgG RAST units). A reference system based on the reference kits in Phadebas IgG-RAST was elaborated. (author)

  7. Determination of specific IgG antibody by crossed radioimmunoelectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Nordvall, S.L. (Dept. of Paediatrics, University Hospital, Uppsala, Sweden); Uhlin, T.; Einarsson, R. (Allergy Research, Pharmacia Diagnostics AB, Uppsala, Sweden)

    1983-01-01

    A crossed radioimmunoelectrophoretic method was developed for detection of honey bee venom specific IgG antibodies in patient sera. At the serum concentration 1/200 the contrast between specific binding and backgroud was the most favourable. The detection limit was fairly low, approximately 30 kU/l(IgG RAST units). A reference system based on the reference kits in Phadebas IgG-RAST was elaborated.

  8. Monoclonal antibodies specific to heat-treated porcine blood.

    Science.gov (United States)

    Raja Nhari, Raja Mohd Hafidz; Hamid, Muhajir; Rasli, Nurmunirah Mohamad; Omar, Abdul Rahman; El Sheikha, Aly Farag; Mustafa, Shuhaimi

    2016-05-01

    Porcine blood is potentially being utilized in food as a binder, gelling agent, emulsifier or colorant. However, for certain communities, the usage of animal blood in food is strictly prohibited owing to religious concerns and health reasons. This study reports the development of monoclonal antibodies (MAbs) against heat-treated soluble proteins (HSPs) of autoclaved porcine blood; characterization of MAbs against blood, non-blood and plasma from different animal species using qualitative indirect non-competitive enzyme-linked immunosorbent assay (ELISA); and immunoblotting of antigenic components in HSPs of porcine blood. Fifteen MAbs are specific to heat-treated and raw porcine blood and not cross-reacted with other animal blood and non-blood proteins (meat and non-meat). Twelve MAbs are specific to porcine plasma, while three MAbs specific to porcine plasma are cross-reacted with chicken plasma. Immunoblotting revealed antigenic protein bands (∼60, ∼85-100 and ∼250 kDa) in porcine blood and plasma recognized by the MAbs. Selection of MAbs that recognized 60 kDa HSPs of porcine blood and plasma as novel monoclonal antibodies would be useful for detection of porcine plasma in processed food using the immunoassay method. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  9. Dengue serotype cross-reactive, anti-E protein antibodies confound specific immune memory for one year after infection

    Directory of Open Access Journals (Sweden)

    Ying Xiu eToh

    2014-08-01

    Full Text Available Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of one year. We found that serotype cross-reactive antibodies peaked three weeks after infection and subsided within one year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

  10. Epitope and functional specificity of monoclonal antibodies to mouse gamma interferon: the synthetic peptide approach

    International Nuclear Information System (INIS)

    Russell, J.K.; Hayes, M.P.; Carter, J.M.; Torres, B.A.; Dunn, B.M.; Johnson, H.M.

    1986-01-01

    Four anti-recombinant mouse gamma interferon (α-IFNγ) monoclonal antibodies were generated using hamster spleen cells. Binding of 125 I-IFNγ by these protein A-bound antibodies was specifically blocked by cold IFNγ. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFNγ, while a corresponding C-terminal (95-133) peptide had no effect on binding. One of the N-terminal specific monoclonal antibodies inhibited both the antiviral and macrophage priming (for tumor cell killing) activities of IFNγ, while the other two had no effect on either biological function. Blocking experiments with cold IFNγ and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFNγ. Polyclonal antibodies to either the N-terminal or C-terminal peptides also inhibited both the antiviral and macrophage priming activities of IFNγ. All of the antibodies that inhibited IFNγ function also blocked binding of IFNγ to membrane receptor on cells, while antibodies that did not inhibit function also did not block binding. The data suggest that both the N-terminal and C-terminal domains of IFNγ play an important role in its antiviral and macrophage priming functions, possibly in a cooperative manner

  11. Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

    Science.gov (United States)

    D’Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie; Erasmus, M. Frank; Hraber, Peter; Bradbury, Andrew R. M.

    2018-01-01

    Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with the same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding. PMID:29568296

  12. 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.

  13. Rituximab selectively suppresses specific islet antibodies.

    Science.gov (United States)

    Yu, Liping; Herold, Kevan; Krause-Steinrauf, Heidi; McGee, Paula L; Bundy, Brian; Pugliese, Alberto; Krischer, Jeff; Eisenbarth, George S

    2011-10-01

    The TrialNet Study Group evaluated rituximab, a B-cell-depleting monoclonal antibody, for its effect in new-onset patients with type 1A diabetes. Rituximab decreased the loss of C-peptide over the first year of follow-up and markedly depleted B lymphocytes for 6 months after administration. This article analyzes the specific effect of rituximab on multiple islet autoantibodies. A total of 87 patients between the ages of 8 and 40 years received either rituximab or a placebo infusion weekly for four doses close to the onset of diabetes. Autoantibodies to insulin (IAAs), GAD65 (GADAs), insulinoma-associated protein 2 (IA2As), and ZnT8 (ZnT8As) were measured with radioimmunoassays. The primary outcome for this autoantibody analysis was the mean level of autoantibodies during follow-up. Rituximab markedly suppressed IAAs compared with the placebo injection but had a much smaller effect on GADAs, IA2As, and ZnT8As. A total of 40% (19 of 48) of rituximab-treated patients who were IAA positive became IAA negative versus 0 of 29 placebo-treated patients (P 1 year in insulin-treated patients. For the patients receiving insulin for >2 weeks prior to rituximab administration, we cannot assess whether rituximab not only blocks the acquisition of insulin antibodies induced by insulin administration and/or also suppresses preformed insulin autoantibodies. Studies in prediabetic non-insulin-treated patients will likely be needed to evaluate the specific effects of rituximab on levels of IAAs.

  14. Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner

    2016-01-01

    BACKGROUND/AIM: Primary tumors display a great level of intra-tumor heterogeneity in breast cancer. The current lack of prognostic and predictive biomarkers limits accurate stratification and the ability to predict response to therapy. The aim of the present study was to select recombinant antibody...... fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against...

  15. Chemokine Receptor-Specific Antibodies in Cancer Immunotherapy: Achievements and Challenges

    Science.gov (United States)

    Vela, Maria; Aris, Mariana; Llorente, Mercedes; Garcia-Sanz, Jose A.; Kremer, Leonor

    2015-01-01

    The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications. PMID:25688243

  16. Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein

    Directory of Open Access Journals (Sweden)

    Denicar Lina Nascimento Fabris Maeda

    2017-09-01

    Full Text Available The heat-labile toxins (LT produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV envelope glycoprotein domain III (EDIII, which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

  17. 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.

  18. Rise and Fall of an Anti-MUC1 Specific Antibody

    Science.gov (United States)

    Li, Jiandong; von Wasielewski, Reinhard; Bastert, Gunther; Schirrmann, Thomas; Esteves, Isabel Tourais; Behrens, Christian K.; Fournes, Bénédict; Fournier, Nathalie; de Romeuf, Christophe; Hust, Michael; Dübel, Stefan

    2011-01-01

    Background So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate. Results A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10−10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells. Conclusions Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these “best in class” binding parameters, the therapeutic success of this antibody was prevented by the target biology. PMID:21264246

  19. Rise and fall of an anti-MUC1 specific antibody.

    Directory of Open Access Journals (Sweden)

    Holger Thie

    2011-01-01

    Full Text Available So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology.

  20. Presence of specific IgG antibody to grain dust does not go with respiratory symptoms.

    Science.gov (United States)

    Park, H S; Suh, C H; Nahm, D H; Kim, H Y

    1999-02-01

    A high prevalence of work-related symptoms in relation to grain dust exposure has been reported in grain dust workers, but the role of the specific IgG antibody is unknown. To study the possible role of specific IgG (sIgG) and specific IgG4 (sIgG4) in the development of work-related symptoms, sIgG and sIgG4 subclass antibodies against grain dust antigens were determined by ELISA in sera from 43 workers and 27 non-exposed controls. They were compared with results of specific IgE antibodies, exposure intensity and the presence of respiratory symptoms. SIgG and sIgG4 antibodies were detectable in almost all sera of exposed workers, and the prevalence were significantly higher than those of controls (pgrain dust exposure and may unlikely play a role in the etiology of respiratory symptoms.

  1. PrP(Sc-specific antibodies with the ability to immunodetect prion oligomers.

    Directory of Open Access Journals (Sweden)

    Mourad Tayebi

    Full Text Available The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0 cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids.

  2. Simultaneous measurements of auto-immune and infectious disease specific antibodies using a high throughput multiplexing tool.

    Directory of Open Access Journals (Sweden)

    Atul Asati

    Full Text Available Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders.

  3. Evaluation of cysticercus-specific IgG (total and subclasses and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

    Full Text Available In the present study, an enzyme-linked immunosorbent assay (ELISA standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses and IgE antibodies in cerebrospinal fluid (CSF samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA=1.17 and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49 and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46 and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12 and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85 and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60 and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.

  4. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies.

    Science.gov (United States)

    Botkjaer, Kenneth A; Fogh, Sarah; Bekes, Erin C; Chen, Zhuo; Blouse, Grant E; Jensen, Janni M; Mortensen, Kim K; Huang, Mingdong; Deryugina, Elena; Quigley, James P; Declerck, Paul J; Andreasen, Peter A

    2011-08-15

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation. © The Authors Journal compilation © 2011 Biochemical Society

  5. HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

    Science.gov (United States)

    Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

    2018-04-01

    While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

  6. Radioimmunoassay for detection of VP1 specific neutralizing antibodies of foot and mouse disease virus

    International Nuclear Information System (INIS)

    Patzer, E.J.; Jackson, M.L.; Moore, D.M.

    1985-01-01

    A solid-phase radioimmunoassay was developed for the detection of antibodies against a specific region of the VP1 protein of the A24 and O1 serotypes of foot and mouth disease virus. The antibody titers from the radioimmunoassay showed a positive correlation with neutralizing antibody titers determined by a mouse protection assay. The specificity of the assay resides in the peptide used as antigen. The assay is rapid, reproducible and does not require the use of whole virions. (orig.)

  7. Epidemiology of myasthenia gravis with anti-muscle specific kinase antibodies in the Netherlands

    NARCIS (Netherlands)

    Niks, Erik H.; Kuks, Jan B. M.; Verschuuren, Jan J. G. M.

    The epidemiology of myasthenia gravis subtypes and the frequency of antibodies to muscle-specific kinase (MuSK) was studied in patients with generalised myasthenia gravis without anti-acetylcholine receptor antibodies who had an onset of symptoms between 1990 and 2004 in a well-defined region in the

  8. 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....

  9. Inflammation-Specific T1 Imaging Using Anti-Intercellular Adhesion Molecule 1 Antibody-Conjugated Gadolinium Diethylenetriaminepentaacetic Acid

    Directory of Open Access Journals (Sweden)

    Kyu-Sil Choi

    2007-03-01

    Full Text Available To examine inflammatory tissue, an initial and common symptom of various types of pathogenesis, we designed inflammation-targeted T1 contrast agents prepared by bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA with anti-intercellular adhesion molecule 1 (ICAM-1 antibody. The anti-ICAM-1 antibody was coupled with DTPA and was then conjugated with Gd. The specific binding of the Gd-DTPA-anti-ICAM-1 antibody complex to the ICAM-1-expressing cells was examined in the cultured endothelial cells where ICAM-1 expression was stimulated. Inflammation-specific T1 imaging was then assessed using a mouse abscess model with the 1.5-Tesla module. The Gd-DTPA-anti-ICAM-1 antibody displayed increased r1, which was two times higher than that of Gd-DTPA and showed predominant binding to cultured endothelial cells, which expressed a high level of ICAM-1. Moreover, the inflammation-specific T1 enhancement was imaged with the Gd-DTPA-anti-ICAM-1 antibody in the mouse acute inflammation model. The Gd-DTPA-anti-ICAM-1 antibody showed significantly increased vascular circulation time, which thereby offered a greater chance for its binding to the target cells. The Gd-DTPA-anti-ICAM-1 antibody displays a potential targeted T1 contrast agent specific to the inflammatory tissue that expresses ICAM-1.

  10. Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

    National Research Council Canada - National Science Library

    Johnson, Erik A; Daugherty, Kelly S

    2006-01-01

    ... behavioral and protein changes due to the absence of guinea pig-specific antibodies. We have developed a procedure to determine the specificity of commercially available, non-guinea pig-specific antibodies in guinea pig lysates...

  11. Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.

    Science.gov (United States)

    Agarwal, Paresh; Bertozzi, Carolyn R

    2015-02-18

    Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecules to create targeted drugs. Despite the simplicity of this concept, generation of clinically successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixtures that cannot be further purified. With advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering.

  12. 125I-Clq-binding and specific antibodies as indicators of pulmonary disease activity in cystic fibrosis

    International Nuclear Information System (INIS)

    Moss, R.B.; Hsu, Y.P.; Lewiston, N.J.

    1981-01-01

    We studied the incidence and levels of circulating immune complexes by the 125 I-Clq-binding assay in patients with cystic fibrosis in relation to clinical respiratory status and specific IgG and IgE antibodies to Pseudomonas aeruginosa. Staphylococcus aureus, Aspergillus fumigatus, and Candida albicans. Overall prevalence of CIC was 43%, but 86% of serially studied patients had evidence of CIC at some time. Patients with acute respiratory exacerbations and deteriorating pulmonary function had a higher incidence of CIC (76%) as compared to stable patients (36%, P less than 0.01), as well as significantly higher levels of CIC. Acute exacerbations were also associated with significant increases in IgG antibody to Pseudomonas (P less than 0.005) but not in other antibodies. CIC did not correlate with Pseudomonas-specific IgG nor with any other specific antibody studied. A variety of age-related differences in specific antibody levels were seen. The episodic appearance of CIC is common in CF and is usually associated with exacerbation of lung disease

  13. 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

  14. Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Juan Pablo Jaworski

    Full Text Available Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig purified from previously infected animals (SHIVIG or control Ig prior to intra-rectal challenge with SHIVSF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased de novo Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2-3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host's ability to eliminate HIV.

  15. Tau passive immunotherapy in mutant P301L mice: antibody affinity versus specificity.

    Directory of Open Access Journals (Sweden)

    Cristina d'Abramo

    Full Text Available The use of antibodies to treat neurodegenerative diseases has undergone rapid development in the past decade. To date, immunotherapeutic approaches to Alzheimer's disease have mostly targeted amyloid beta as it is a secreted protein that can be found in plasma and CSF and is consequently accessible to circulating antibodies. Few recent publications have suggested the utility of treatment of tau pathology with monoclonal antibodies to tau. Our laboratory has begun a systematic study of different classes of tau monoclonal antibodies using mutant P301L mice. Three or seven months old mutant tau mice were inoculated weekly with tau monoclonal antibodies at a dose of 10 mg/Kg, until seven or ten months of age were reached respectively. Our data strongly support the notion that in P301L animals treated with MC1, a conformational monoclonal antibody specific for PHF-tau, the rate of development of tau pathology is effectively reduced, while injecting DA31, a high affinity tau sequence antibody, does not exert such benefit. MC1 appears superior to DA31 in overall effects, suggesting that specificity is more important than affinity in therapeutic applications. Unfortunately the survival rate of the P301L treated mice was not improved when immunizing either with MC1 or PHF1, a high affinity phospho-tau antibody previously reported to be efficacious in reducing pathological tau. These data demonstrate that passive immunotherapy in mutant tau models may be efficacious in reducing the development of tau pathology, but a great deal of work remains to be done to carefully select the tau epitopes to target.

  16. HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Simone I Richardson

    2018-04-01

    Full Text Available While the induction of broadly neutralizing antibodies (bNAbs is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP, complement deposition (ADCD, cellular cytotoxicity (ADCC and cellular trogocytosis (ADCT were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

  17. Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

    Directory of Open Access Journals (Sweden)

    Heger Christopher D

    2010-12-01

    Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

  18. Tus-Ter-lock immuno-PCR assays for the sensitive detection of tropomyosin-specific IgE antibodies.

    Science.gov (United States)

    Johnston, Elecia B; Kamath, Sandip D; Lopata, Andreas L; Schaeffer, Patrick M

    2014-02-01

    The increasing prevalence of food allergies requires development of specific and sensitive tests capable of identifying the allergen responsible for the disease. The development of serologic tests that can detect specific IgE antibodies to allergenic proteins would, therefore, be highly received. Here we present two new quantitative immuno-PCR assays for the sensitive detection of antibodies specific to the shrimp allergen tropomyosin. Both assays are based on the self-assembling Tus-Ter-lock protein-DNA conjugation system. Significantly elevated levels of tropomyosin-specific IgE were detected in sera from patients allergic to shrimp. This is the first time an allergenic protein has been fused with Tus to enable specific IgE antibody detection in human sera by quantitative immuno-PCR.

  19. Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

    NARCIS (Netherlands)

    West, Christopher M.; Van Der Wel, Hanke; Chinoy, Zoiesha; Boons, Geert Jan; Gauthier, Ted J.; Taylor, Carol M.; Xu, Yuechi

    2015-01-01

    Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1,

  20. A Potent Virus-Specific Antibody-Secreting Cell Response to Acute Enterovirus 71 Infection in Children.

    Science.gov (United States)

    Huang, Kuan-Ying Arthur; Lin, Jainn-Jim; Chiu, Cheng-Hsun; Yang, Shuan; Tsao, Kuo-Chien; Huang, Yhu-Chering; Lin, Tzou-Yien

    2015-09-01

    Enterovirus 71 (EV71) remains a leading pathogen for acute infectious diseases in children, especially in Asia. The cellular basis for establishing a virus-specific antibody response to acute EV71 infections is unclear in children. We studied the magnitude of virus-specific antibody-secreting B cells (ASCs) and its relationship with serological response, clinical parameters, and virological parameters among children with laboratory-confirmed EV71 infection. A potent EV71 genogroup B- and virus-specific ASC response was detected in the first week of illness among genotype B5 EV71-infected children. The cross-reactive EV71-specific ASC response to genogroup C viral antigens composed about 10% of the response. The EV71-specific ASC response in children aged ≥3 years produced immunoglobulin G predominantly, but immunoglobulin M was predominant in younger children. Proliferation marker was expressed by the majority of circulating ASCs in the acute phase of EV71 infection. Virus-specific ASC responses significantly correlated with throat viral load, fever duration, and serological genogroup-specific neutralization titer. The presence of a virus-specific ASC response serves an early cellular marker of an EV71-specific antibody response. Further detailed study of EV71-specific ASCs at the monoclonal level is crucial to delineate the specificity and function of antibody immunity in children. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Wildtype p53-specific Antibody and T-Cell Responses in Cancer Patients

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Stryhn, Anette; Justesen, Sune

    2011-01-01

    patients. Detection of antibodies against wt p53 protein has been used as a diagnostic and prognostic marker and discovery of new T-cell epitopes has enabled design of cancer vaccination protocols with promising results. Here, we identified wt p53-specific antibodies in various cancer patients......(264-272) in breast cancer patients and against HLA-A*01:01 binding peptide wt p53(226-234) and HLA-B*07:02 binding peptide wt p53(74-82) in renal cell cancer and breast cancer patients, respectively. Finally, we analyzed antibody and T-cell responses against wt p53 15-mer peptides in patients with metastatic renal...

  2. Radioimmunoassay for the detection of virus-specific IgA antibodies in saliva

    International Nuclear Information System (INIS)

    Friedman, M.G.

    1982-01-01

    The use of a sensitive and versatile radioimmunoassay (RIA) for detection of mumps-specific IgA and measles-specific IgA in unconcentrated saliva samples is described. The samples were obtained either by expectoration or by swabbing of the oral cavity, with or without stimulation of secretion, and were inactivated and clarified before testing. Mumps-specific IgA antibodies were detected as early as one day after onset of illness and peaked at 1-2 weeks after onset. Measles-specific salivary IgA antibodies were detected in 15-month old children 2-3 weeks after immunization. These results suggest that the RIA technique may be useful for early diagnosis of viral infections and for confirmation of response to immunization without the need for a blood sample, as well as for the study of the secretory immune response in very young and older subjects. (Auth.)

  3. Rhinovirus-induced VP1-specific Antibodies are Group-specific and Associated With Severity of Respiratory Symptoms

    Directory of Open Access Journals (Sweden)

    Katarzyna Niespodziana

    2015-01-01

    Interpretation: Our results demonstrate that increases of antibodies towards the VP1 N-terminus are group-specific and associated with severity of respiratory symptoms and suggest that it may be possible to develop serological tests for identifying causative RV groups.

  4. Sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Sikora, K; Alderson, T St.J.; Ellis, J [Ludwig Institute for Cancer Research, Cambridge (UK)

    1983-02-25

    A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants.

  5. Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

    Science.gov (United States)

    2006-05-01

    guinea pig model does present a significant problem...trying to correlate behavioral and protein changes due to the absence of guinea pig -specific antibodies. We...have developed a procedure to determine the specificity of commercially available, non- guinea pig -specific antibodies in guinea pig lysates.

  6. The effects of variations in the specificities of the antibody components on a two-site immunoradiometric assay for ferritin

    International Nuclear Information System (INIS)

    Cowan, S.I.; Stagg, B.H.; Niemann, E.

    1977-01-01

    Variations in the sub-unit antigenic structure of ferritins derived from various human tissues are reflected in the differing specificities of antisera raised against these ferritin preparations. In this study it was shown that antibody specificity played an important role in determining the sensitivity and overall binding of labelled antibody in a two-site immunoradiometric assay for ferritin. Homologous assay systems, in which solid phase and radiolabelled antibodies were of similar specificities, were generally less sensitive and showed lower binding than heterologous assay systems, in which solid phase and labelled antibodies were of different specificities. The source of the ferritin which was used as assay standard also played an important part in determining the sensitivity and overall binding in homologous antibody systems, spleen ferritin standards yielding assays superior to those obtained with placenta or liver ferritin standards. However, these differences between standards were not seen in a heterologous system employing solid phase antibodies directed against liver ferritin and labelled antibodies directed against placenta ferritin. The nature of the ferritin used to prepare immunoadsorbant for the purification of antibodies prior to radioiodination also affected the assay characteristics; antibodies prepared on spleen ferritin immunoadsorbant being more reactive than antibodies prepared on placenta ferritin immunoadsorbant, which in turn were more reactive then antibodies prepared on liver ferritin immunoadsorbant. (orig.) [de

  7. Analysis of the relations between allergen specific LgG antibody and allergic dermatosis of 14 kinds foods

    Directory of Open Access Journals (Sweden)

    Yin’e Hu

    2015-01-01

    Full Text Available To use food-specific IgG antibody detection to explore its application in the allergy dermatoses. 181 patients were included from January 2014 to September 2014. Fourteen food-specific IgG antibodies were detected by ELISA. The positive rates of IgG antibody of the patient group and the healthy group were significantly different. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. There was mild and moderate intolerance of food in the allergic dermatitis group while there was no distribution difference of food intolerance in urticaria group and eczema group. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults while there was no significant difference between children and adults among eczema patients with positive antibody. Allergy dermatoses are closely related to food-specific IgG antibody and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  8. Llama-Derived Single Domain Antibodies Specific for Abrus Agglutinin

    Science.gov (United States)

    Goldman, Ellen R.; Anderson, George P.; Zabetakis, Dan; Walper, Scott; Liu, Jinny L.; Bernstein, Rachael; Calm, Alena; Carney, James P.; O’Brien, Thomas W.; Walker, Jennifer L.; Garber, Eric A. E.

    2011-01-01

    Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations. PMID:22174977

  9. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C

    2011-01-01

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types......, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active u......PA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems...

  10. Subtype-Specific Influenza A Virus Antibodies in Canada Geese (Branta canadensis)

    Science.gov (United States)

    Kistler, Whitney M.; Stallknecht, David E.; DeLiberto, Thomas J.; Van Why, Kyle; Yabsley, Michael J.

    2015-01-01

    Historically, surveillance for influenza A viruses (IAVs) in wild birds has relied on viral detection assays. This was largely due to poor performance of serological assays in wild birds; however, recently developed commercial serological assays have improved the ability to detect IAV antibodies in wild birds. Serological surveillance for IAV antibodies in Canada geese (Branta canadensis) has shown that, despite a low prevalence of virus isolations, Canada geese are frequently exposed to IAVs and that exposure increases with latitude, which follows virus isolation prevalence patterns observed in dabbling ducks. The objectives of this study were to further evaluate IAV antibodies in Canada geese using a subtype-specific serological assay to determine if Canada geese are exposed to subtypes that commonly circulate in dabbling ducks. We collected serum samples from Canada geese in Minnesota, New Jersey, Pennsylvania, and Wisconsin and tested for antibodies to IAVs using a blocking ELISA. Positive samples were further tested by hemagglutination inhibition for 10 hemagglutinin IAV subtypes (H1–H10). Overall, we detected antibodies to NP in 24% (714/2,919) of geese. Antibodies to H3, H4, H5, and H6 subtypes predominated, with H5 being detected most frequently. A decrease in H5 HI antibody prevalence and titers was observed from 2009 to 2012. We also detected similar exposure pattern in Canada geese from New Jersey, Minnesota, Washington and Wisconsin. Based on the published literature, H3, H4, and H6 viruses are the most commonly reported IAVs from dabbling ducks. These results indicate that Canada geese also are frequently exposed to viruses of the same HA subtypes; however, the high prevalence of antibodies to H5 viruses was not expected as H5 IAVs are generally not well represented in reported isolates from ducks. PMID:25845755

  11. Shape-specific perceptual learning in a figure-ground segregation task.

    Science.gov (United States)

    Yi, Do-Joon; Olson, Ingrid R; Chun, Marvin M

    2006-03-01

    What does perceptual experience contribute to figure-ground segregation? To study this question, we trained observers to search for symmetric dot patterns embedded in random dot backgrounds. Training improved shape segmentation, but learning did not completely transfer either to untrained locations or to untrained shapes. Such partial specificity persisted for a month after training. Interestingly, training on shapes in empty backgrounds did not help segmentation of the trained shapes in noisy backgrounds. Our results suggest that perceptual training increases the involvement of early sensory neurons in the segmentation of trained shapes, and that successful segmentation requires perceptual skills beyond shape recognition alone.

  12. Indirect micro-immunofluorescence test for detecting type-specific antibodies to herpes simplex virus.

    Science.gov (United States)

    Forsey, T; Darougar, S

    1980-02-01

    A rapid indirect micro-immunofluorescence test capable of detecting and differentiating type-specific antibodies to herpes simplex virus is described. The test proved highly sensitive and, in 80 patients with active herpes ocular infection, antibody was detected in 94%. No anti-herpes antibody was detected in a control group of 20 patients with adenovirus infections. Testing of animal sera prepared against herpes simplex virus types 1 and 2 and of human sera from cases of ocular and genital herpes infections showed that the test can differentiate antibodies to the infecting serotypes. Specimens of whole blood, taken by fingerprick, and eye secretions, both collected on cellulose sponges, could be tested by indirect micro-immunofluorescence. Anti-herpes IgG, IgM, and IgA can also be detected.

  13. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

    International Nuclear Information System (INIS)

    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C.

    1990-01-01

    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with 131 I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author)

  14. Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

    Science.gov (United States)

    Tanaka, Kento; Yoshimura, Chigusa; Shiina, Tetsuo; Terauchi, Tomoko; Yoshitomi, Tomomi; Hirahara, Kazuki

    2017-04-01

    CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

  15. Quality control of radiolabeled antibodies through simultaneous determination of antibody concentration and specific activity using time-resolved interaction analysis and reverse kinetic fit

    International Nuclear Information System (INIS)

    Andersson, K.; Mihaylova, D.; Wang, E.; Abrahamsen, L.; Buijs, J.; Bjoerkelund, H.

    2015-01-01

    Full text of publication follows. With the advent of efficient methods for producing proteins that bind to a defined target, the number of radiolabeled proteins, and in particular antibodies, used for medical imaging and cancer therapy is increasing rapidly. In line with this increase, focus should be put on methods for the quality control (QC). Proper antibody quality is of fundamental importance to guarantee safety and consistent efficacy for the patient. Adequate QC procedures exist for small radiolabeled synthetic compounds like FDG, but antibody based radiopharmaceuticals are different. Proteins are much more complex and fragile than the synthetic compounds, and hence require new methods for adequate characterization and QC. Yet another complication is the labeling where there is a risk that a subpopulation of the protein is damaged to the level that it no longer binds the target. Therefore, a new toolbox is required to fulfill the quality characterization of radiolabeled antibodies. We have developed a QC assay for the simultaneous determination of antibody function, concentration and specific activity. The assay is based on time-resolved detection of the antibody interaction with antigen-coated magnetic beads in LigandTracer instruments. The resulting binding curve is evaluated using reverse kinetic fits, where the known interaction parameters of the antibody-antigen interaction are set constant while as the concentration and signal level are fitted. The assay takes approximately 2 hours and the majority of the time constitutes automated data collection in the instrument. The QC assay has been tested on multiple antibody-antigen interactions and consistently provides repeatable results for concentration and specific activity, both with coefficient of variation (CV) less than 15%. We believe that this QC assay can improve the quality of radiolabeled therapeutic antibodies. (authors)

  16. 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

  17. Antibody index and specific antibody quotient in horses after intragastric administration of Sarcocystis neurona sporocysts.

    Science.gov (United States)

    Heskett, Katherine A; Mackay, Robert J

    2008-03-01

    To investigate the use of a specific antibody index (AI) that relates Sarcocystis neurona-specific IgG quotient (Q(SN)) to total IgG quotient (Q(IgG)) for the detection of the anti-S neurona antibody fraction of CNS origin in CSF samples obtained from horses after intragastric administration of S neurona sporocysts. 18 adult horses. 14 horses underwent intragastric inoculation (day 0) with S neurona sporocysts, and 4 horses remained unchallenged; blood and CSF samples were collected on days - 1 and 84. For purposes of another study, some challenged horses received intermittent administration of ponazuril (20 mg/kg, PO). Sarcocystis neurona-specific IgG concentrations in CSF (SN(CSF)) and plasma (SN(plasma)) were measured via a direct ELISA involving merozoite lysate antigen and reported as ELISA units (EUs; arbitrary units based on a nominal titer for undiluted immune plasma of 100,000 EUs/mL). Total IgG concentrations in CSF (IgG(CSF)) and plasma (IgG(plasma)) were quantified via a sandwich ELISA and a radial immunodiffusion assay, respectively; Q(SN), Q(IgG), and AI were calculated. Following sporocyst challenge, mean +/- SEM SN(CSF) and SN(plasma) increased significantly (from 8.8 +/- 1.0 EUs/mL to 270.0 +/- 112.7 EUs/mL and from 1,737 +/- 245 EUs/mL to 43,169 +/- 13,770 EUs/mL, respectively). Challenge did not affect total IgG concentration, Q(SN), Q(IgG), or AI. S neurona-specific IgG detected in CSF samples from sporocyst-challenged horses appeared to be extraneural in origin; thus, this experimental challenge may not reliably result in CNS infection. Calculation of a specific AI may have application to the diagnosis of S neurona-associated myeloencephalitis in horses.

  18. Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

    NARCIS (Netherlands)

    Back, J.W.; Frisch, C.; Van Pee, K.; Boschert, V.; van Vught, R.; Puijk, W.; Mueller, T. D.; Knappik, A.; Timmerman, P.

    2012-01-01

    Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides

  19. 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.

  20. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    Directory of Open Access Journals (Sweden)

    Yanan Sun

    Full Text Available Single chain variable fragments (scFvs against diethylstilbestrol (DES were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3 for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR analysis was used to determine binding kinetics of one clone (30-1. The measured K(D was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

  1. Site-Specific Antibody Functionalization Using Tetrazine-Styrene Cycloaddition.

    Science.gov (United States)

    Umlauf, Benjamin J; Mix, Kalie A; Grosskopf, Vanessa A; Raines, Ronald T; Shusta, Eric V

    2018-05-03

    Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.

  2. Site-Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne-Azide Cycloaddition Reactions.

    Science.gov (United States)

    Perols, Anna; Arcos Famme, Melina; Eriksson Karlström, Amelie

    2015-11-01

    Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z32 BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z32 BPA and azide-Z32 BPA for Cu(I) -catalyzed cycloaddition, as well as DBCO-Z32 BPA for Cu-free strain-promoted cycloaddition. The Z32 BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The schistosoma-specific antibody response after treatment in non-immune travellers

    DEFF Research Database (Denmark)

    Duus, Liv Marie; Christensen, Anders Vittrup; Navntoft, Dorte

    2009-01-01

    Egg detection is the gold standard in diagnosing and controlling treatment in schistosomiasis, but sensitivity is poor in lightly infected individuals, whereas Schistosoma-specific antibodies are more sensitive. The purpose of the study was to evaluate use of Gut Associated Antigen (GAA...

  4. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C. (Nottingham Univ. (UK))

    1990-05-01

    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with {sup 131}I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author).

  5. Site-specific chemical modification of antibody fragments using traceless cleavable linkers.

    Science.gov (United States)

    Bernardes, Gonçalo J L; Steiner, Martina; Hartmann, Isabelle; Neri, Dario; Casi, Giulio

    2013-11-01

    Antibody-drug conjugates (ADCs) are promising agents for the selective delivery of cytotoxic drugs to specific cells (for example, tumors). In this protocol, we describe two strategies for the precise modification at engineered C- or N-terminal cysteines of antibodies in IgG, diabody and small immunoprotein (SIP) formats that yield homogenous ADCs. In this protocol, cemadotin derivatives are used as model drugs, as these agents have a potent cytotoxic activity and are easy to synthesize. However, other drugs with similar functional groups could be considered. In the first approach, a cemadotin derivative containing a sulfhydryl group results in a mixed disulfide linkage. In the second approach, a cemadotin derivative containing an aldehyde group is joined via a thiazolidine linkage. The procedures outlined are robust, enabling the preparation of ADCs with a defined number of drugs per antibody in a time frame between 7 and 24 h.

  6. Prevalence of parvovirus B19 specific antibody in pregnant women with spontaneous abortion.

    Science.gov (United States)

    Rahbar, Nahid; Vali Zadeh, Saeid; Ghorbani, Raheb; Kheradmand, Pegah

    2015-01-01

    Human parvovirus B19 is a very common viral infection especially in school-aged children. The infection during pregnancy can affect the fetus due to lack of mother's immunity. Although, there is still no evidence of fetal teratogenic effects with parvovirus B19, but non-immune fetal hydrops and abortion may be caused by vertical transmission of the virus during pregnancy. This study was aimed to assess the prevalence of parvovirus B19-specific antibody (IgM) in pregnant women who had a spontaneous abortion. This cross-sectional study was carried out in all pregnant women who referred due to a spontaneous abortion. All demographic information such as age, occupation, and gestational age, last history of abortion, gravity, and presence of children below the age of six was recorded and a blood sample was provided for all the women. Then, the blood samples were tested to assay parvovirus B19-specific antibody (IgM) by EuroImmune ELISA kit. Among 94 pregnant women with the mean age of 28.4 years who had a spontaneous abortion, parvovirus B19 specific antibody (IgM) was detected in 17 participants (18.1%). Meanwhile, 14 women (14.9%) were suspected for presence of the antibody in their blood sample. There was no significant difference between the presence of antibody and age of pregnant women, occupation, gestational age, number of previous abortion, presence of children below the age of six and number of pregnancy. These findings revealed that a high percentage of pregnant women are probably non-immune against parvovirus B19, and also there might be a number of spontaneous abortions in which parvovirus infection caused fetal death.  However, more studies are needed to prove the absolute role of parvovirus B19 in these abortions.

  7. Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium's Adaptive Mechanisms of Intramacrophage Survival and Replication.

    Directory of Open Access Journals (Sweden)

    Swarmistha Devi Aribam

    Full Text Available Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

  8. Complement-dependent pathogenicity of brain-specific antibodies in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Asgari, Nasrin; Khorooshi, Reza; Lillevang, Søren T

    2013-01-01

    The specificity and potential pathogenicity of autoantibodies vary between neurological diseases. It is often unclear whether their detection in cerebrospinal fluid (CSF) is a consequence or a cause of pathology. The goal was to test whether administration of brain-specific antibodies into CSF...... would be sufficient for pathology. Purified immunoglobulin G from a neuromyelitis optica patient was injected intrathecally with complement to naive mice. Histopathological analysis at 7 days revealed damage to the ependyma, disruption of the CSF parenchymal barrier and pathologic lesions, distant from...

  9. 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

  10. Value of serum TORCH-specific antibody detection in assessment of neonatal jaundice

    Directory of Open Access Journals (Sweden)

    Guang-Hua Dai

    2016-06-01

    Full Text Available Objective: To study the value of serum TORCH-specific antibody detection in assessment of neonatal jaundice. Methods: A total of 70 cases of children with neonatal jaundice were selected as jaundice group, 70 cases of healthy newborn were the control group, and serum serum TORCH-specific antibody content as well as heart function, liver function, kidney function and nerve function indicators were detected. Results: Serum TOX-IgM, RV-IgM, CMV-IgM and HSV-IgM positive rate and content of jaundice group were significantly higher than those of control group; serum CK-MB, cTnI, AST, ALT, Cys-C, RBP, MBP, S100β and NSE content of TORCH-positive children were significantly higher than those of TORCHnegative children, and BDNF, NT-3, NT-4 and NGF content were significantly lower than those of TORCH-negative children; T1WI signal of pallidum MRI of TORCH-positive children was significantly higher than that of TORCH-negative children. Conclusions: Serum TORCHspecific antibodies significantly increase in children with neonatal jaundice and can assess the degree of bilirubin metabolism disorder and the degree of target organ damage.

  11. The clinical syndrome of specific antibody deficiency in children.

    Science.gov (United States)

    Boyle, R J; Le, C; Balloch, A; Tang, M L-K

    2006-12-01

    Specific antibody deficiency (SAD) is an immune deficiency which has been reported in adults and children with recurrent respiratory tract infections; however, the clinical features of SAD are not well described. This study evaluated formally the clinical syndrome of SAD, by comparing the clinical features of children with SAD and those of children with recurrent infection but normal immune function tests. SAD was defined as an adequate IgG antibody response to less than 50% of 12 pneumococcal serotypes tested following 23-valent unconjugated pneumococcal immunization. An adequate IgG antibody response was defined as a post-immunization titre of >or= 1.3 microg/ml or >or= four times the preimmunization value. Seventy-four children with recurrent infection were evaluated where immune deficiencies other than SAD had been excluded. Eleven (14.9%) of these children had SAD. Clinical features differed between the group with SAD and the group with normal antibody responses. A history of otitis media, particularly in association with chronic otorrhoea was associated with SAD [relative risk (RR) of SAD in those with chronic otorrhoea 4.64 (P = 0.02)]. SAD was associated with allergic disease, particularly allergic rhinitis [RR of SAD in those with allergic rhinitis 3.77 (P = 0.04)]. These two clinical associations of SAD were independent in this study [RR of chronic otorrhoea in those with allergic rhinitis 0.85 (P = 0.28)]. SAD was not an age-related phenomenon in this population. SAD has a distinct clinical phenotype, presenting as recurrent infection associated with chronic otorrhoea and/or allergic disease, and the condition should be sought in children with these features.

  12. Micro solid-phase radioimmunoassay for detection of herpesvirus type-specific antibody: parameters involved in standardization

    Energy Technology Data Exchange (ETDEWEB)

    Matson, D.O.; Adler-Storthz, K.; Adam, E.; Dreesman, G.R. (Baylor Univ., Houston, TX (USA). Coll. of Medicine)

    1983-02-01

    A micro solid-phase radioimmunoassay (micro-SPRIA) was developed to demonstrate type-specific antibodies to herpes simplex virus types 1 and 2 (HSV1 and HSV2). Glycoproteins from the 123,000 dalton region of HSV1 (VP123) and the 119,000 dalton region of HSV2 (VP119) were isolated on preparative polyacrylamide gels for use as antigens in the micro-SPRIA. Human sera selected from clinical samples by virological history and appropriate microneutralization data were used to standardize the micro-SPRIA. Optimization of the assay required the use of siliconized microtiter wells for adsorption of antigen. Maximized results were highly dependent on the concentrations of antigen, primary antibody, and secondary antibody as well as the diluents used for these principal test reagents. Incorporation of HSV glycoproteins of each respective type with the optimal condition established in this study facilitates the direct detection of type-specific antibody in human sera.

  13. A sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    International Nuclear Information System (INIS)

    Sikora, K.; Alderson, T.St.J.; Ellis, J.

    1983-01-01

    A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants. (Auth.)

  14. Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods

    DEFF Research Database (Denmark)

    Pressler, T.; Karpati, F.; Granstrom, M.

    2008-01-01

    to characterize patients with different infection status. Elevated levels of specific anti-Pseudomonas antibodies showed to be the risk factor for developing chronic Pa infection. Due to the specificity of the tests, antibiotic treatment based on serology might be considered in selected cases. There is a window...... of opportunity for suppression and eradication of initial P. aeruginosa infection making measurement of specific anti-Pseudomonas antibodies helpful Udgivelsesdato: 2009/1...

  15. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  16. Diagnostic Value of ELISA Tests for the Detection of Specific Antibodies in Cats and Rabbits with Dermatophytosis

    Directory of Open Access Journals (Sweden)

    Marinka Drobnič-Košorok

    2002-01-01

    Full Text Available Two indirect ELISA tests developed for the detection of specific IgG in cats and rabbits, infected with M. canis and T. mentagrophytes, respectively, were evaluated and compared. The levels of specific antibodies were determined in sera of 20 cats and 25 rabbits naturally infected with M. canis and T. mentagrophytes, respectively. Infection was confirmed by the results of fungal culture. Blood samples from 12 cats and 17 rabbits, previously unexposed to dermatophytes, served as negative controls. A significant increase in the level of specific antibodies in groups of infected animals was demonstrated. Sensitivity, specificity and predictive values of a positive and a negative test were determined to evaluate the diagnostic potential. ELISA for the detection of specific antibodies in cats infected with M. canis (ELISA-cats test exhibited 75.0 % of sensitivity at 91.7 % of specificity, whereas the test for the detection of specific antibodies in rabbits, infected with T. mentagrophytes (ELISA-rabbits test is highly sensitive (96.0 % and highly specific (94.1 %, confirming its encouraging diagnostic potential. The cross-reactivity of fungal antigens was tested by performing the assays with antigens M. canis, T. mentagrophytes, M. pachydermatis and A. fumigatus. There were no significant indications of cross-reactions in the test T. mentagrophytes-rabbits, whereas strong cross-reaction between dermatophyte antigens was observed in the test M. canis-cats.

  17. Re-engineering therapeutic antibodies for Alzheimer's disease as blood-brain barrier penetrating bi-specific antibodies.

    Science.gov (United States)

    Pardridge, William M

    2016-12-01

    Therapeutic antibodies are large molecule drugs that do not cross the blood-brain barrier (BBB). Therefore, drug development of therapeutic antibodies for Alzheimer's disease (AD) requires that these molecules be re-engineered to enable BBB delivery. This is possible by joining the therapeutic antibody with a transporter antibody, resulting in the engineering of a BBB-penetrating bispecific antibody (BSA). Areas covered: The manuscript covers transporter antibodies that cross the BBB via receptor-mediated transport systems on the BBB, such as the insulin receptor or transferrin receptor. Furthermore, it highlights therapeutic antibodies for AD that target the Abeta amyloid peptide, beta secretase-1, or the metabotropic glutamate receptor-1. BSAs are comprised of both the transporter antibody and the therapeutic antibody, as well as IgG constant region, which can induce immune tolerance or trigger transport via Fc receptors. Expert opinion: Multiple types of BSA molecular designs have been used to engineer BBB-penetrating BSAs, which differ in valency and spatial orientation of the transporter and therapeutic domains of the BSA. The plasma pharmacokinetics and dosing regimens of BSAs differ from that of conventional therapeutic antibodies. BBB-penetrating BSAs may be engineered in the future as new treatments of AD, as well as other neural disorders.

  18. Patient-specific estimation of detailed cochlear shape from clinical CT images

    DEFF Research Database (Denmark)

    Kjer, H Martin; Fagertun, Jens; Wimmer, Wilhelm

    2018-01-01

    of the detailed patient-specific cochlear shape from CT images. From a collection of temporal bone [Formula: see text]CT images, we build a cochlear statistical deformation model (SDM), which is a description of how a human cochlea deforms to represent the observed anatomical variability. The model is used...... for regularization of a non-rigid image registration procedure between a patient CT scan and a [Formula: see text]CT image, allowing us to estimate the detailed patient-specific cochlear shape. We test the accuracy and precision of the predicted cochlear shape using both [Formula: see text]CT and CT images...

  19. Donor-Specific Anti-HLA Antibodies in Huntington's Disease Recipients of Human Fetal Striatal Grafts.

    Science.gov (United States)

    Porfirio, Berardino; Paganini, Marco; Mazzanti, Benedetta; Bagnoli, Silvia; Bucciantini, Sandra; Ghelli, Elena; Nacmias, Benedetta; Putignano, Anna Laura; Rombolà, Giovanni; Saccardi, Riccardo; Lombardini, Letizia; Di Lorenzo, Nicola; Vannelli, Gabriella B; Gallina, Pasquale

    2015-01-01

    Fetal grafting in a human diseased brain was thought to be less immunogenic than other solid organ transplants, hence the minor impact on the efficacy of the transplant. How much prophylactic immune protection is required for neural allotransplantation is also debated. High-sensitive anti-HLA antibody screening in this field has never been reported. Sixteen patients with Huntington's disease underwent human fetal striatal transplantation in the frame of an open-label observational trial, which is being carried out at Florence University. All patients had both brain hemispheres grafted in two separate robotic-stereotactic procedures. The trial started in February 2006 with the first graft to the first patient (R1). R16 was given his second graft on March 2011. All patients received triple immunosuppressive treatment. Pre- and posttransplant sera were analyzed for the presence of anti-HLA antibodies using the multiplexed microsphere-based suspension array Luminex xMAP technology. Median follow-up was 38.5 months (range 13-85). Six patients developed anti-HLA antibodies, which turned out to be donor specific. Alloimmunization occurred in a time window of 0-49 months after the first neurosurgical procedure. The immunogenic determinants were non-self-epitopes from mismatched HLA antigens. These determinants were both public epitopes shared by two or more HLA molecules and private epitopes unique to individual HLA molecules. One patient had non-donor-specific anti-HLA antibodies in her pretransplant serum sample, possibly due to previous sensitization events. Although the clinical significance of donor-specific antibodies is far from being established, particularly in the setting of neuronal transplantation, these findings underline the need of careful pre- and posttransplant immunogenetic evaluation of patients with intracerebral grafts.

  20. Influenza A virus H5-specific antibodies in mute swans (Cygnus olor) in the USA.

    Science.gov (United States)

    Kistler, Whitney M; Stallknecht, David E; Lebarbenchon, Camille; Pedersen, Kerri; Marks, David R; Mickley, Randy; DeLiberto, Thomas J; Yabsley, Michael J

    2015-04-01

    The use of serologic assays for influenza A virus (IAV) surveillance in wild birds has increased because of the availability of commercial enzyme-linked immunosorbent assays (ELISAs). Recently, an H5-specific blocking ELISA (bELISA) was shown to reliably detect H5-specific antibodies to low- and high-pathogenic H5 viruses in experimentally infected waterfowl. Mute Swans (Cygnus olor) were frequently associated with highly pathogenic H5N1 outbreaks in Europe and may have a similar role if highly pathogenic H5N1 is introduced into North America. We measured the prevalence of antibodies to the nucleoprotein and H5 protein in Mute Swans using three serologic assays. We collected 340 serum samples from Mute Swans in Michigan, New Jersey, New York, and Rhode Island, US. We detected antibodies to the IAV nucleoprotein in 66.2% (225/340) of the samples. We detected H5-specific antibodies in 62.9% (214/340) and 18.8% (64/340) using a modified H5 bELISA protocol and hemagglutination inhibition (HI) assay, respectively. The modified H5 bELISA protocol detected significantly more positive samples than did the manufacturer's protocol. We also tested 46 samples using virus neutralization. Neutralization results had high agreement with the modified H5 bELISA protocol and detected a higher prevalence than did the HI assay. These results indicate that North American Mute Swans have high nucleoprotein and H5 antibody prevalences.

  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. A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

    Science.gov (United States)

    Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

    2018-04-01

    Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

  3. Specificity of antibodies directed against the cytolethal distending toxin of Haemophilus ducreyi in patients with chancroid.

    Science.gov (United States)

    Mbwana, Judica; Ahmed, Hinda J; Ahlman, Karin; Sundaeus, Vivian; Dahlén, Gunnar; Lyamuya, Eligius; Lagergård, Teresa

    2003-09-01

    Antibodies specific for the cytolethal-distending toxin of Haemophilus ducreyi (HdCDT) complex and for the CdtA, CdtB, and CdtC components were measured by ELISA in the sera of 50 patients with culture and/or PCR proven chancroid, 42 patients with periodontitis, 50 blood donors from Tanzania, 50 blood donors from Sweden. In addition, the biological activity e.g. neutralization capacity of the sera were tested. Our results demonstrate that majority of chancroid patients and healthy individuals had detectable levels of serum antibodies to HdCDT complex and to separate toxin components. However, high levels (> or =100 units) of antibodies to HdCDT complex were significantly more prevalent in the sera of patients with both chancroid and periodontitis than in the sera of the corresponding controls (P=0.001 and P=0.04, respectively). In the sera of the 50 patients with chancroid, antibodies to CdtA, CdtB, and CdtC were detected in 50, 35, and 34 individuals, respectively. Antibodies to CdtC, being less frequently detected than the antibodies to other components, show a good correlation with the neutralizing capacity of sera. High levels of neutralizing antibodies (> or =160) were detected in only 22 and 2% of the patients with chancroid and periodontitis, respectively. The data suggest that the low levels of anti-HdCDT antibodies, which include neutralizing antibodies, may contribute to limited protection in chancroid and since anti-HdCDT antibodies, may be detected in healthy individuals and in patients with certain disease conditions (e.g. periodontitis), they may not be specific markers for chancroid infection.

  4. Comparison of Six Automated Treponema-Specific Antibody Assays.

    Science.gov (United States)

    Park, Borae G; Yoon, Jihoon G; Rim, John Hoon; Lee, Anna; Kim, Hyon-Suk

    2016-01-01

    Six different Treponema (TP)-specific immunoassays were compared to the fluorescent treponemal antibody absorption (FTA-ABS) test. A total of 615 samples were tested. The overall percent agreement, analytical sensitivity, and analytical specificity of each assay compared to the FTA-ABS test were as follows: Architect Syphilis TP, 99.2%, 96.8%, and 100%; Cobas Syphilis, 99.8%, 99.4%, and 100%; ADVIA Centaur Syphilis, 99.8%, 99.4%, and 100%; HISCL Anti-TP assay kit, 99.7%, 98.7%, and 100%; Immunoticles Auto3 TP, 99.0%, 97.5%, and 99.6%; Mediace TPLA, 98.0%, 98.1%, and 98.0%. All results that were discrepant between the TP-specific assays were associated with samples from noninfectious cases (11 immunoassay false positives and 7 from previous syphilis cases). Our study demonstrated that TP-specific immunoassays generally showed high sensitivities, specificities, and percentages of agreement compared to FTA-ABS, with rare cases of false-positive or false-negative results. Therefore, most TP-specific immunoassays are acceptable for use in screening for syphilis. However, it is important to perform a thorough review of a patient's clinical and treatment history for interpreting the results of syphilis serology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. A Comprehensive Overview on Myositis-Specific Antibodies: New and Old Biomarkers in Idiopathic Inflammatory Myopathy

    Science.gov (United States)

    Satoh, Minoru; Tanaka, Shin; Ceribelli, Angela; Calise, S. John; Chan, Edward K. L.

    2018-01-01

    Autoantibodies specific for idiopathic inflammatory myopathy (myositis-specific autoantibodies (MSAs)) are clinically useful biomarkers to help the diagnosis of polymyositis/dermatomyositis (PM/DM). Many of these are also associated with a unique clinical subset of PM/DM, making them useful in predicting and monitoring certain clinical manifestations. Classic MSAs known for over 30 years include antibodies to Jo-1 (histidyl transfer RNA (tRNA) synthetase) and other aminoacyl tRNA synthetases (ARS), anti-Mi-2, and anti-signal recognition particle (SRP). Anti-Jo-1 is the first autoantibodies to ARS detected in 15–25 % of patients. In addition to anti-Jo-1, antibodies to seven other aminoacyl tRNA synthetases (ARS) have been reported with prevalence, usually 1–5 % or lower. Patients with any antiARS antibodies are associated with anti-synthetase syndrome characterized by myositis, interstitial lung disease (ILD), arthritis, Raynaud’s phenomenon, and others. Several recent studies suggested heterogeneity in clinical features among different anti-ARS antibody-positive patients and anti-ARS may also be found in idiopathic ILD without myositis. Anti-Mi-2 is a classic marker for DM and associated with good response to steroid treatment and good prognosis. Anti-SRP is specific for PM and associated with treatment-resistant myopathy histologically characterized as necrotizing myopathy. In addition to classic MSAs, several new autoantibodies with strong clinical significance have been described in DM. Antibodies to transcription intermediary factor 1γ/α (TIF1γ/α, p155/140) are frequently found in DM associated with malignancy while anti-melanoma differentiation-associated gene 5 (MDA5; CADM140) are associated with clinically amyopathic DM (CADM) complicated by rapidly progressive ILD. Also, anti-MJ/nuclear matrix protein 2 (NXP-2) and anti-small ubiquitin-like modifier-1 (SUMO-1) activating enzyme (SAE) are recognized as new DM-specific autoantibodies. Addition of

  6. Sensory memory of structure-from-motion is shape-specific.

    Science.gov (United States)

    Pastukhov, Alexander; Füllekrug, Jana; Braun, Jochen

    2013-08-01

    Perceptual priming can stabilize the phenomenal appearance of multistable visual displays (Leopold, Wilke, Maier, & Logothetis, Nature Neuroscience, 5, 605-609, 2002). Prior exposure to such displays induces a sensory memory of their appearance, which persists over long intervals and intervening stimulation, and which facilitates renewed perception of the same appearance. Here, we investigated perceptual priming for the apparent rotation in depth of ambiguous structure-from-motion (SFM) displays. Specifically, we generated SFM objects with different three-dimensional shapes and presented them in random order and with intervening blank periods. To assess perceptual priming, we established the probability that a perceived direction of rotation would persist between successive objects. In general, persistence was greatest between identical objects, intermediate between similar objects, and negligible between dissimilar objects. These results demonstrate unequivocally that sensory memory for apparent rotation is specific to three-dimensional shape, contrary to previous reports (e.g., Maier, Wilke, Logothetis, & Leopold, Current Biology, 13, 1076-1085, 2003). Because persistence did not depend on presentation order for any pair of objects, it provides a commutative measure for the similarity of object shapes. However, it is not clear exactly which features or aspects of object shape determine similarity. At least, we did not find simple, low-level features (such as volume overlap, heterogeneity, or rotational symmetry) that could have accounted for all observations. Accordingly, it seems that sensory memory of SFM (which underlies priming of ambiguous rotation) engages higher-level representations of object surface and shape.

  7. Neutralizing antibody response during human immunodeficiency virus type 1 infection: type and group specificity and viral escape

    DEFF Research Database (Denmark)

    Arendrup, M; Sönnerborg, A; Svennerholm, B

    1993-01-01

    The paradox that group-specific neutralizing antibodies (NA) exist in the majority of human immunodeficiency virus type 1 (HIV-1)-infected patients, whereas the NA response against autologous HIV-1 virus isolates is highly type-specific, motivated us to study the type- and group-specific NA...... demonstrated, suggesting that the majority of the change in neutralization sensitivity is driven by the selective pressure of type-specific NA. Furthermore, no differences were observed in sensitivity to neutralization by anti-carbohydrate neutralizing monoclonal antibodies or the lectin concanavalin A...

  8. 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.

  9. Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting.

    Science.gov (United States)

    Pan, Lucy Yan; Salas-Solano, Oscar; Valliere-Douglass, John F

    Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C H 2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.

  10. HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

    Science.gov (United States)

    2018-01-01

    ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the

  11. Seroprevalence of toxoplasma-specific antibodies in patients suspected to have active toxoplasmosis: A cross-sectional survey

    Directory of Open Access Journals (Sweden)

    Abbas Ali Eskandarian

    2014-01-01

    Full Text Available Background: The aim of this study was to investigate the presence and distribution of anti-toxoplasma-specific IgM and IgG tantibodies in patients suspected to have toxoplasmosis and investigate for any association between IgM and IgG antibodies and some toxoplasmosis risk factors as well. Materials and Methods: In a comparative cross-sectional study, 70 patients suspected to had active toxoplasmosis and 30 control volunteers, who gave informed consent, entered the study. In each group, patient age, sex, signs of appearance, education level, residency status (urban / rural, occupation, frequency of toxoplasma-specific IgG and IgM antibodies, abortion history, and some risk factors (Direct cat exposure, Occupational exposure to raw meat, and Raw vegetable consumption were recorded. The enzyme-linked immunosorbent assay (ELISA kits (EUROIMMUN®, United Kingdom were used for the evaluation of anti-toxoplasma IgG and IgM antibodies according to the manufacturer›s instructions. All analyses were done using SPSS-20. Results: The frequency of toxoplasma-specific IgG and IgM antibodies like: Direct cat exposures, Occupational exposure to raw meat, and Raw vegetable consumption were not statistically significant between the two groups (P > 0.05. The history of previous abortions in women in the toxoplasmosis-suspected group was significantly higher than that in the controls (31.4% versus 6.7%; P = 0.009. Conclusion: The frequency of specific IgM and IgG antibodies in toxoplasmosis suspected in the toxoplasmosis and control groups was not statistically significant.

  12. Potential role of specific antibodies as important vaccine induced protective mechanism against Aeromonas salmonicida in rainbow trout.

    Directory of Open Access Journals (Sweden)

    Kasper Rømer Villumsen

    Full Text Available Furunculosis caused by infection with Aeromonas salmonicida subsp. salmonicida has been a known threat to aquaculture for more than a century. Efficient prophylactic approaches against this disease are essential for continued growth of salmonid aquaculture. Since the introduction of successful oil-adjuvanted vaccines in the early 1990's, a number of studies have been published on the protective as well as adverse effects of these vaccines. Most studies focus on vaccination of salmon (Salmo salar. However, rainbow trout (Oncorhynchus mykiss are also very susceptible to infection and are vaccinated accordingly. In this study we have examined the protection against infection with a Danish strain of A. salmonicida in both vaccinated and non-vaccinated rainbow trout. A commercial and an experimental auto-vaccine were tested. The protective effects of the vaccines were evaluated through an A. salmonicida challenge 18 weeks post vaccination. Both vaccines resulted in a significantly increased survival in the vaccinated fish during a 28 day challenge period relative to non-vaccinated fish (P = 0.01 and P = 0.001 for the commercial and experimental vaccine, respectively. Throughout the entire experiment, the presence of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred in vaccinated fish. A positive correlation was found between mean levels of specific antibodies pre challenge and overall survival. This correlation, along with the observed depletion of antibodies during the initial phase of infection, suggests that specific antibodies play an essential role in vaccine mediated protection against A. salmonicida in rainbow trout.

  13. Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

    Science.gov (United States)

    Williams, Y J; Rea, S M; Popovski, S; Skillman, L C; Wright, A-D G

    2014-12-01

    Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

  14. Development of a dipstick assay for detection of Leishmania-specific canine antibodies

    NARCIS (Netherlands)

    Schallig, Henk D. F. H.; Cardoso, Luís; Hommers, Marieke; Kroon, Nel; Belling, Guus; Rodrigues, Manuela; Semião-Santos, Saul J.; Vetter, Hans

    2004-01-01

    A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the

  15. Radioimmunoassay of total IgE and allergen-specific IgE antibodies with a uniform indicator system in allergies of childhood

    International Nuclear Information System (INIS)

    Struy, H.; Schuster, R.; Sollich, V.; Thal, W.; Morenz, J.

    1984-01-01

    Solid-phase radioimmunoassays for the determination of allergen-specific and total IgE have been developed. In an indirect solid-phase radioimmunoassay for the measurement of allergen-specific antibodies PVC blisters coated with allergens and in a sandwich solid-phase radioimmunoassay blisters coated with antihuman IgE antibodies are incubated sequentially with patient serum, unlabelled antihuman IgE from rabbits purified by affinity chromatography, and finally with antirabbitglobulin from sheep. Antirabbitglobuline was purified by immunoadsorption. The 125 I-labelled antibody with a specific activity of 30 kBq/μg antibody protein could be used universally for the determination of antibodies of each immunoglobulin class. In 160 patients mostly with seasonal asthma these assays supported RAST and PRIST kits and were helpful in the diagnosis of atopic diseases. (author)

  16. Immunoradiometric assay for cytomegalovirus-specific IgG antibodies; Assay development and evaluation in blood transfusion practice

    Energy Technology Data Exchange (ETDEWEB)

    Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J. (Medical School, Manchester (United Kingdom). Department of Medical microbiology, Virology Unit); Morell, G. (Regional Blood Transfusion Centre, manchester (United Kingdom))

    1990-03-01

    An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 (human cytomegalovirus (CMV)) has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab.

  17. Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Eszter Szarka

    2018-01-01

    Full Text Available Background: In rheumatoid arthritis (RA, anti-citrullinated protein/peptide antibodies (ACPAs are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

  18. Many de novo donor‐specific antibodies recognize β2‐microglobulin‐free, but not intact HLA heterodimers

    Science.gov (United States)

    Michel, K.; Santella, R.; Steers, J.; Sahajpal, A.; Downey, F. X.; Thohan, V.

    2016-01-01

    Abstract Solid‐phase single antigen bead (SAB) assays are standard of care for detection and identification of donor‐specific antibody (DSA) in patients who receive solid organ transplantation (SOT). While several studies have documented the reproducibility and sensitivity of SAB testing for DSA, there are little data available concerning its specificity. This study describes the identification of antibodies to β2‐microglobulin‐free human leukocyte antigen (β2‐m‐fHLA) heavy chains on SAB arrays and provides a reassessment of the clinical relevance of DSA testing by this platform. Post‐transplant sera from 55 patients who were positive for de novo donor‐specific antibodies on a SAB solid‐phase immunoassay were tested under denaturing conditions in order to identify antibodies reactive with β2‐m‐fHLA or native HLA (nHLA). Antibodies to β2‐m‐fHLA were present in nearly half of patients being monitored in the post‐transplant period. The frequency of antibodies to β2‐m‐fHLA was similar among DSA and HLA antigens that were irrelevant to the transplant (non‐DSA). Among the seven patients with clinical or pathologic antibody‐mediated rejection (AMR), none had antibodies to β2‐m‐fHLA exclusively; thus, the clinical relevance of β2‐m‐fHLA is unclear. Our data suggests that SAB testing produces false positive reactions due to the presence of β2‐m‐fHLA and these can lead to inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post‐transplant period. PMID:27060279

  19. Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6

    Directory of Open Access Journals (Sweden)

    Sotiropoulou Georgia

    2012-12-01

    Full Text Available Abstract Background Human kallikrein-related peptidase 6 (KLK6 has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6. Results Chicken were immunized with recombinant KLK6 and antibodies Y (IgYs were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins. Conclusions Based on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples.

  20. Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide.

    Science.gov (United States)

    Schlageter, A M; Kozel, T R

    1990-06-01

    A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.

  1. Development of Strongylus vulgaris-specific serum antibodies in naturally infected foals.

    Science.gov (United States)

    Nielsen, M K; Vidyashankar, A N; Gravatte, H S; Bellaw, J; Lyons, E T; Andersen, U V

    2014-03-01

    Strongylus vulgaris is regarded as the most pathogenic helminth parasite infecting horses. Migrating larvae cause pronounced endarteritis and thrombosis in the cranial mesenteric artery and adjacent branches, and thromboembolism can lead to ischemia and infarction of large intestinal segments. A recently developed serum ELISA allows detection of S. vulgaris-specific antibodies during the six-month-long prepatent period. A population of horses has been maintained at the University of Kentucky without anthelmintic intervention since 1979, and S. vulgaris has been documented to be highly prevalent. In 2012, 12 foals were born in this population, and were studied during a 12-month period (March-March). Weekly serum samples were collected to monitor S. vulgaris specific antibodies with the ELISA. Nine colts underwent necropsy at different time points between 90 and 300 days of age. At necropsy, Strongylus spp. and Parascaris equorum were identified to species and stage and enumerated. Initial statistical findings indicate a significant interaction between foal age and ELISA results (pvulgaris-directed maternal antibodies transferred in the colostrum, but then remained ELISA negative during their first three months of life. Foals born in February and March became ELISA positive at about 12 weeks of age, while those born in April and May went positive at about 15 and 21 weeks, respectively. Foal date of birth was significantly associated with ELISA results (pvulgaris burdens (pvulgaris, S. edentatus, and P. equorum burdens (pvulgaris larvae leaving the bloodstream and migrating back to the intestine. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Application of 125I radioimmunoassay to measure inhibition of precipitin reactions using carbohydrate-specific antibodies

    International Nuclear Information System (INIS)

    Boullanger, P.H.; Nagpurkar, A.; Noujaim, A.A.; Lemieux, R.U.

    1978-01-01

    Antibodies raised to an artificial antigen with β-D-galactopyranosyl groups as antigenic determinants were purified using an immunoadsorbent prepared from the hapten involved in the synthesis of the antigen. In order to study the specificity of these antibodies, 125 I radiolabelling of either the artificial antigen or the antibody was used in the study of inhibitions of the precipitin reaction. The method, involving labelling of the artificial antigen and counting radioactivity in the supernatant, was found to be more accurate and faster than the usual methods based on measuring the amount of protein precipitated by chemical or spectroscopic methods. (author)

  3. Rat Monoclonal Antibodies Specific for LST1 Proteins

    OpenAIRE

    Schiller, Christian; Nitschké, Maximilian J. E.; Seidl, Alexander; Kremmer, Elisabeth; Weiss, Elisabeth H.

    2009-01-01

    The LST1 gene is located in the human MHC class III region and encodes transmembrane and soluble isoforms that have been suggested to play a role in the regulation of the immune response and are associated with inflammatory diseases such as rheumatoid arthritis. Here we describe the generation and characterization of the first monoclonal antibodies against LST1. Two hybridoma lines secreting monoclonal antibodies designated 7E2 and 8D12 were established. The 7E2 antibody detects recombinant a...

  4. Gag- and env-specific serum antibodies in cats after natural and experimental infection with feline immunodeficiency virus.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); C.H.J. Siebelink (Kees); H. Broos; G.A. Drost; K. Weijer (Kees); R. van Herwijnen (Rob); A.D.M.E. Osterhaus (Albert)

    1994-01-01

    textabstractIn order to monitor the antibody response to feline immunodeficiency virus (FIV) in cats, following experimental and natural infection, enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant env and gag proteins and p24-specific monoclonal antibodies. It was shown

  5. A Novel, Rapid Assay for Detection and Differentiation of Serotype-Specific Antibodies to Venezuelan Equine Encephalitis Complex Alphaviruses

    National Research Council Canada - National Science Library

    Wang, Eryu; Paessler, Slobodan; Smith, Darci R; Coffey, Lark L; Kang, Wenli; Estrada-Franco, Jose; Weaver, Scott C; Aguilar, Patricia V; Pfeffer, Martin; Olson, James

    2005-01-01

    ... of Venezuelan equine encephalitis (VEE) virus. Two monoclonal antibodies that differentially recognize epizootic versus enzootic VEE virus epitopes were used to measure the serotype-specific blocking abilities of antibodies in sera of naturally...

  6. 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.

  7. Donor-specific Anti-HLA antibodies in allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Sarah Morin-Zorman

    2016-08-01

    Full Text Available Allogeneic Hematopoietic Stem Cell Transplantation (AHSCT is a curative treatment for a wide variety of hematological diseases. In 30% of the cases, a geno-identical donor is available. Any other situation displays some level of Human Leukocyte Antigen (HLA incompatibility between donor and recipient. Deleterious effects of anti-HLA immunization have long been recognized in solid organ transplant recipients. More recently, anti-HLA immunization was shown to increase the risk of Primary Graft Failure (PGF, a severe complication of AHSCT that occurs in 3 to 4% of matched unrelated donor transplantation and up to 15% in cord blood transplantation and T-cell depleted haplo-identical stem cell transplantation. Rates of PGF in patients with DSA were reported to be between 24 to 83% with the highest rates in haplo-identical and cord blood transplantation recipients. This led to the recommendation of anti-HLA antibody screening to detect Donor Specific Antibodies (DSA in recipients prior to AHSCT. In this review, we highlight the role of anti-HLA antibodies in AHSCT and the mechanisms that may lead to PGF in patients with DSA, and discuss current issues in the field.

  8. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

    Directory of Open Access Journals (Sweden)

    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  9. Detection of specific antibody producing cells in porcine colostrum by in ovo translation of their mRNA

    International Nuclear Information System (INIS)

    Kortbeek-Jacobs, N.; Donk, H. van der

    1978-01-01

    An improved method is described for the determination of antibody producing cells in sows colostrum. The test system comprises in ovo translation of mRNA from swine colostral cells and analysis of the translation products by radioimmunoassay with specific antibodies and antigen. (C.F.)

  10. Screening for hen's egg and chicken meat specific IgE antibodies in ...

    African Journals Online (AJOL)

    Background: Allergy to hen's egg and meat contributes significantly to the manifestations of food allergy all over the world. Objectives: This study was performed to assess the presence of hen's egg and meat specific IgE antibodies among patients investigated for various allergic disorders. Methods. This is a retrospective ...

  11. Screening for hen's egg and chicken meat specific IgE antibodies in ...

    African Journals Online (AJOL)

    Abstract. Background: Allergy to hen's egg and meat contributes significantly to the manifestations of food allergy all over the world. Objectives: This study was performed to assess the presence of hen's egg and meat specific IgE antibodies among patients investigated for various allergic disorders. Methods. This is a ...

  12. Usefulness of the nonself-self algorithm of HLA epitope immunogenicity in the specificity analysis of monospecific antibodies induced during pregnancy

    Directory of Open Access Journals (Sweden)

    Rene J Duquesnoy

    2015-05-01

    Full Text Available Background HLAMatchmaker is a program to analyze the epitope specificities of HLA antibodies. It considers each HLA allele as a string of eplets. Intralocus and interlocus comparisons between donor and recipient alleles offer a structural assessment of compatibility and an analysis of allele panel reactivity patterns can generate information about epitope specificities of HLA antibodies. However, HLAMatchmaker cannot always generate conclusive interpretations of reactivity patterns of all monospecific antibodies which by definition recognize single epitopes. Hypothesis We have therefore developed a new antibody analysis approach that utilizes the nonself-self algorithm of HLA epitope immunogenicity. It is based in the concept that HLA antibodies originate from B-cells with immunoglobulin receptors to self HLA epitopes on one given allele and which can be activated by epitopes defined by a few nonself residue differences whereas the remainder of the structural epitope of the immunizing allele consists of self residues. Methods Three human monoclonal class I antibodies from HLA typed women sensitized during pregnancy were tested in Ig-binding assays with single alleles on a Luminex platformFindings Three new HLA epitopes were identified; they are defined by combinations of nonself and self residues for one allele of the antibody producer. Conclusion The nonself-self paradigm of HLA epitope immunogenicity offers a second approach to analyze HLA antibody specificities.

  13. Radioimmunoassay of class-specific antibodies to Streptococcus mutans in monkey serum and saliva

    International Nuclear Information System (INIS)

    Walker, J.; Colman, G.; Huges, M.

    1979-01-01

    A radioimmunoassay (RIA) has been developed to measure class-specific antibodies to Streptococcus mutans in the serum and saliva of monkeys (Macaca fascicularis). Antihuman immunoglobulin antibodies purified by affinity chromatography on immobilised monkey immoglobulins and labelled with 125 I were employed. Formalised cells of S. mutans and an extract of culture supernatant adsorbed to polystyrene wells were used as solid-phase antigens. The coefficients of variation of IgG, IgA, and IgM assays were less than or equal to 10% for both antigen systems. It is shown that this RIA is a sensitive, reproducible and quantitative method. (Auth.)

  14. Monoclonal antibody-dendrimer conjugates enable radiolabeling of antibody with markedly high specific activity with minimal loss of immunoreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, H.; Togashi, K. [Kyoto Univ. (Japan). School of Medicine; Sato, N.; Saga, T.; Nakamoto, Y.; Ishimori, T.; Konishi, J. [Dept. of Nuclear Medicine and Medical Imaging, Kyoto Univ. Graduate School of Medicine, Kyoto (Japan); Toyama, S. [Inst. for Virus Research, Kyoto Univ., Kyoto (Japan); Brechbiel, M.W. [Chemistry Section, Radiation Oncology Branch, National Cancer Inst., National Inst. of Health, Bethesda, Md. (United States)

    2000-09-01

    For the purpose of radioimmunotherapy, labelling of monoclonal antibody with high specific activity is often necessary, especially when using a radionuclide with a shorter half-life. Polyamine dendrimers (PAMAM) are novel synthetic polymeric molecules with large numbers of amine residues on their spherical surface. In order to bind large numbers of radiometals to single antibody molecules, the generation-4 PAMAM (G4), which has 64 amines, was conjugated with 43 molecules of 2-(p-isothiocyanatobenzyl)-6-methyl-diethylene triamine penta-acetic acid (1B4M), a derivative of DTPA. This product [G4-(1B4M){sub 43}] was then conjugated with OST7, a murine monoclonal IgG{sub 1}. We evaluated the achievable specific activity for {sup 111}In labeling, immunore-activity, biodistribution, and tumor targeting in mice of the {sup 111}In- or {sup 153}Gd-OST7-G4-(1B4M){sub 43} as compared with radiolabeled OST7-1B4M or 56C-1B4M. The maximum specific activity of {sup 111}In-OST7-G4-(1B4M){sub 43} and {sup 111}In-OST7-1B4M was 470 and 8.7 GBq/mg (12,700 and 263 mCi/mg), respectively. Immunoreactivity of radiolabeled OST7-G4-(1B4M){sub 43} and OST7-1B4M, as determined by the binding to KT005 cells expressing the antigen, was respectively 91% and 84% of that of {sup 125}I-labelled OST7. Biodistribution studies for preparations with maximum specific activity in normal mice 3 h after injection showed that {sup 111}In- or {sup 153}Gd-OST7-G4-(1B4M){sub 43} cleared faster from the blood and accumulated more in the liver than did {sup 111}In- or {sup 153}Gd-OST7-1B4M. The dendrimer 1B4M [G4-(1B4M){sub 64}] itself showed similar saturation effects with metals. The radioactivity in all the other organs reflected the rapid clearance of radioactivity from the blood. {sup 153}Gd-OST7-G4-(1B4M){sub 43} showed specific accumulation in the KT005 tumor. In conclusion, we could successfully bind 49 times as many metal atoms to an antibody molecule as is possible with conventional metal labeling for

  15. Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

    Science.gov (United States)

    Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

    2014-11-13

    To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

  16. Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

    International Nuclear Information System (INIS)

    Katus, H.A.; Hurrell, J.G.; Matsueda, G.R.; Ehrlich, P.; Zurawski, V.R. Jr.; Khaw, B.-A.; Haber, E.

    1982-01-01

    An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to differential between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (lC5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125 I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, lC5 and 2B9 were 25% cross reactive together, lC5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive. (author)

  17. Lip-Synching Using Speaker-Specific Articulation, Shape and Appearance Models

    Directory of Open Access Journals (Sweden)

    Gaspard Breton

    2009-01-01

    Full Text Available We describe here the control, shape and appearance models that are built using an original photogrammetric method to capture characteristics of speaker-specific facial articulation, anatomy, and texture. Two original contributions are put forward here: the trainable trajectory formation model that predicts articulatory trajectories of a talking face from phonetic input and the texture model that computes a texture for each 3D facial shape according to articulation. Using motion capture data from different speakers and module-specific evaluation procedures, we show here that this cloning system restores detailed idiosyncrasies and the global coherence of visible articulation. Results of a subjective evaluation of the global system with competing trajectory formation models are further presented and commented.

  18. Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains

    International Nuclear Information System (INIS)

    Leger, J.; Chevalier, J.; Larue, C.; Gautier, P.; Planchenault, J.; Aumaitre, E.; Messner, P.; Puech, P.; Saccavini, J.C.; Pau, B.

    1991-01-01

    The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited but significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis

  19. 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...

  20. Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.

    Directory of Open Access Journals (Sweden)

    Yan Gong

    Full Text Available β-Catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may

  1. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Science.gov (United States)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  2. Human antibody technology and the development of antibodies against cytomegalovirus.

    Science.gov (United States)

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Hormesis of specific IgG antibody to rabies virus in serum of mice irradiated with low dose γ-rays

    International Nuclear Information System (INIS)

    Liu Qingjie; Chen Deqing

    1998-01-01

    Objective: To explore the effect of low dose ionizing radiation on specific antibody in mouse serum. Methods: Kunming strain male mice, weighing 18-22 g, aged 6-8 weeks, were immunized intraperitoneally with rabies vaccine after exposure to cobalt-60 γ-rays. The specific IgG antibody against rabies virus in mouse serum was measured. Results: (1) The serum levels of specific IgG in mice irradiated with 5-30 cGy γ-rays were significantly elevated in comparison with those in control mice (P<0.01), the optimum stimulating dose being 10 cGy. (2) Exposure to 10 cGy caused significant enhancement and earlier emergence of the peak level of specific IgG in serum. (3) The hormesis of specific IgG to rabies virus induced by 10 cGy γ-rays could last one week at least. Conclusion: Low dose ionizing radiation can enhance the level of specific antibody in mouse serum, and this effect can last for one week at least

  4. 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

  5. Using an improved phagocytosis assay to evaluate the effect of HIV on specific antibodies to pregnancy-associated malaria.

    Science.gov (United States)

    Ataíde, Ricardo; Hasang, Wina; Wilson, Danny W; Beeson, James G; Mwapasa, Victor; Molyneux, Malcolm E; Meshnick, Steven R; Rogerson, Stephen J

    2010-05-25

    Pregnant women residing in malaria endemic areas are highly susceptible to Plasmodium falciparum malaria, particularly during their first pregnancy, resulting in low birth weight babies and maternal anaemia. This susceptibility is associated with placental sequestration of parasitised red blood cells expressing pregnancy-specific variant surface antigens. Acquisition of antibodies against these variant surface antigens may protect women and their offspring. Functions of such antibodies may include prevention of placental sequestration or opsonisation of parasitised cells for phagocytic clearance. Here we report the development and optimisation of a new high-throughput flow cytometry-based phagocytosis assay using undifferentiated Thp-1 cells to quantitate the amount of opsonizing antibody in patient sera, and apply this assay to measure the impact of HIV on the levels of antibodies to a pregnancy malaria-associated parasite line in a cohort of Malawian primigravid women. The assay showed high reproducibility, with inter-experimental correlation of r(2) = 0.99. In primigravid women, concurrent malaria infection was associated with significantly increased antibodies, whereas HIV decreased the ability to acquire opsonising antibodies (Mann-Whitney ranksum: p = 0.013). This decrease was correlated with HIV-induced immunosuppression, with women with less than 350 x 10(6) CD4+ T- cells/L having less opsonising antibodies (coef: -11.95,P = 0.002). Levels of antibodies were not associated with protection from low birth weight or anaemia. This flow cytometry-based phagocytosis assay proved to be efficient and accurate for the measurement of Fc-receptor mediated phagocytosis-inducing antibodies in large cohorts. HIV was found to affect mainly the acquisition of antibodies to pregnancy-specific malaria in primigravidae. Further studies of the relationship between opsonising antibodies to malaria in pregnancy and HIV are indicated.

  6. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.

    1987-01-01

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  7. CD44v10, osteopontin and lymphoma growth retardation by a CD44v10-specific antibody.

    Science.gov (United States)

    Megaptche, Amelie Pajip; Erb, Ulrike; Büchler, Markus Wolfgang; Zöller, Margot

    2014-09-01

    Blockade of CD44 is considered a therapeutic option for the elimination of leukemia-initiating cells. However, the application of anti-panCD44 can be burdened by severe side effects. We determined whether these side effects could be avoided by replacing anti-panCD44 with CD44 variant isoform (CD44v)-specific antibodies in CD44v-positive hematological malignancies using the EL4 thymoma and CD44v10-transfected EL4 (EL4-v10) as models. Subcutaneous growth of EL4 and EL4-v10 was equally well inhibited by the anti-panCD44 and anti-CD44v10 antibodies, respectively. Ex vivo analysis indicated that natural killer cytotoxicity and antibody-dependent cellular cytotoxicity were the main effector mechanisms. Under local inflammation, the efficacy of anti-CD44v10 prolonged the survival time twofold compared with untreated, EL4-v10 tumor-bearing mice, and this was due to inflammation-induced expression of osteopontin (OPN). A high level of OPN in EL4-v10 tumors supported leukocyte recruitment and tumor-infiltrating T-cell activation. Taken together, in hematological malignancies expressing CD44v, anti-panCD44 can be replaced by CD44v-specific antibodies without a loss in efficacy. Furthermore, CD44v10-specific antibodies appear particularly advantageous in cutaneous leukemia therapy, as CD44v10 binding of OPN drives leukocyte recruitment and activation.

  8. Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins

    DEFF Research Database (Denmark)

    Uttenthal, Åse; Larsen, Lars Erik; Philipsen, J.S.

    2000-01-01

    Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection. Most calves experienced an increase in the specific IgM and IgG1 titres about 6-10 days after infection with BRSV. The Ig......M titre was transient showing positive titres for only 5-10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection. In two closed herds, repeated blood......, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence...

  9. An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

    Science.gov (United States)

    Weir, C.; Vesey, G.; Slade, M.; Ferrari, B.; Veal, D. A.; Williams, K.

    2000-01-01

    The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies. PMID:10973448

  10. Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

    International Nuclear Information System (INIS)

    Kurachi, H.; Wakimoto, H.; Sakumoto, T.; Aono, T.; Kurachi, K.

    1984-01-01

    The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using 125 I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodies in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women

  11. Differential neutralizing activities of a single domain camelid antibody (VHH specific for ricin toxin's binding subunit (RTB.

    Directory of Open Access Journals (Sweden)

    Cristina Herrera

    Full Text Available Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody" specific for ricin's enzymatic (RTA and binding (RTB subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50 that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

  12. A novel antihuman C3d monoclonal antibody with specificity to the C3d complement split product

    DEFF Research Database (Denmark)

    Rasmussen, Karina Juhl; Skjødt, Mikkel-Ole; Vitved, Lars

    2017-01-01

    The complement component C3 and the cleavage products of C3b/iC3b, C3c and C3d are used as biomarkers in clinical diagnostics. Currently, no specific antibodies are able to differentiate C3d from other fragments, although such a distinction could be very valuable considering that they may reflect...... different pathophysiological mechanisms. We have developed a rat antihuman C3d monoclonal antibody with specificity to the end sequence of the N-terminal region of C3d. The antibody can therefore only bind to C3d when it manifests itself as the final end product of cleaved C3. We believe...

  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. Immunomodulatory activity of andrographolide on macrophage activation and specific antibody response

    Science.gov (United States)

    Wang, Wei; Wang, Jing; Dong, Sheng-fu; Liu, Chun-hong; Italiani, Paola; Sun, Shu-hui; Xu, Jing; Boraschi, Diana; Ma, Shi-ping; Qu, Di

    2010-01-01

    Aim: To investigate the immunomodulatory effects of andrographolide on both innate and adaptive immune responses. Methods: Andrographolide (10 μg/mL in vitro or 1 mg/kg in vivo) was used to modulate LPS-induced classical activated (M1) or IL-4-induced alternative activated (M2) macrophages in vitro and humor immune response to HBsAg in vivo. Cytokine gene expression profile (M1 vs M2) was measured by real-time PCR, IL-12/IL-10 level was detected by ELISA, and surface antigen expression was evaluated by flow cytometry, whereas phosphorylation level of ERK 1/2 and AKT was determined by Western blot. The level of anti-HBs antibodies in HBsAg immunized mice was detected by ELISA, and the number of HBsAg specific IL-4-producing splenocyte was enumerated by ELISPOT. Results: Andrographolide treatment in vitro attenuated either LPS or IL-4 induced macrophage activation, inhibited both M1 and M2 cytokines expression and decreased IL-12/IL-10 ratio (the ratio of M1/M2 polarization). Andrographolide down-regulated the expression of mannose receptor (CD206) in IL-4 induced macrophages and major histocompability complex/costimulatory molecules (MHC I, CD40, CD80, CD86) in LPS-induced macrophages. Correspondingly, anti-HBs antibody production and the number of IL-4-producing splenocytes were reduced by in vivo administration of andrographolide. Reduced phosphorylation levels of ERK1/2 and AKT were observed in macrophages treated with andrographolide. Conclusion: Andrographolide can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and Ag-specific antibody production. MAPK and PI3K signaling pathways may participate in the mechanisms of andrographolide regulating macrophage activation and polarization. PMID:20139902

  15. Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains.

    Science.gov (United States)

    Perols, Anna; Karlström, Amelie Eriksson

    2014-03-19

    Site-specific labeling of antibodies can be performed using the immunoglobulin-binding Z domain, derived from staphylococcal protein A (SpA), which has a well-characterized binding site in the Fc region of antibodies. By introducing a photoactivable probe in the Z domain, a covalent bond can be formed between the Z domain and the antibody by irradiation with UV light. The aim of this study was to improve the conjugation yield for labeling of different subclasses of IgG having different sequence composition, using a photoactivated Z domain variant. Four different variants of the Z domain (Z5BPA, Z5BBA, Z32BPA, and Z32BBA) were synthesized to investigate the influence of the position of the photoactivable probe and the presence of a flexible linker between the probe and the protein. For two of the variants, the photoreactive benzophenone group was introduced as part of an amino acid side chain by incorporation of the unnatural amino acid benzoylphenylalanine (BPA) during peptide synthesis. For the other two variants, the photoreactive benzophenone group was attached via a flexible linker by coupling of benzoylbenzoic acid (BBA) to the ε-amino group of a selectively deprotected lysine residue. Photoconjugation experiments using human IgG1, mouse IgG1, and mouse IgG2A demonstrated efficient conjugation for all antibodies. It was shown that differences in linker length had a large impact on the conjugation efficiency for labeling of mouse IgG1, whereas the positioning of the photoactivable probe in the sequence of the protein had a larger effect for mouse IgG2A. Conjugation to human IgG1 was only to a minor extent affected by position or linker length. For each subclass of antibody, the best variant tested using a standard conjugation protocol resulted in conjugation efficiencies of 41-66%, which corresponds to on average approximately one Z domain attached to each antibody. As a combination of the two best performing variants, Z5BBA and Z32BPA, a Z domain variant with

  16. Higher Plasma Concentration of Food-Specific Antibodies in Persons with Autistic Disorder in Comparison to Their Siblings

    Science.gov (United States)

    Trajkovski, Vladimir; Petlichkovski, Aleksandar; Efinska-Mladenovska, Olivija; Trajkov, Dejan; Arsov, Todor; Strezova, Ana; Ajdinski, Ljubomir; Spiroski, Mirko

    2008-01-01

    Specific IgA, IgG, and IgE antibodies to food antigens in 35 participants with autistic disorder and 21 of their siblings in the Republic of Macedonia were examined. Statistically significant higher plasma concentration of IgA antibodies against alpha-lactalbumin, beta-lactoglobulin, casein, and gliadin were found in the children with autistic…

  17. Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue

    Science.gov (United States)

    Korzeniowska-Kowal, Agnieszka; Kochman, Agata; Gamian, Elżbieta; Lis-Nawara, Anna; Lipiński, Tomasz; Seweryn, Ewa; Ziółkowski, Piotr; Gamian, Andrzej

    2015-01-01

    Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker

  18. Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

    Science.gov (United States)

    Sun, Yingli; Du, Fengxia

    2017-01-01

    The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

  19. Detection of specific IgA antibodies against a novel deamidated 8-Mer gliadin peptide in blood plasma samples from celiac patients.

    Directory of Open Access Journals (Sweden)

    Sara Vallejo-Diez

    Full Text Available We studied whether celiac disease (CD patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD, 17 CD patients on a gluten-free diet (GFD, 10 healthy controls (C and 23 patients with other gastrointestinal pathology (GP and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients. Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA anti-DGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD.

  20. Serotype Specificity of Antibodies against Foot-and-Mouth Disease Virus in Cattle in Selected Districts in Uganda

    DEFF Research Database (Denmark)

    Mwiine, F.N.; Ayebazibwe, C.; Olaho-Mukani, W.

    2010-01-01

    Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non-structural......Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non......-structural proteins and structural proteins. Three hundred and forty-nine cattle sera were collected from seven districts in Uganda, and 65% of these were found positive for antibodies against the non-structural proteins of FMDV. A subset of these samples were analysed for serotype specificity of the identified...... antibodies. High prevalences of antibodies against non-structural proteins and structural proteins of FMDV serotype O were demonstrated in herds with typical visible clinical signs of FMD, while prevalences were low in herds without clinical signs of FMD. Antibody titres were higher against serotype O than...

  1. Tumor-specific binding of radiolabeled G-22 monoclonal antibody in glioma patients

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Jun; Wakabayashi, Toshihiko; Mizuno, Masaaki; Sugita, Kenichiro; Oshima, Motoo; Tadokoro, Masanori; Sakuma, Sadayuki [Nagoya Univ. (Japan). Faculty of Medicine; Seo, Hisao

    1992-03-01

    Iodine-131-labeled G-22 monoclonal antibody F(ab'){sub 2} fragment reacting specifically with a glioma-associated surface glycoprotein was administered to 12 glioma patients to investigate its use in radioimaging of intracranial gliomas. No immediate or delayed side effects were attributable to antibody injection. Nine patients received the radiolabeled complex intravenously. The images of low-grade gliomas were generally poor and disappeared within 4 days. High-contrast images were obtained beyond the 7th day in high-grade gliomas except one case in the pineal region. Three patients received intraventricular or intratumoral administration. Clear images of all tumors were demonstrated from the 2nd until later than the 7th day. One patient with cerebrospinal fluid (CSF) dissemination of brainstem glioma demonstrated negative CSF cytology after intraventricular administration. (author).

  2. Summary of workshop findings for porcine T-lymphocyte-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Saalmuller, A.; Kuebart, G.; Hollemweguer, E.

    2001-01-01

    antibodies, 37 data sets were used for the clustering of the respective mAb. Using the LTDB4 program, 19 preliminary clusters could be defined. Two clusters (C3 and C7) with 4 mAb showed no labelling of resting T-lymphocytes. Seven clusters (CI, C2, C4, C5, C6, C11, and C12) contain mAb (in total: 16 mAb...... to recognise CD45R. Cluster C17 is composed of different standards directed against CD2, CD3, CD5 and wCD6. Two additional mAb recognising the CD2a-epitope could be enclosed. CIS contains two mAb directed against SWC2.......Fifty-seven monoclonal antibodies (mAb) selected after the first round analyses in the Third International Swine CD workshop for their possible reactivity with T-lymphocyte specific antigens were further analysed in a second round. As target cells for flow cytometric analyses served peripheral...

  3. Effect of maternal dry period length on colostrum immunoglobulin content and natural and specific antibody titers in calves

    NARCIS (Netherlands)

    Mayasari, N.; Vries Reilingh, de G.; Nieuwland, M.G.B.; Remmelink, G.J.; Parmentier, H.K.; Kemp, B.; Knegsel, van A.T.M.

    2015-01-01

    The objective was to study the effect of dry period length in dairy cows on immunoglobulin content and natural antibodies (NAb) titers in colostrum, growth, and plasma natural and specific antibody titers in plasma of calves. Holstein-Friesian dairy cows (n = 167) were randomly assigned to 3 dry

  4. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    Directory of Open Access Journals (Sweden)

    Michał Śmiga

    Full Text Available Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins and T. forsythia (Tfo protein and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

  5. Specific IgG and its subclass antibodies after immunotherapy with gynandropsis gynandra

    Directory of Open Access Journals (Sweden)

    Latha G

    2005-01-01

    Full Text Available Background : About 10 to 15 % of the Indian population is known to suffer from major allergic disorders such as Asthma, Rhinitis, Atopic Dermatitis and Urticaria. Aeroallergens play a major role in the pathogenesis of respiratory allergic diseases. Among the aeroallergens, pollens are major causative agents. The predominance of pollen allergens necessitate the need to assess the specific immunotherapy (SIT in allergic patients. Objective : To evaluate the effect of immunotherapy based on the presence of IgG and its subclass antibodies towards whole pollen antigen of Gynandropsis gynandra (G.gynandra and its fractions. Material and Methods : A study was conducted in 30 bronchial asthma patients on immunotherapy, by assessing the levels of IgG and its subclasses specific to G. gynandra pollen. Results : There was a significant increase in IgG and its subclass antibodies to whole pollen antigen and its fractions i.e.> 90kD, 46-37kD and 36-32kD after the course of IT. Conclusion : The use of peptide fractions may be more appropriate instead of the whole pollen antigen to test the effect of immunotherapy.

  6. Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

    Science.gov (United States)

    Lim, Theam Soon; Chan, Soo Khim

    2016-01-01

    Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    International Nuclear Information System (INIS)

    Guo Junqing; Chen Liu; Ai Hongwu; Jing Jiannian; Zhou Jiyong; Zhang Chuyu; You Shangyou

    2004-01-01

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-γ inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V H region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo

  8. Evaluation of a direct immunofluorescent antibody (difma test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Martha E. Chico

    1995-06-01

    Full Text Available A direct immunofluorescent antibody (DIFMA test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

  9. Specific IgE antibodies to vespids in the course of immunotherapy with Vespula germanica administered to patients sensitized to Polistes dominulus.

    Science.gov (United States)

    Juarez, C; Blanca, M; Miranda, A; Sanchez, F; Carmona, M J; Avila, M J; Fernandez, S; Fernandez, J; Terrados, S

    1992-08-01

    Sera from a group of 12 patients with anaphylactic reactions to vespids were studied. Field observations and RAST values suggested that the offending insect was Polistes dominulus (PD). Specific IgE antibodies to PD appeared in all cases and to Vespula germanica (VG) in nine. Absorption studies in these basal sera showed that IgE antibodies to VG were due to cross-reactivity with PD. The RAST value to both venoms was higher after immunotherapy (IT) in six cases. IgE antibodies increased to determinants common to both vespids, and in 41% of the cases to specific epitopes of VG venom allergens not initially detected in the basal sera. In one case antibodies increased only to VG without a corresponding rise to PD. These results indicate that if the correct venom to which the individuals are sensitized is not administered IgE antibodies may appear which were not initially detected in the patients' sera. The levels of these antibodies declined during the course of IT.

  10. Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets.

    Science.gov (United States)

    McCutcheon, Krista M; Gray, Julia; Chen, Natalie Y; Liu, Keyi; Park, Minha; Ellsworth, Stote; Tripp, Ralph A; Tompkins, S Mark; Johnson, Scott K; Samet, Shelly; Pereira, Lenore; Kauvar, Lawrence M

    2014-01-01

    Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

  11. Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses.

    Directory of Open Access Journals (Sweden)

    Stefan W Metz

    2016-10-01

    Full Text Available Dengue virus (DENV is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE, purified and adsorbed to poly (lactic-co-glycolic acid (PLGA nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.

  12. Analysis of Individuals from a Dengue-Endemic Region Helps Define the Footprint and Repertoire of Antibodies Targeting Dengue Virus 3 Type-Specific Epitopes.

    Science.gov (United States)

    Andrade, Daniela V; Katzelnick, Leah C; Widman, Doug G; Balmaseda, Angel; de Silva, Aravinda M; Baric, Ralph S; Harris, Eva

    2017-09-19

    The four dengue virus serotypes (DENV1 to 4) cause dengue, a major public health problem worldwide. Individuals exposed to primary DENV infections develop serotype-specific neutralizing antibodies, including strongly neutralizing antibodies targeting quaternary epitopes. To date, no studies have measured the levels and kinetics of serum antibodies directed to such epitopes among populations in regions where dengue is endemic. Here, we use a recombinant DENV4 (rDENV4/3-M14) displaying a major DENV3 type-specific quaternary epitope recognized by human monoclonal antibody 5J7 to measure the proportion, magnitude, and kinetics of DENV3 type-specific neutralizing antibody responses targeting this epitope. Primary DENV3 sera from 30 individuals in a dengue hospital-based study in Nicaragua were studied 3, 6, 12, and 18 months post-infection, alongside samples collected annually 1 to 4 years post-primary DENV3 infection from 10 individuals in a cohort study in Nicaragua. We found substantial individual variation in the proportion of DENV3 type-specific neutralizing antibody titers attributed to the 5J7 epitope (range, 0 to 100%), with the mean significantly increasing from 22.6% to 41.4% from 3 to 18 months. We extended the transplanted DENV3 5J7 epitope on the virion (rDENV4/3-M16), resulting in increased recognition in several individuals, helping define the footprint of the epitope. However, 37% and 13% of the subjects still showed little to no recognition of the 5J7 epitope at 3 and 18 months, respectively, indicating that one or more additional DENV3 type-specific epitopes exist. Overall, this study demonstrates how DENV-immune plasma from populations from areas of endemicity, when coupled with structurally guided recombinant viruses, can help characterize the epitope-specific neutralizing antibody response in natural DENV infections, with direct implications for design and evaluation of dengue vaccines. IMPORTANCE The four serotypes of dengue virus cause dengue

  13. Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

    Science.gov (United States)

    Dorvignit, Denise; García-Martínez, Liliana; Rossin, Aurélie; Sosa, Katya; Viera, Justo; Hernández, Tays; Mateo, Cristina; Hueber, Anne-Odile; Mesa, Circe; López-Requena, Alejandro

    2015-12-01

    Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors. Copyright © 2015 Elsevier GmbH. All rights reserved.

  14. Tissue-specific and pathogen-inducible expression of a fusion protein containing a Fusarium-specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins.

    Science.gov (United States)

    Cheng, Wei; Li, He-Ping; Zhang, Jing-Bo; Du, Hong-Jie; Wei, Qi-Yong; Huang, Tao; Yang, Peng; Kong, Xian-Wei; Liao, Yu-Cai

    2015-06-01

    Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.-specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea-specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea-specific in transgenic wheat. Single-floret inoculation of independent transgenic wheat lines of the T3 to T6 generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography-mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB-susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real-time PCR analysis revealed that the tissue-specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue-specific and pathogen-inducible expression of this Fusarium-specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

    Science.gov (United States)

    Rossano, M G; Mansfield, L S; Kaneene, J B; Murphy, A J; Brown, C M; Schott, H C; Fox, J C

    2000-01-01

    Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (Pblot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

  16. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  17. 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.

  18. 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

  19. The in vivo fate of a 211At labelled monoclonal antibody with known specificity in a murine system

    International Nuclear Information System (INIS)

    Vaughan, A.T.M.; Bateman, W.J.; Fisher, D.R.

    1982-01-01

    A monoclonal antibody reactive against the human transferrin receptor has been labelled with the alpha and X ray emitting isotope Astatine 211. The labelling procedure does not affect the ability of the product to bind to the transferrin receptor on the human leukemic cell line HL60. Using a direct binding assay, 211 At labelled antibody can be specifically inhibited from binding to its target cells by excess unlabelled antibody. Furthermore, the binding inhibition demonstrated in this system correlates to enhanced clonogenic survival of these cells, indicating that very few atoms of 211 At/cell are required for cell death. Data obtained from labelled antibody injected into mice show that the labelled product in serum retains the ability to bind to HL60 cells in vitro, although tissue distributions of the injected activity implies that some of the radiolabel is lost from the protein. Despite this loss of label, preliminary experiments on the localization of labelled antibody to HL60 cells growing s/c in nude mice show that tumor tissue has a higher specific activity than all other tissues, other than blood, after 12 hours. This suggests that further work on the nature of label degradation in vivo is warranted in the context of potential therapeutic and diagnostic studies

  20. ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-06-01

    Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

  1. 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

  2. Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

    Science.gov (United States)

    Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

    2010-08-01

    Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

  3. Type-specific IgG and IgA antibodies in old lymphogranuloma venerum determined by solid-phase radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Meurman, O.; Terho, P.; Sonck, C.E.

    1981-01-01

    A solid-phase radioimmunoassay (RIA) using egg-grown purified Chlamydia trachomatis lymphogranuloma venereum (LGV) serotypes L1, L2, and L3 as antigen was used to measure type-specific IgG and IgA antibodies in sera of 36 patients who had contracted LGV infection about 40 years ago. The RIA test gave compatible results with the standard microimmunofluorescence test, and by RIA it was possible to identify the infecting serotype in 30 out of 36 patients studied. In 28 cases this was L2 and in two cases L1. Each patient had IgG antibodies and most of them (80%) IgA antibodies to at least one of the LGV serotypes. The antibody titers were still high 40 years after the acute infection, being higher than in male patients with a recent chlamydial urethritis. Highest antibody titers were detected in LGV patients who had a severe disease with intestinal involvement.

  4. Type-specific IgG and IgA antibodies in old lymphogranuloma venerum determined by solid-phase radioimmunoassay

    International Nuclear Information System (INIS)

    Meurman, O.; Terho, P.; Sonck, C.E.

    1981-01-01

    A solid-phase radioimmunoassay (RIA) using egg-grown purified Chlamydia trachomatis lymphogranuloma venereum (LGV) serotypes L1, L2, and L3 as antigen was used to measure type-specific IgG and IgA antibodies in sera of 36 patients who had contracted LGV infection about 40 years ago. The RIA test gave compatible results with the standard microimmunofluorescence test, and by RIA it was possible to identify the infecting serotype in 30 out of 36 patients studied. In 28 cases this was L2 and in two cases L1. Each patient had IgG antibodies and most of them (80%) IgA antibodies to at least one of the LGV serotypes. The antibody titers were still high 40 years after the acute infection, being higher than in male patients with a recent chlamydial urethritis. Highest antibody titers were detected in LGV patients who had a severe disease with intestinal involvement. (orig.)

  5. NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

    Directory of Open Access Journals (Sweden)

    Saeko Yanaka

    2017-09-01

    Full Text Available Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

  6. HIV-1 specific antibody titers and neutralization among chronically infected patients on long-term suppressive antiretroviral therapy (ART: a cross-sectional study.

    Directory of Open Access Journals (Sweden)

    Johannes S Gach

    Full Text Available The majority of potent and broadly neutralizing antibodies against HIV-1 have been isolated from untreated patients with acute or chronic infection. To assess the extent of HIV-1 specific antibody response and neutralization after many years of virologic suppression from potent combination ART, we examined antibody binding titers and neutralization of 51 patients with chronic HIV-1 infection on suppressive ART for at least three years. In this cross-sectional analysis, we found high antibody titers against gp120, gp41, and the membrane proximal external region (MPER in 59%, 43%, and 27% of patients, respectively. We observed significantly higher endpoint binding titers for gp120 and gp41 for patients with >10 compared to ≤ 10 years of detectable HIV RNA. Additionally, we observed higher median gp120 and gp41 antibody titers in patients with HIV RNA 10 years of detectable HIV RNA (8/20 [40.0%] versus 3/31 [9.7%] for ≤ 10 years, p = 0.02 and a trend toward greater neutralization in patients with ≤ 5 years of HIV RNA 5 years, p = 0.08. All patients with neutralizing activity mediated successful phagocytosis of VLPs by THP-1 cells after antibody opsonization. Our findings of highly specific antibodies to several structural epitopes of HIV-1 with antibody effector functions and neutralizing activity after long-term suppressive ART, suggest continuous antigenic stimulation and evolution of HIV-specific antibody response occurs before and after suppression with ART. These patients, particularly those with slower HIV progression and more time with detectable viremia prior to initiation of suppressive ART, are a promising population to identify and further study functional antibodies against HIV-1.

  7. Characterization of specific antibodies against cytomegalovirus (CMV)-encoded interleukin 10 produced by 28 % of CMV-seropositive blood donors

    DEFF Research Database (Denmark)

    de Lemos Rieper, Carina; Galle, Pia Søndergaard; Pedersen, Bente Klarlund

    2011-01-01

    -10 nor with Epstein-Barr virus-encoded IL-10. Anti-cmvIL-10 antibodies potently inhibited the binding of cmvIL-10 to cellular receptors, and they specifically inhibited cmvIL-10-induced JAK-STAT signalling. Ultimately, anti-cmvIL-10 antibodies blocked the inhibitory effect of cmvIL-10...... percent of plasma samples from 3200 Danish blood donors (corresponding to 28¿% of the anti-CMV IgG-positive donors) contained substantial levels of anti-cmvIL-10 IgG antibodies, as measured by a radioimmunoassay for human anti-cmvIL-10 antibodies. The antibodies neither cross-reacted with native human IL...... on lipopolysaccharide-induced tumour necrosis factor alpha and IL-1ß from blood mononuclear cells. Taken together, our data signify that cmvIL-10 has been produced during CMV infection, and that anti-cmvIL-10 IgG antibodies represent an effective immunological counter reaction against cmvIL-10....

  8. Acrylic microspheres in vivo. X. Elimination of circulating cells by active targeting using specific monoclonal antibodies bound to microparticles

    International Nuclear Information System (INIS)

    Laakso, T.; Andersson, J.; Artursson, P.; Edman, P.; Sjoeholm, I.

    1986-01-01

    The elimination from the blood of 51 Cr-labelled mouse erythrocytes modified with trinitrophenyl (TNP) groups was followed in mice. After 24 hours, when a stable concentration of the labelled erythrocytes has been attained, monoclonal anti-TNP-antibodies were given intravenously, either in free, soluble form, or bound to microparticles containing immobilized protein A. The anti-TNP-antibodies induced a rapid elimination of the TNP- and 51 Cr-labelled erythrocytes. Over the 8-hours time period studied, the elimination rate was significantly faster when the antibodies were administered bound to the particles. After the elimination of the target cells, the radioactivity was found in the liver, spleen and bone marrow. These results and relevant control experiments indicate that a solid carrier (1) can be directed to a specific target cell with a specific antibody and (2) can induce a rapid elimination of the target cell from the circulation. 31 references, 1 figure, 2 tables

  9. 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.

  10. Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies

    Directory of Open Access Journals (Sweden)

    Tristan Legris

    2016-08-01

    Full Text Available Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs. The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK cells as innate immune effectors of antibody-dependent cellular cytotoxicity, but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years. Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate (eGFR over a 1-year period (hazard ratio: 2.83. In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration

  11. Anti-idiotypic antibody specific to GAD65 autoantibody prevents type 1 diabetes in the NOD mouse.

    Directory of Open Access Journals (Sweden)

    Xin Wang

    Full Text Available Overt autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab are a characteristic in patients with Type 1 diabetes (T1D. Anti-idiotypic antibodies (anti-Id directed to GAD65Ab effectively prevent the binding of GAD65 to GAD65Ab in healthy individuals. Levels of GAD65Ab-specific anti-Id are significantly lower in patients with T1D, leading to overt GAD65Ab in these patients. To determine the possible protective role of GAD65Ab-specific anti-Id in T1D pathogenesis, we developed the monoclonal anti-Id MAb 8E6G4 specifically targeting human monoclonal GAD65Ab b96.11. MAb 8E6G4 was demonstrated as a specific anti-Id directed to the antigen binding site of b96.11. MAb 8E6G4 recognized human antibodies in sera from healthy individuals, T2D patients, and T1D patients as established by ELISA. We confirmed these MAb 8E6G4-bound human antibodies to contain GAD65Ab by testing the eluted antibodies for binding to GAD65 in radioligand binding assays. These findings confirm that GAD65Ab are present in sera of individuals, who test GAD65Ab-negative in conventional detection assays. To test our hypothesis that GAD65Ab-specific anti-Id have an immune modulatory role in T1D, we injected young Non Obese Diabetic (NOD mice with MAb 8E6G4. The animals were carefully monitored for development of T1D for 40 weeks. Infiltration of pancreatic islets by mononuclear cells (insulitis was determined to establish the extent of an autoimmune attack on the pancreatic islets. Administration of MAb 8E6G4 significantly reduced the cumulative incidence rate of T1D and delayed the time of onset. Insulitis was significantly less severe in animals that received MAb 8E6G4 as compared to control animals. These results support our hypothesis that anti-Id specific to GAD65Ab have a protective role in T1D.

  12. Construction of a humanized antibody to hepatitis B surface antigen by specificity-determining residues (SDR)-grafting and de-immunization.

    Science.gov (United States)

    Kim, Keun-Soo; Kim, Hyun-Jung; Han, Byung Woo; Myung, Pyung-Keun; Hong, Hyo Jeong

    2010-05-28

    We previously constructed a humanized antibody, HuS10, by grafting the complementarity-determining regions (CDRs) of a parental murine monoclonal antibody into the homologous human antibody sequences. This process is termed CDR grafting. Some residues that were thought to affect the CDR loops and stabilize the structure of the variable regions were retained in the framework region. HuS10 exhibited in vivo virus-neutralizing activity, but its murine content had the potential to elicit immune responses in patients. In this study, to minimize the immunogenic potential of HuS10, we replaced 17 mouse residues in HuS10 with the comparable human residues using specificity-determining residue (SDR)-grafting and de-immunization methods. The resultant humanized antibody, HzS-III, had the same affinity and epitope specificity as HuS10 and had reduced immunogenic potential, as assessed by T-cell epitope analysis. Thus, SDR grafting in combination with de-immunization may be a useful strategy for minimizing the immunogenicity of humanized antibodies. In addition, HzS-III may be a good candidate for immunoprophylaxis of HBV infection. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  13. 2012 annual literature review of donor-specific HLA antibodies after organ transplantation.

    Science.gov (United States)

    Kaneku, Hugo

    2012-01-01

    From the articles reviewed in the present chapter, we observed: 1. The frequency of de novo donor-specific human leukocyte antigen (HLA) antibodies (DSA) detection in different organs is very similar: ranging between 15% and 23% in kidney, 23% in pancreas, and 18% in intestinal transplant patients. Apparently, all organs can elicit humoral responses after transplantation at comparable rates. 2. Although rates of de novo DSA formation after kidney transplantation are very similar across different centers--between 15% and 23%--, the mean time to the first detection of de novo DSA is markedly variable between centers (from 8 months to 4 years). Some differences found in the studies that may account for this could be the age of patients (studies including pediatric patients tend to show longer time to DSA detection compared to studies only including adults patients), patients' race, and maintenance immunosuppression regimens. 3. In most organs, alloantibodies against class II HLA--and especially against HLA-DQ antigens--are the most common DSA detected. This finding supports previous studies, but the explanation remains unclear. Poor HLA-DQ matching, paucity of class II HLA antigen expression on cell surface, and technical factors related to the detection of these antibodies (mean fluorescence intensity cutoff, multiple beads with the same antigen, denatured protein on single antigen beads) are some of the potential explanations that need further investigation. 4. Recent focus on histological changes during rejection in the presence of DSA that are independent of C4d deposition may change how antibody-mediated rejection is diagnosed in the near future. 5. More studies are looking into the importance of DSA in non-kidney transplants and now evidence shows that DSA may not only affect survival and rejection rates, but may also be associated with organ-specific lesions like fibrosis and biliary complications in livers or capillaritis in lungs.

  14. 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.

  15. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    Energy Technology Data Exchange (ETDEWEB)

    Junqing, Guo; Liu, Chen; Hongwu, Ai; Jiannian, Jing; Jiyong, Zhou; Chuyu, Zhang; Shangyou, You

    2004-07-23

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-{gamma} inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V{sub H} region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.

  16. [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.

  17. Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  18. Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms.

    Science.gov (United States)

    Roncolato, Eduardo Crosara; Pucca, Manuela Berto; Funayama, Jaqueline Carlos; Bertolini, Thaís Barboza; Campos, Lucas Benício; Barbosa, José Elpidio

    2013-01-01

    Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA₂) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.

  19. Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

    Science.gov (United States)

    Um, Jihye; Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun; Park, Jun-Sun; Kim, Su Yeon

    2017-12-01

    Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, prabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

  20. 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

    Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein, is the only prophylactic agent approved for use for the treatment of RSV infection. However, its relatively low neutralizing potency and high cost have limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high-potency neutralizing antibody, 5C4, that specifically recognizes antigenic site Ø at the apex of the pre-F protein trimer. We compared in vitro and in vivo the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 had similar pre-F protein binding affinities, the 5C4 neutralizing activity was nearly 50-fold greater than that of 1129 in vitro In BALB/c mice, 5C4 reduced the peak titers of RSV 1,000-fold more than 1129 did in both the upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site Ø are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site Ø-specific antibodies may be useful for the prevention or treatment of RSV infection and support the use of the pre-F protein as a vaccine antigen. IMPORTANCE There is no vaccine yet available to prevent RSV infection. The use of the licensed antibody palivizumab, which recognizes site II on both the pre-F and post-F proteins, is restricted to prophylaxis in neonates at high risk of severe RSV disease. Recommendations for using passive immunization in the general population or for therapy in immunocompromised persons with

  1. Broadly reactive antibodies specific for Plasmodium falciparum MSP-119 are associated with the protection of naturally exposed children against infection

    Directory of Open Access Journals (Sweden)

    Dent Arlene E

    2012-08-01

    Full Text Available Abstract Background The 19 kDa C-terminal region of Plasmodium falciparum Merozoite Surface Protein-1 is a known target of naturally acquired humoral immunity and a malaria vaccine candidate. MSP-119 has four predominant haplotypes resulting in amino acid changes labelled EKNG, QKNG, QTSR and ETSR. IgG antibodies directed against all four variants have been detected, but it is not known if these variant specific antibodies are associated with haplotype-specific protection from infection. Methods Blood samples from 201 healthy Kenyan adults and children who participated in a 12-week treatment time-to-infection study were evaluated. Venous blood drawn at baseline (week 0 was examined for functional and serologic antibodies to MSP-119 and MSP-142 variants. MSP-119 haplotypes were detected by a multiplex PCR assay at baseline and weekly throughout the study. Generalized linear models controlling for age, baseline MSP-119 haplotype and parasite density were used to determine the relationship between infecting P. falciparum MSP-119 haplotype and variant-specific antibodies. Results A total of 964 infections resulting in 1,533 MSP-119 haplotypes detected were examined. The most common haplotypes were EKNG and QKNG, followed by ETSR and QTSR. Children had higher parasite densities, greater complexity of infection (>1 haplotype, and more frequent changes in haplotypes over time compared to adults. Infecting MSP-119 haplotype at baseline (week 0 had no influence on haplotypes detected over the subsequent 11 weeks among children or adults. Children but not adults with MSP-119 and some MSP-142 variant antibodies detected by serology at baseline had delayed time-to-infection. There was no significant association of variant-specific serology or functional antibodies at baseline with infecting haplotype at baseline or during 11 weeks of follow up among children or adults. Conclusions Variant transcending IgG antibodies to MSP-119 are associated with protection

  2. Directional Selection for Specific Sheep Cell Antibody Responses Affects Natural Rabbit Agglutinins of Chickens

    NARCIS (Netherlands)

    Cotter, P.F.; Ayoub, J.; Parmentier, H.K.

    2005-01-01

    Agglutination data from generations 8 through 19 indicate that bidirectional selection for specific SRBC antibody responses was successful in a line cross of ISA × Warren medium heavy layers. After 11 generations titers of the high SRBC selected line (H line) were nearly 1:32,000; those of the low

  3. [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

  4. Impact of donor-specific HLA antibodies in transplantation, a review of the literature published in the last three years.

    Science.gov (United States)

    Kaneku, Hugo

    2010-01-01

    This chapter summarizes some of the recent findings published on the role in organ transplantation of HLA antibodies, and--more important--donor-specific HLA antibodies. The negative impact of both, preformed and de novo DSA is now better recognized in recipients of kidney, heart, lung, liver, pancreas, islet cells and bone marrow transplants. An appropriate design of a schedule to monitor HLA antibodies may identify patients at higher risk for immunological events earlier and allow interventions to avoid later graft loss. The value of strategies like preemptive treatment of antibodies and the use of new agents like bortezomib and eculizumab are of interest and need further investigation.

  5. Gender differences in colour naming performance for gender specific body shape images.

    Science.gov (United States)

    Elliman, N A; Green, M W; Wan, W K

    1998-03-01

    Males are increasingly subjected to pressures to conform to aesthetic body stereotypes. There is, however, comparatively little published research on the aetiology of male body shape concerns. Two experiments are presented, which investigate the relationship between gender specific body shape concerns and colour-naming performance. Each study comprised a between subject design, in which each subject was tested on a single occasion. A pictorial version of a modified Stroop task was used in both studies. Subjects colour-named gender specific obese and thin body shape images and semantically homogeneous neutral images (birds) presented in a blocked format. The first experiment investigated female subjects (N = 68) and the second investigated males (N = 56). Subjects also completed a self-report measure of eating behaviour. Currently dieting female subjects exhibited significant colour-naming differences between obese and neutral images. A similar pattern of colour-naming performance was found to be related to external eating in the male subjects.

  6. 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.

  7. Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

    NARCIS (Netherlands)

    Burgess, Janette K.; Lopez, Jose A.; Gaudry, Leonie E.; Chong, Beng H.

    2000-01-01

    The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because

  8. 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.

  9. High-affinity monoclonal antibodies specific for deoxynucleosides structurally modified by alkylating agents: Applications for immunoanalysis

    International Nuclear Information System (INIS)

    Adamkiewicz, J.; Ahrens, O.; Rajewsky, M.F.

    1984-01-01

    So far the results of attempts to use monoclonal antibodies for the demonstration of carcinogen-DNA adducts in cells by immunostaining have been promising. Thus the authors have established a standardized procedure for the quantitation of specific alkyl-deoxynucleosides in the nuclear DNA of individual cells by direct immunofluorescence, using tetramethylrhodamine isothiocyanate-labeled monoclonal antibodies and a computer-based image analysis of electronically intensified fluorescence signals. With a fluorescent anti-(O/sup 6/-EtdGuo) monoclonal antibody, the present detection limit for O/sup 6/-Etd-Guo in the nuclei of individual cells previously exposed to an ethylating N-nitroso compound (e.g., N-ethyl-N-nitrosourea) is -- 700 O/sup 6/-EtdGuo molecules per diploid genome, i.e., similar to the detection limit for the same ethylation product in a hydrolysate of (O/sup 6/-EtdGuo)-containing DNA analyzed by competitive RIA

  10. Characterization of novel peptide-specific antibodies against the translation elongation factor eEF1A2 and their application for cancer research

    Directory of Open Access Journals (Sweden)

    Shalak V. F.

    2014-11-01

    Full Text Available Aim. We intend to characterize the new peptide-specific antibodies against the isoform 2 of translation elongation factor 1A (eEF1A2 and determine its presence in the postoperative samples of human breast, lung and stomach tumor tissues. Methods. The analysis of antibody specificity was performed by enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry were used for the determination of the eEF1A2 in the human tumor samples, as well as in the samples of normal tissues surrounding tumors. Results. The antibodies obtained against the eEF1A2 specifically recognized this protein in the cell extracts and histological sections and did not cross-react with the elongation factor 1A isoform 1. eEF1A2 was revealed in the postoperative samples of breast, lung and stomach tumors as well as in the putative normal tissues surrounding tumors. Conclusions. The antibodies obtained against eEF1A2 are highly specific for the antigen and can be used for the immunological studies of tumors.

  11. Lack of gender-specific antibody recognition of products from domains of a var gene implicated in pregnancy-associated Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Jensen, Anja T R; Zornig, Hanne D; Buhmann, Caecilie

    2003-01-01

    Gender-specific and parity-dependent acquired antibody recognition is characteristic of variant surface antigens (VSA) expressed by chondroitin sulfate A (CSA)-adherent Plasmodium falciparum involved in pregnancy-associated malaria (PAM). However, antibody recognition of recombinant products...

  12. 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.

  13. 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.

  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. High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis.

    Science.gov (United States)

    Weiss, Eric R; Alter, Galit; Ogembo, Javier Gordon; Henderson, Jennifer L; Tabak, Barbara; Bakiş, Yasin; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Luzuriaga, Katherine

    2017-01-01

    The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a

  16. Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Abe, Shinji; Nishioka, Yasuhiko; Kunita, Akiko; Fukayama, Masashi; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C-type lectin-like receptor 2 (CLEC-2). The overexpression of hPDPN is involved in invasion and metastasis. Anti-hPDPN monoclonal antibodies (mAbs) such as NZ-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-2, using the cancer-specific mAb (CasMab) technology. In this study we developed chLpMab-2, a human-mouse chimeric anti-hPDPN antibody, derived from LpMab-2. chLpMab-2 was produced using fucosyltransferase 8-knockout (KO) Chinese hamster ovary (CHO)-S cell lines. By flow cytometry, chLpMab-2 reacted with hPDPN-expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-2 exhibited high antibody-dependent cellular cytotoxicity (ADCC) against PDPN-expressing cells, despite its low complement-dependent cytotoxicity. Furthermore, treatment with chLpMab-2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab-2 suppressed tumor development via ADCC. In conclusion, chLpMab-2 could be useful as a novel antibody-based therapy against hPDPN-expressing tumors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  17. Impaired Epstein-Barr Virus-Specific Neutralizing Antibody Response during Acute Infectious Mononucleosis Is Coincident with Global B-Cell Dysfunction.

    Science.gov (United States)

    Panikkar, Archana; Smith, Corey; Hislop, Andrew; Tellam, Nick; Dasari, Vijayendra; Hogquist, Kristin A; Wykes, Michelle; Moss, Denis J; Rickinson, Alan; Balfour, Henry H; Khanna, Rajiv

    2015-09-01

    Here we present evidence for previously unappreciated B-cell immune dysregulation during acute Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBV-specific neutralizing antibodies and gp350-specific B-cell responses, which were associated with a significant reduction in memory B cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumor necrosis factor family members BAFF and APRIL and increased expression of FAS on circulating B cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

    Science.gov (United States)

    Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

    2017-01-01

    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

  19. A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anke K Trilling

    Full Text Available BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA tests and soluble antigen by surface plasmon resonance (SPR analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp. The highest affinity VHH had a dissociation constant (KD of 4 × 10(-10 M. CONCLUSIONS/SIGNIFICANCE: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

  20. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

  1. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    Directory of Open Access Journals (Sweden)

    Edyta Petters

    2015-08-01

    Full Text Available Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer.

  2. Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

    DEFF Research Database (Denmark)

    Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette

    2003-01-01

    During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...... epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding...... was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide...

  3. Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors.

    Science.gov (United States)

    Taniguchi, K; Yoshihara, S; Maruya, E; Ikegame, K; Kaida, K; Hayashi, K; Kato, R; Inoue, T; Fujioka, T; Tamaki, H; Okada, M; Onuma, T; Fujii, N; Kusunoki, Y; Soma, T; Saji, H; Ogawa, H

    2012-10-01

    Pre-existing donor-specific HLA antibodies in patients undergoing HLA-mismatched SCT have increasingly been recognized as a risk factor for primary graft failure. However, the clinical implications of the presence of HLA antibodies in donors remain unknown. We prospectively examined 123 related donors for the presence of HLA antibodies by using a Luminex-based single antigen assay. Of these, 1/57 (1.8%) male, 6/27 (22%) parous female and 0/39 (0%) nonparous female donors were HLA antibody-positive. Then, we determined the presence of HLA antibodies in seven patients who received SCT from antibody-positive donors. Of these, four became HLA antibody-positive after SCT. The specificities of the antibodies that emerged in the patients closely resembled those of the antibodies found in the donors, indicating their production by donor-derived plasma cells. Moreover, the kinetics of the HLA antibody levels were similar in all four patients: levels started increasing within 1 week after SCT and peaked at days 10-21, followed by a gradual decrease. These results suggest that donor-derived HLA antibody production frequently occurs in patients undergoing SCT from antibody-positive donors. Further studies are warranted for clarifying the clinical significance of donor-derived HLA antibodies, including the role of these antibodies in post transplant platelet transfusion refractoriness.

  4. Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis

    Directory of Open Access Journals (Sweden)

    Swetha Tati

    2017-09-01

    Full Text Available JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11. Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1 suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

  5. 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.

  6. Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies

    NARCIS (Netherlands)

    J. Groen (Jan); P. Koraka (Penelope); J. Velzing (Jans); C. Copra (Cederick); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractThe performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid

  7. Simple and efficient generation of virus-specific T cells for adoptive therapy using anti-4-1BB antibody.

    Science.gov (United States)

    Imahashi, Nobuhiko; Nishida, Tetsuya; Goto, Tatsunori; Terakura, Seitaro; Watanabe, Keisuke; Hanajiri, Ryo; Sakemura, Reona; Imai, Misa; Kiyoi, Hitoshi; Naoe, Tomoki; Murata, Makoto

    2015-01-01

    Although recent studies of virus-specific T-cell (VST) therapy for viral infections after allogeneic hematopoietic stem cell transplantation have shown promising results, simple and less time-intensive and labor-intensive methods are required to generate VSTs for the wider application of VST therapy. We investigated the efficacy of anti-CD28 and anti-4-1BB antibodies, which can provide T cells with costimulatory signals similar in strength to those of antigen-presenting cells, in generating VSTs. When peripheral blood mononuclear cells were stimulated with viral peptides together with isotype control, anti-CD28, or anti-4-1BB antibodies, anti-4-1BB antibodies yielded the highest numbers of VSTs, which were on an average 7.9 times higher than those generated with isotype control antibody. The combination of anti-CD28 and anti-4-1BB antibodies did not result in increased numbers of VSTs compared with anti-4-1BB antibody alone. Importantly, the positive effect of anti-4-1BB antibody was observed regardless of the epitopes of the VSTs. In contrast, the capacity of dendritic cells (DCs) to generate VSTs differed considerably depending on the epitopes of the VSTs. Furthermore, the numbers of VSTs generated with DCs were at most similar to those generated with the anti-4-1BB antibody. Generation of VSTs with anti-4-1BB antibody did not result in excessive differentiation or deteriorated function of the generated VSTs compared with those generated with control antibody or DCs. In conclusion, VSTs can be generated rapidly and efficiently by simply stimulating peripheral blood mononuclear cells with viral peptide and anti-4-1BB antibody without using antigen-presenting cells. We propose using anti-4-1BB antibody as a novel strategy to generate VSTs for adoptive therapy.

  8. Rotavirus specific maternal antibodies and immune response to RV3-BB neonatal rotavirus vaccine in New Zealand

    OpenAIRE

    Chen, Mee-Yew; Kirkwood, Carl D.; Bines, Julie; Cowley, Daniel; Pavlic, Daniel; Lee, Katherine J.; Orsini, Francesca; Watts, Emma; Barnes, Graeme; Danchin, Margaret

    2017-01-01

    Background: Maternal antibodies, acquired passively via placenta and/or breast milk, may contribute to the reduced efficacy of oral rotavirus vaccines observed in children in developing countries. This study aimed to investigate the effect of rotavirus specific maternal antibodies on the serum IgA response or stool excretion of vaccine virus after any dose of an oral rotavirus vaccine, RV3-BB, in parallel to a Phase IIa clinical trial conducted at Dunedin Hospital, New Zealand. At the time o...

  9. Antibody-mediated modulation of cytokinins in tobacco: Organ-specific changes in cytokinin homeostasis

    Czech Academy of Sciences Publication Activity Database

    Gelová, Z.; Hoopen, P.; Novák, Ondřej; Motyka, Václav; Pernisová, M.; Dabravolski, S.; Didi, V.; Tillack, F.; Oklešťková, Jana; Strnad, Miroslav; Hause, B.; Haruštiaková, D.; Conrad, U.; Janda, L.; Hejátko, J.

    2018-01-01

    Roč. 69, č. 3 (2018), s. 441-454 ISSN 0022-0957 R&D Projects: GA MŠk(CZ) LQ1601; GA MŠk(CZ) LO1204; GA MŠk(CZ) LM2015062; GA ČR(CZ) GA16-14649S Institutional support: RVO:61389030 Keywords : Antibody-mediated modulation * biosynthesis * ckx * cytokinin * homeostasis * organ specificity * tobacco Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 5.830, year: 2016

  10. The transferrin receptor of Actinobacillus pleuropneumoniae: Quantitation of expression and structural characterization using a peptide-specific monoclonal antibody

    DEFF Research Database (Denmark)

    Bøg, Yang S.; Andresen, Lars Ole; Bastholm, L.

    2001-01-01

    transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70 kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron...... expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found...

  11. 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

  12. Monoclonal antibody form and function: manufacturing the right antibodies for treating drug abuse.

    Science.gov (United States)

    Peterson, Eric; Owens, S Michael; Henry, Ralph L

    2006-05-26

    Drug abuse continues to be a major national and worldwide problem, and effective treatment strategies are badly needed. Antibodies are promising therapies for the treatment of medical problems caused by drug abuse, with several candidates in preclinical and early clinical trials. Monoclonal antibodies can be designed that have customized affinity and specificity against drugs of abuse, and because antibodies can be designed in various forms, in vivo pharmacokinetic characteristics can be tailored to suit specific clinical applications (eg, long-acting for relapse prevention, or short-acting for overdose). Passive immunization with antibodies against drugs of abuse has several advantages over active immunization, but because large doses of monoclonal antibodies may be needed for each patient, efficient antibody production technology is essential. In this minireview we discuss some of the antibody forms that may be effective clinical treatments for drug abuse, as well as several current and emerging production systems that could bridge the gap from discovery to patient use.

  13. Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a

    DEFF Research Database (Denmark)

    Hecker, J.; Diethers, A.; Seismann, H.

    2011-01-01

    The scarcity of monoclonal human IgE antibodies with specificity for defined allergens is a bottleneck for the molecular characterisation of allergens and their epitopes. Insights into the characteristics of such antibodies may allow for analyses of the molecular basis underlying allergenicity an...

  14. Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany.

    Science.gov (United States)

    Weis, Sonia; Rettinger, Anna; Bergmann, Michele; Llewellyn, Julia R; Pantchev, Nikola; Straubinger, Reinhard K; Hartmann, Katrin

    2017-04-01

    Objectives Clinical manifestation of infection with Leptospira species in cats is rare. Nevertheless, cats can develop specific antibodies against the spirochetes after infection. In Canada, Taiwan and the USA it was recently demonstrated that naturally infected cats can also shed DNA from pathogenic Leptospira species in their urine, but the zoonotic potential of infected cats is still unclear. The objective of this study was to demonstrate if outdoor cats in Germany shed DNA from pathogenic Leptospira species in their urine. As a second aim, antibody prevalence was determined. Methods Two hundred and fifteen outdoor cats were prospectively recruited. Urine samples were tested by real-time PCR targeting the lipL32 gene of pathogenic Leptospira species. Antibody titres against eight serovars (Australis, Autumnalis, Bratislava, Canicola, Copenhageni, Grippotyphosa, Pomona, Saxkoebing) belonging to seven serogroups (Australis, Autumnalis, Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona, Sejroe) were determined by microscopic agglutination test. Results Urine samples from 7/215 cats (3.3%; 95% confidence interval [CI] 0.9-5.7) were PCR-positive. Specific antibodies were detected in 35/195 cats (17.9%; 95% CI: 12.5-23.3) with titres ranging from 1:100 to 1:6400. Australis, Bratislava and Grippotyphosa were the most common serovars. Conclusions and relevance Outdoor cats in Germany can shed DNA from pathogenic Leptospira species. Therefore, outdoor cats should be considered as a possible source of infection for dogs or humans. Further studies are needed to determine the role of Leptospira species as a cause of disease in cats.

  15. A novel whole-bacterial enzyme linked-immunosorbant assay to quantify Chlamydia trachomatis specific antibodies reveals distinct differences between systemic and genital compartments.

    Directory of Open Access Journals (Sweden)

    Hannah L Albritton

    Full Text Available Chlamydia trachomatis (CT is the leading sexually transmitted bacterial infection. The continued global burden of CT infection strongly predicates the need for a vaccine to supplement current chlamydial control programs. The correlates of protection against CT are currently unknown, but they must be carefully defined to guide vaccine design. The localized nature of chlamydial infection in columnar epithelial cells of the genital tract necessitates investigation of immunity at the site of infection. The purpose of this study was to develop a sensitive whole bacterial enzyme-linked immunosorbent assay (ELISA to quantify and compare CT-specific IgG and IgA in sera and genital secretions from CT-infected women. To achieve this, elementary bodies (EBs from two of the most common genital serovars (D and E were attached to poly-L-lysine-coated microtiter plates with glutaraldehyde. EB attachment and integrity were verified by the presence of outer membrane antigens and the absence of bacterial cytoplasmic antigens. EB-specific IgG and IgA standards were developed by pooling sera with high titers of CT-specific antibodies from infected women. Serum, endocervical and vaginal secretions, and endocervical cytobrush specimens from CT-infected women were used to quantify CT-specific IgG and IgA which were then normalized to total IgG and IgA, respectively. Analyses of paired serum and genital samples revealed significantly higher proportions of EB-specific antibodies in genital secretions compared to sera. Cervical and vaginal secretions and cytobrush specimens had similar proportions of EB-specific antibodies, suggesting any one of these genital sampling techniques could be used to quantify CT-specific antibodies when appropriate normalization methodologies are implemented. Overall, these results illustrate the need to investigate genital tract CT antibody responses, and our assay provides a useful quantitative tool to assess natural immunity in defined

  16. Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

    Science.gov (United States)

    Bobeck, Elizabeth A; Hellestad, Erica M; Sand, Jordan M; Piccione, Michelle L; Bishop, Jeff W; Helvig, Christian; Petkovich, Martin; Cook, Mark E

    2015-06-01

    Hyperimmunized hens are an effective means of generating large quantities of antigen specific egg antibodies that have use as oral supplements. In this study, we attempted to create a peptide specific antibody that produced outcomes similar to those of the human pharmaceutical, sevelamer HCl, used in the treatment of hyperphosphatemia (a sequela of chronic renal disease). Egg antibodies were generated against 8 different human intestinal sodium-dependent phosphate cotransporter 2b (NaPi2b) peptides, and hNaPi2b peptide egg antibodies were screened for their ability to inhibit phosphate transport in human intestinal Caco-2 cell line. Antibody produced against human peptide sequence TSPSLCWT (anti-h16) was specific for its peptide sequence, and significantly reduced phosphate transport in human Caco-2 cells to 25.3±11.5% of control nonspecific antibody, when compared to nicotinamide, a known inhibitor of phosphate transport (P≤0.05). Antibody was then produced against the mouse-specific peptide h16 counterpart (mouse sequence TSPSYCWT, anti-m16) for further analysis in a murine model. When anti-m16 was fed to mice (1% of diet as dried egg yolk powder), egg yolk immunoglobulin (IgY) was detected using immunohistochemical staining in mouse ileum, and egg anti-m16 IgY colocalized with a commercial goat anti-NaPi2b antibody. The effectiveness of anti-m16 egg antibody in reducing serum phosphate, when compared to sevelamer HCl, was determined in a mouse feeding study. Serum phosphate was reduced 18% (Pegg yolk powder) and 30% (Pegg immunoglobulin. The methods described and the findings reported show that oral egg antibodies are useful and easy to prepare reagents for the study and possible treatment of select diseases. © 2015 Poultry Science Association Inc.

  17. 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

  18. Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

    Science.gov (United States)

    Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

    2002-10-01

    We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

  19. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  20. Hexon and fiber of adenovirus type 14 and 55 are major targets of neutralizing antibody but only fiber-specific antibody contributes to cross-neutralizing activity.

    Science.gov (United States)

    Feng, Ying; Sun, Xikui; Ye, Xianmiao; Feng, Yupeng; Wang, Jinlin; Zheng, Xuehua; Liu, Xinglong; Yi, Changhua; Hao, Mingli; Wang, Qian; Li, Feng; Xu, Wei; Li, Liang; Li, Chufang; Zhou, Rong; Chen, Ling; Feng, Liqiang

    2018-05-01

    Re-emerging human adenoviruses type 14 (HAdV14) and 55 (HAdV55) represent two highly virulent adenoviruses. The neutralizing antibody (nAb) responses elicited by infection or immunization remain largely unknown. Herein, we generated hexon-chimeric HAdV14 viruses harboring each single or entire hexon hyper-variable-regions (HVR) from HAdV55, and determined the neutralizing epitopes of human and mouse nAbs. In human sera, hexon-targeting nAbs are type-specific and mainly recognize HVR2, 5, and 7. Fiber-targeting nAbs are only detectable in sera cross-neutralizing HAdV14 and HAdV55 and contribute substantially to cross-neutralization. Penton-binding antibodies, however, show no significant neutralizing activities. In mice immunized with HAdV14 or HAdV55, a single immunization mainly elicited hexon-specific nAbs, which recognized HAdV14 HVR1, 2, and 7 and HAdV55 HVR1 and 2, respectively. After a booster immunization, cross-neutralizing fiber-specific nAbs became detectable. These results indicated that hexon elicits type-specific nAbs whereas fiber induces cross-neutralizing nAbs to HAdV14 and HAdV55, which are of significance in vaccine development. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    Science.gov (United States)

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  2. In-depth analysis of subclass-specific conformational preferences of IgG antibodies

    DEFF Research Database (Denmark)

    Tian, Xinsheng; Vestergaard, Bente; Thorolfsson, Matthias

    2015-01-01

    IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designe...... properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering....

  3. Simultaneous radioimmunoassay for specific antibodies to members of the human herpesvirus group

    International Nuclear Information System (INIS)

    Gehle, W.D.; Smith, K.O.; Fuccillo, D.A.; Perry, A.; Andrese, A.P.

    1979-01-01

    A method is described for the simultaneous radioimmunoassay (RIA) for antibody to members of the human herpesvirus group. The RIA is compared with some of the conventional serologic techniques used to quantitate antibody to these viruses (Epstein-Barr virus, cytomegalovirus, herpesvirus type 1 and varicella-zoster virus). Color-coded beads, each coated with the antigens of a different herpesvirus, were simultaneously placed in a well which contained a human serum to be assayed for antibody to each of these 4 viruses. The results of this test were compared with the results obtained when the serum was assayed for antibody to the 4 viruses in 4 separate tests. We conclude that the antigen-antibody reactions do not significantly interfere with each other when a serum is assayed for antibody to the 4 viruses simultaneously. A comparison of the RIA with conventional serologic techniques shows excellent correlation in the antibody titers obtained. Features of the solid-phase RIA allow significant savings of time, reagents and space, and thus make it feasible for the small laboratory to screen large numbers of sera for antibody to a variety of antigens. (Auth.)

  4. Specific binding of large aggregates of amphiphilic molecules to the respective antibodies.

    Science.gov (United States)

    Nabok, Alexei; Tsargorodskaya, Anna; Holloway, Alan; Starodub, Nikolay F; Demchenko, Anna

    2007-07-31

    The Binding of nonylphenol to respective antibodies immobilized on solid substrates was studied with the methods of total internal reflection ellipsometry (TIRE) and QCM (quartz crystal microbalance) impedance spectroscopy. The binding reaction was proved to be highly specific having an association constant of KA=1.6x10(6) mol(-1) L and resulted in an increase in both the adsorbed layer thickness of 23 nm and the added mass of 18.3 microg/cm2 at saturation. The obtained responses of both TIRE and QCM methods are substantially higher than anticipated for the immune binding of single molecules of nonylphenol. The mechanism of binding of large aggregates of nonylphenol was suggested instead. Modeling of the micelle of amphiphilic nonylphenol molecules in aqueous solutions yielded a micelle size of about 38 nm. The mechanism of binding of large molecular aggregates to respective antibodies can be extended to other hydrophobic low-molecular-weight toxins such as T-2 mycotoxin. The formation of large molecular aggregates of nonylphenol and T-2 mycotoxin molecules on the surface was proved by the AFM study.

  5. 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...

  6. α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid β42 accumulation in the mouse brain to impair memory.

    Directory of Open Access Journals (Sweden)

    Olena Lykhmus

    Full Text Available Nicotinic acetylcholine receptors (nAChRs expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ42, memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208 or injected with bacterial lipopolysaccharide (LPS for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ40 and Aβ42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208 resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1 neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ42 accumulation and memory impairment in mice and (2 α7(1-208 nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.

  7. The usefulness of casein-specific IgE and IgG4 antibodies in cow's milk allergic children

    Directory of Open Access Journals (Sweden)

    Ito Komei

    2012-01-01

    Full Text Available Abstract Background Cow's milk allergy is one of the most common food allergies among younger children. We investigated IgE antibodies to milk, and IgE and IgG4 antibodies to casein, α-lactalbumin and β-lactoglobulin in cow's milk allergic (CMA and non-allergic (non-CMA children in order to study their clinical usefulness. Methods Eighty-three children with suspected milk allergy (median age: 3.5 years, range: 0.8-15.8 years were diagnosed as CMA (n = 61 or non-CMA (n = 22 based on an open milk challenge or convincing clinical history. Their serum concentrations of allergen-specific (s IgE and IgG4 antibodies were measured using ImmunoCAP®. For the sIgG4 analysis, 28 atopic and 31 non-atopic control children were additionally included (all non-milk sensitized. Results The CMA group had significantly higher levels of milk-, casein- and β-lactoglobulin-sIgE antibodies as compared to the non-CMA group. The casein test showed the best discriminating performance with a clinical decision point of 6.6 kUA/L corresponding to 100% specificity. All but one of the CMA children aged > 5 years had casein-sIgE levels > 6.6 kUA/L. The non-CMA group had significantly higher sIgG4 levels against all three milk allergens compared to the CMA group. This was most pronounced for casein-sIgG4 in non-CMA children without history of previous milk allergy. These children had significantly higher casein-sIgG4 levels compared to any other group, including the non-milk sensitized control children. Conclusions High levels of casein-sIgE antibodies are strongly associated with milk allergy in children and might be associated with prolonged allergy. Elevated casein-sIgG4 levels in milk-sensitized individuals on normal diet indicate a modified Th2 response. However, the protective role of IgG4 antibodies in milk allergy is unclear.

  8. Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

    Science.gov (United States)

    Krawczyk, Adalbert; Dirks, Miriam; Kasper, Maren; Buch, Anna; Dittmer, Ulf; Giebel, Bernd; Wildschütz, Lena; Busch, Martin; Goergens, Andre; Schneweis, Karl E.; Eis-Hübinger, Anna M.; Sodeik, Beate; Heiligenhaus, Arnd; Roggendorf, Michael; Bauer, Dirk

    2015-01-01

    The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans. PMID:25587898

  9. Prevention of herpes simplex virus induced stromal keratitis by a glycoprotein B-specific monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Adalbert Krawczyk

    Full Text Available The increasing incidence of acyclovir (ACV and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis or 24, 40, and 56 hours after infection (post-exposure immunotherapy. Topical treatment was performed by periodical inoculations (5 times per day of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

  10. Disease-specific monoclonal antibodies targeting glutamate decarboxylase impair GABAergic neurotransmission and affect motor learning and behavioral functions

    Directory of Open Access Journals (Sweden)

    Mario U Manto

    2015-03-01

    Full Text Available Autoantibodies to the smaller isoform of glutamate decarboxylase can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The detection of disease-specific autoantibody epitopes led to the hypothesis that distinct glutamate decarboxylase autoantibodies may elicit specific neurological phenotypes. We explored the in vitro/in vivo effects of well-characterized monoclonal glutamate decarboxylase antibodies. We found that glutamate decarboxylase autoantibodies present in patients with stiff person syndrome (n = 7 and cerebellar ataxia (n = 15 recognized an epitope distinct from that recognized by glutamate decarboxylase autoantibodies present in patients with type 1 diabetes mellitus (n = 10 or limbic encephalitis (n = 4. We demonstrated that the administration of a monoclonal glutamate decarboxylase antibody representing this epitope specificity (1 disrupted in vitro the association of glutamate decarboxylase with γ-Aminobutyric acid containing synaptic vesicles, (2 depressed the inhibitory synaptic transmission in cerebellar slices with a gradual time course and a lasting suppressive effect, (3 significantly decreased conditioned eyelid responses evoked in mice, with no modification of learning curves in the classical eyeblink-conditioning task, (4 markedly impaired the facilitatory effect exerted by the premotor cortex over the motor cortex in a paired-pulse stimulation paradigm, and (5 induced decreased exploratory behavior and impaired locomotor function in rats. These findings support the specific targeting of glutamate decarboxylase by its autoantibodies in the pathogenesis of stiff-person syndrome and cerebellar ataxia. Therapies of these disorders based on selective removal of such glutamate decarboxylase antibodies could be envisioned.

  11. 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

  12. 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

  13. Genus and species-specific IgG and IgM antibodies pulmonary tuberculosis

    International Nuclear Information System (INIS)

    Butt, T.; Abbassi, S.A.; Ahmad, R.N.; Mahmood, A.; Karamat, K.A; Malik, H.S.; Anwar, M.

    2004-01-01

    Objective: To evaluate three different enzyme immunoassays for serological diagnosis of pulmonary tuberculosis and to compare their diagnostic accuracy in different combinations. Subjects and Methods: Sera from patients suffering from pulmonary tuberculosis (n=94) with sputum positive for acid fast bacilli (AFB) and sera from control group of healthy individuals (n=90) with sputum negative for AFB were tested by Pathozyme-Myco G EIA, Pathozyme-TB Complex Plus EIA and Pathozyme Myco M EIA kits for the genus-specific IgG and IgM, and the species-specific IgG antibodies against antigens of Mycobacterium tuberculosis. Results: The detection of IgG against genus-specific antigens by Pathozyme-Myco G had a sensitivity of 46% and a specificity of 93%, of IgG against species-specific antigens by Pathozyme- TB Complex Plus had a sensitivity of 64% and specificity of 97% and of IgM against genus-specific antigens by Pathozyme Myco M had a sensitivity of 67% and specificity of 98%. When the results of these immunoassays were evaluated in combination, their sensitivity improved. Combination of genus-specific IgM and species-specific IgG yielded best results with a sensitivity of 87% and specificity of 93%. Conclusion: The sensitivity of serological diagnosis of tuberculosis is low, but it can be increased by utilizing a combination of several antigens. (author)

  14. Evidence of a pro-apoptotic effect of specific antibodies in a bovine macrophage model of infection with Mycobacterium avium subsp. paratuberculosis.

    Science.gov (United States)

    Jolly, Ana; Lompardía, Silvina; Hajos, Silvia E; Mundo, Silvia L

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Seroprevalence and placental transmission of maternal antibodies specific for Neisseria meningitidis Serogroups A, C, Y and W135 and influence of maternal antibodies on the immune response to a primary course of MenACWY-CRM vaccine in the United Kingdom.

    Science.gov (United States)

    Blanchard-Rohner, Geraldine; Snape, Matthew D; Kelly, Dominic F; O'Connor, Daniel; John, Tessa; Kibwana, Elizabeth; Parks, Hannah; Ford, Karen; Dull, Peter M; Pollard, Andrew J

    2013-07-01

    Maternal antibodies give neonates some protection against bacterial infection. We measured antibodies against Neisseria meningitidis serogroups A, C, Y and W135 in mothers and their 2-month-old infants at study enrollment. We also assessed the impact of maternal antibody present at 2 months of age on the immune response to a primary course of quadrivalent meningococcal conjugate vaccine (MenACWY-CRM197) given at 2 and 4 months of age. This was a single-center, open-label, randomized study undertaken in Oxford, United Kingdom. Two hundred sixteen healthy infants were enrolled in the study and vaccinated with MenACWY-CRM197 at 2 and 4 months of age. Blood was obtained from all mothers, in a subset of infants at 2 months and all infants at 5 months. Antibody and memory B-cell responses at 5 months were correlated with maternal antibodies. Mothers had low IgG antibodies against serogroups C, W135 and Y polysaccharides, but high serogroup A antibody, whereas 61-78% had protective human complement serum bactericidal activity (hSBA) (≥1:4) for serogroups C, W135 and Y but only 31% for serogroup A. Only 9%, 32%, 45% and 19% of 2-month-old infants had hSBA ≥1:4 for serogroups A, C, W135 and Y, respectively. Maternal antibody had little association on responses to MenACWY-CRM197, except a moderate negative association between MenC-specific bactericidal antibody at 2 and 5 months (r = -0.5, P = 0.006, n = 28) and between carrier-specific IgG antibody at 2 months and MenC-specific hSBA/IgG antibody at 5 months (r = -0.4, P = 0.02 and 0.04, n = 32 and 23). Nonetheless, 90% of infants achieved protective MenC-hSBA titers after vaccination at 2 and 4 months of age. The levels of serogroup-specific meningococcal antibodies were low in mothers and 2-month-old infants. Immunizing mothers before or during pregnancy with meningococcal conjugate vaccines might increase antibody levels in early infancy and provide protection against infection due to N. meningitidis.

  16. Site-specifically {sup 89}Zr-labeled monoclonal antibodies for ImmunoPET

    Energy Technology Data Exchange (ETDEWEB)

    Tinianow, Jeff N.; Gill, Herman S.; Ogasawara, Annie; Flores, Judith E.; Vanderbilt, Alexander N.; Luis, Elizabeth; Vandlen, Richard; Darwish, Martine; Junutula, Jagath R.; Williams, Simon-P. [Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080 (United States); Marik, Jan [Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080 (United States)], E-mail: marik.jan@gene.com

    2010-04-15

    Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled {sup 89}Zr-Df-thio-trastuzumab conjugates were investigated. Methods: The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N-hydroxysuccinimidyl iodoacetate, or N-hydroxysuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with {sup 89}Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model. Results: The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with {sup 89}Zr in yields exceeding 75%. {sup 89}Zr-Df-Ac-thio-trastuzumab and {sup 89}Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20-25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1-7.1. Conclusions: The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with {sup 89}Zr. The site-specifically {sup 89}Zr-labeled thio-antibodies

  17. The specific heat of Cu-Al-Ni shape memory alloys

    International Nuclear Information System (INIS)

    Ruiz-Larrea, I.; Lopez-Echarri, A.; Bocanegra, E.H.; No, M.L.; San Juan, J.M.

    2006-01-01

    The specific heat of Cu 81.8 Al 13.7 Ni 4.5 (AK10) shape memory alloy has been studied by means of conventional DSC and adiabatic calorimetry techniques. The transformation temperatures and the shape of the calorimetric curves obtained by adiabatic calorimetry do not show any noticeable dependence on the temperature measurement rates, contrarily to what is observed by other calorimetric techniques. The dynamical character of the various experimental methods together with the influence of the latent heat associated to the first order character of these phase transitions are discussed. The specific heat of AK10 has been measured from 50 to 350 K which covers the phase transformation temperature range. The forward and reverse martensitic transformation peaks were found at 299.5 and 304.6 K, showing a thermal hysteresis of 5.1 deg. C. The C p accuracy can be estimated in 0.1% of C p and permits a reliable assignment of the following values to the phase transition thermodynamic functions: ΔH = 7.4 ± 0.2 J/g and ΔS = 0.025 ± 0.001 J/gK

  18. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

    Directory of Open Access Journals (Sweden)

    Rasool Suhail

    2007-09-01

    Full Text Available Abstract Background Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD, amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils. Results We immunized rabbits with a morphologically homogeneous population of Aβ42 fibrils. The resulting immune serum (OC specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 × G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type

  19. Determining Vaccination Frequency in Farmed Rainbow Trout Using Vibrio anguillarum O1 Specific Serum Antibody Measurements

    DEFF Research Database (Denmark)

    Holten-Andersen, Lars; Dalsgaard, Inger; Nylén, Jørgen

    2012-01-01

    Background Despite vaccination with a commercial vaccine with a documented protective effect against Vibrio anguillarum O1 disease outbreaks caused by this bacterium have been registered among rainbow trout at Danish fish farms. The present study examined specific serum antibody levels as a valid...

  20. Maintenance plasma exchange treatment for muscle specific kinase antibody positive myasthenia gravis patients.

    Science.gov (United States)

    Yamada, Chisa; Teener, James W; Davenport, Robertson D; Cooling, Laura

    2015-10-01

    Anti-muscle specific kinase antibody positive myasthenia gravis (MuSK MG) is often characterized by a relatively severe and progressive course, refractoriness to standard myasthenia gravis (MG) medications, and an increased risk of myasthenic crisis. We report here successful management of three MuSK MG patients using maintenance therapeutic plasma exchange (TPE) treatment for up to 4.5 years. The study was a 5-year retrospective review of all MG patients treated with TPE between 2008 and 2013 at University of Michigan. Inclusion criteria of MuSK MG were positive for anti-MuSK antibodies and a diagnosis of MuSK MG by staff neurologists. Patient data included age, gender, diagnostic testing results, medications, and the dates and response to TPE treatments. A total of 153 MG patients underwent at least one course of TPE between 2008 and 2013. A total of 12 patients (7.8%) were positive for anti-MuSK antibodies. Patients were predominantly female (83.3%) and a median age of onset was 46-years old. Three MuSK MG patients were successfully managed with maintenance TPE. Maintenance TPE may be an effective option for MuSK MG patients. The key of successful maintenance treatment at our institution has been to tailor the TPE frequency for each individual, and to modify the treatment interval in conjunction with medical management. © 2014 Wiley Periodicals, Inc.

  1. 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

  2. [Comparative study of immunoglobulins and specific antibodies in the sera of chronic brucellosis patients].

    Science.gov (United States)

    Kichieva, B N; Chernysheva, M I; Zheludkov, M M; Musaeva, N B

    1982-04-01

    The data on the IgA, IgM and IgG levels in the sera of 89 patients with chronic brucellosis lasting for 1-10 years and longer are presented. The chronic form of brucellosis is characterized by the normal or low level of immunoglobulins. No correlation between the levels of IgG, IgM and the titer of specific antibodies has been established.

  3. ETAC reagents: A new class of sulfhydryl site-specific radiolabelling probes for antibodies

    International Nuclear Information System (INIS)

    del Rosario, R.B.; Brocchini, S.J.; Baron, L.A.; Smith, R.H.; Lawton, R.G.; Wahl, R.L.

    1990-01-01

    A new class of bis-alkylating Michael reagents, equilibrium transfer crosslink reagents, 'ETAC', which combine the techniques of crosslinking with tethering have been synthesized. Following a succession of Michael and retro-Michael additions and elimination of the arylsulfone groups, reduced heavy-heavy and heavy-light disulfide links of an anti-ovarian IgG2a monoclonal antibody, 5G6.4, were site-specifically re-annealed via a 3-carbon bridge having a tether branch containing a designated label

  4. Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Ramos Franco Antonia Maria

    1997-01-01

    Full Text Available A large number of Endotrypanum stocks (representing an heterogeneous population of strains have been screened against a panel of monoclonal antibodies (MAbs derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12 or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5 reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent, but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

  5. Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies.

    Directory of Open Access Journals (Sweden)

    K Laurence Jost

    Full Text Available Methyl CpG binding protein 2 (MeCP2 binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro.

  6. STUDY ON SEROPREVALENCE OF MUMPS - SPECIFIC IgG ANTIBODIES IN A HEALTHY POPULATION

    Directory of Open Access Journals (Sweden)

    Milena Karcheva

    2010-09-01

    Full Text Available Mumps is a vaccine preventable viral infection. Its typical clinical manifestations are characterized by pain and swelling of the salivary glands, fever, and fatigue. Often other organs are affected - testes in males after puberty (orchitis, ovaries in women (ooforitis, pancreas (pancreatitis, central nervous system (meningities. The use of specific immune prophylaxis led to a significant success in the fight against mumps, but there are still unresolved issues related to the immunological and epidemiological effectiveness of the vaccines. The disease continues to interest researchers today. The main issues being tackled are related to the conduct of virological, clinical and sero-epidemiological studies in different countries. Objectives of the study is to determine the frequency distribution of mumps-specific IgG antibodies in healthy populations in the region of Pleven, Bulgaria. Methods: a cross-sectional sero - epidemiological representative population - based survey in the area was made. Enzyme immunoassay method was used for an indirect proof of mumps - specific IgG serum antibodies. 410 people were examined at an average age of 25 (1 to 84. Of these, 250 (61 % were women and 160 (39 % - men. Results: Of all test results, the negative were 72 (19 %, the borderline were 12 (3 %, the positive were 182 (44 %, and highly positive were 144 (35 %. The vaccination status showed that 242 (69 % of all surveyed were immunized with a vaccine against mumps. According to the immunization schedule in Bulgaria, 132 (33 % people were immunized with monovaccine during the years - 1 intake, 80 (20 % with trivaccine - 1 intake, and 64 (16 % - 2 doses. Conclusion: We believe that despite the specific immunprophylaxis carried out against mumps decades on end, the necessary level of protection leading to its elimination has not yet been reached.

  7. Kinetics of the avian influenza-specific humoral responses in lung are indicative of local antibody production

    NARCIS (Netherlands)

    Geus, de E.D.; Rebel, J.M.J.; Vervelde, L.

    2012-01-01

    The role and kinetics of respiratory immunoglobulins in AIV infection has not been investigated. In this study we determined the numbers of both total antibody secreting cells (ASC) and virus-specific ASC in lung, spleen, blood and bone marrow (BM) following low-pathogenic AIV infection. Antiviral

  8. Human immunodeficiency virus (HIV) specific antibodies among ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-03-20

    Mar 20, 2009 ... Key words: HIV-1/2 antibody prevalence, pregnant women, commercial sex workers, risk factors, Nigeria. INTRODUCTION. There are two .... Africa. However, among Japanese and Chilean female. SWs, Miyazaki et al. .... STIs (P = 0.0001, OR = 6.0), level of education (P = 0.0001, OR = 40.7) and age (P ...

  9. Annual literature review of donor-specific HLA antibodies after organ transplantation.

    Science.gov (United States)

    Kaneku, Hugo

    2011-01-01

    The literature review of post-transplant DSA published in 2011 shows: Observations after kidney and lung transplant in non-sensitized transplant recipients show that monitoring post-transplant HLA antibodies offers limited benefit in predicting acute rejection episodes. It remains to be seen if a different monitoring schedule and/ or studying other organs may show otherwise. Nevertheless, others have shown that monitoring post-transplant antibodies does identify patients at higher risk for chronic rejection. Studies in kidney, heart, and liver patients transplanted in the presence of preformed DSA show that detecting these antibodies early after transplant identifies a group of patients with greater risk for allograft dysfunction. New and larger studies using bortezomib and eculizumab to treat acute antibody-mediated rejection confirm earlier observations that these two therapies are effective in treating and preventing rejections. In general, identification of HLAantibodies and DSA after transplant is associated with higher rates of rejection and poor allograft survival in all organs examined. IgM antibodies appear to play an important role after lung transplants.

  10. 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

  11. The Clinical Significance of Specific Antibody Deficiency (SAD) Severity in Chronic Rhinosinusitis (CRS).

    Science.gov (United States)

    Keswani, Anjeni; Dunn, Neha M; Manzur, Angelica; Kashani, Sara; Bossuyt, Xavier; Grammer, Leslie C; Conley, David B; Tan, Bruce K; Kern, Robert C; Schleimer, Robert P; Peters, Anju T

    Despite the increased identification of specific antibody deficiency (SAD) in chronic rhinosinusitis (CRS), little is known about the relationship between SAD severity and the severity and comorbidities of CRS. The prevalence of an impaired antibody response in the general population is also unknown. The objective of this study was to determine if the SAD severity stratification applies to real-life data of patients with CRS. An electronic health record database was used to identify patients with CRS evaluated for humoral immunodeficiency with quantitative immunoglobulins and Streptococcus pneumoniae antibody titers before and after pneumococcal vaccine. SAD severity was defined, according to the guidelines, based on the numbers of titers ≥1.3 μg/dL after vaccination: severe (≤2 serotypes), moderate (3-6 serotypes), and mild (7-10 serotypes). Comorbidities and therapeutic response were assessed. The prevalence of an impaired antibody response in a normal population was assessed. Twenty-four percent of the patients with CRS evaluated for immunodeficiency had SAD, whereas 11% of a normal population had an impaired immune response to polysaccharide vaccination (P SAD. Twenty-four (10%) had severe SAD, 120 (50%) had moderate SAD, and 95 (40%) had mild SAD. Patients with moderate-to-severe SAD had worse asthma, a greater likelihood of pneumonia, and more antibiotic courses in the 2 years after vaccination than patients with mild SAD. This study provides real world data supporting stratification of SAD by severity, demonstrating a significant increase in the comorbid severity of asthma and infections in CRS patients with moderate-to-severe SAD compared with those with mild SAD and those without SAD. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  12. Selective assay for CyPA and CyPB in human blood using highly specific anti-peptide antibodies.

    Science.gov (United States)

    Allain, F; Boutillon, C; Mariller, C; Spik, G

    1995-01-13

    Cyclophilins A and B (CyPA and CyPB) are known to be the main binding proteins for cyclosporin A (CsA), a potent immunosuppressive drug. Due to the high homology between the two proteins, antibodies to CyPB were found to cross-react with CyPA. In order to avoid this phenomenon, we raised specific antibodies against peptides copying the most divergent parts of the two sequences. These antibodies allowed us to develop an ELISA capture assay selective for either isotype. Thus, we showed that leukocyte CyPB concentration was almost ten times lower than that of CyPA, and that in contrast to the results described in the literature, only CyPB was released in plasma. Moreover, CyPB levels in leukocytes and plasma were found to correlate for the same donor, but no relationship was found with CyPA level.

  13. 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...

  14. SENSITIVITY AND SPECIFICITY OF MONOCLONAL ANTIBODY DSSE10 IN HEAD SQUASH TOXORHYNCHITES SPLENDENS USING IMMUNOHISTOCHEMICAL PEROXIDASE TECHNIQUE

    Directory of Open Access Journals (Sweden)

    Tika Fiona Sari

    2014-06-01

    Full Text Available Dengue virus are transmitted from human to human by the bites of infective female Aedesmosquitoes from subgenus Stegomyia. One of the way to detect Dengue virus antigen is by usingimmunohistochemical technique. This method was reported to detect dengue vims antigen in lowlevels. The aims of this study is to measure sensitivity and specificity of monoclonal antibodyDSSE10 using SBPC to detect antigen Dengue virus in head squash Toxorhynchites splendenswere infected with dengue patient serum and RT-PCR as gold standart. Artificially-infected Tx.splendens mosquitoes with serum positif dengue virus were used as infectious samples and noninfectedTx. splendens mosquitoes were used as control negative. The immunohistochemichalSBPC assay using monoclonal antibody DSSE10 then applied in mosquitoes head squash todetect Dengue vims antigen. RT-PCR as a gold standart was applied in each mosquito thorax.The result were analyzed by descriptive stasistic test and 2x2 diagnostic test table. Monoclonalantibody DSSE10 using immunohistochemical SBPC assay in head squash Tx. splendens wasgave sensitivity 87,09% and specificity 92,5%. Conclussion of this study is DSSE10 Monoclonalantibodies can be used as primary antibodies for the detection of dengue vims antigen inmosquito head squashKeywords: Dengue viruses, SBPC, antibodies DSSE10, head squash, Toxorhynchitessplendens' Virus Dengue ditularkan dari orang ke orang melalui gigitan nyamuk Aedes dari subgenusStegomyia. Salah satu cara untuk mendeteksi antigen vims Dengue adalah dengan menggunakanteknik imunohistokimia. Metode imunohistokimia dilaporkan dapat mendeteksi antigen vimsDengue dalam kadar yang rendah. Tujuan penelitian ini adalah melakukan evaluasi sensitivitasdan spesifitas antibodi monoklonal DSSE10 dengan metode imunohistokimia Streptavidin BiotinPeroxidase Complex (SBPC untuk mendeteksi antigen Dengue melalui scdiaan head squashnyamuk Toxorhynchites splendens yang diinfeksi dengan scrum penderita

  15. Preclinical Analysis of JAA-F11, a Specific Anti–Thomsen-Friedenreich Antibody via Immunohistochemistry and In Vivo Imaging

    Directory of Open Access Journals (Sweden)

    Loukia G. Karacosta

    2018-04-01

    Full Text Available The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked, has been comprehensively studied by in vitro immunohistochemical (IHC staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by micro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG3 (control, was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untargetable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper “Humanization of JAA-F11, a Highly Specific Anti–Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis” (Neoplasia 19: 716-733, 2017, and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124I- JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need.

  16. 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.

  17. Association of Escherichia coli J5-specific serum antibody responses with clinical mastitis outcome for J5 vaccinate and control dairy cattle.

    Science.gov (United States)

    Wilson, David J; Mallard, Bonnie A; Burton, Jeanne L; Schukken, Ynte H; Grohn, Yrjo T

    2009-02-01

    Dairy cattle in two commercial Holstein herds were randomly selected to be vaccinated twice with J5, at approximately 60 days and 28 days before the expected calving date, or to be untreated controls. Based on whether milk production changed following clinical mastitis or whether cows were culled or died within 30 days after onset, 51 mastitis cases were classified as severe or mild. J5-specific antibody responses were evaluated by enzyme-linked immunosorbent assay of all 32 severe and 19 mild cases. The amounts of J5-specific immunoglobulin M (IgM), IgG1, and IgG2 antibodies in sera from the 27 J5 vaccinates were compared with those of the 24 controls. At drying off (before J5 vaccination), all cows had similar amounts of J5-specific antibody. Immediately after calving (approximately 28 days after the second vaccination), J5 vaccinates had significantly higher production of J5-specific IgG1 and IgG2 than controls. When cows were tested following clinical mastitis, none of the three antibody classes differed significantly between the controls and the vaccinates. Vaccinates that contracted Escherichia coli mastitis had 75% less milk loss than controls. The cows that contracted clinical mastitis later in lactation, the unvaccinated controls, and those infected with E. coli had more milk loss following mastitis. The hazards of being culled for all reasons and of being culled for mastitis were significantly lower for J5 vaccinates. Vaccination with J5 was associated with protection against milk production loss and culling following clinical mastitis, and it was also significantly associated with changes in J5-specific IgM, IgG1, and IgG2 antibodies in sera of vaccinated cows.

  18. Human immunodeficiency virus (HIV) specific antibodies among ...

    African Journals Online (AJOL)

    obtained from each sample was tested using parallel testing algorithm with DETERMINE® HIV-1/2 and HIV-1/2 STAT-PAK® test was used for statistical analysis of the data. The overall prevalence of HIV-1/2 antibodies was 29.1% (n = 199). Seroprevalence of 39.4 and 19.0% were observed for the CSWs and the PW, ...

  19. Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen

    DEFF Research Database (Denmark)

    Boesen, Henriette T.; Jensen, Tim Kåre; Jungersen, Gregers

    2005-01-01

    Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic...... (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis...

  20. Identifying long-term memory B-cells in vaccinated children despite waning antibody levels specific for Bordetella pertussis proteins.

    Science.gov (United States)

    Hendrikx, Lotte H; Oztürk, Kemal; de Rond, Lia G H; Veenhoven, Reinier H; Sanders, Elisabeth A M; Berbers, Guy A M; Buisman, Anne-Marie

    2011-02-04

    Whooping cough is a respiratory disease caused by Bordetella pertussis. Since the 1950s in developed countries pertussis vaccinations are included in the national immunization program. However, antibody levels rapidly wane after both whole cell and acellular pertussis vaccination. Therefore protection against pertussis may depend largely on long-term B- and T-cell immunities. We investigated long-term pertussis-specific memory B-cell responses in children who were primed at infant age with the Dutch wP-vaccine (ISRCTN65428640). Purified B-cells were characterized by FACS-analysis and after polyclonal stimulation memory B-cells were detected by ELISPOT-assays specific for pertussis toxin, filamentous haemagglutinin, pertactin and tetanus. In addition, plasma IgG levels directed to the same antigens were measured by a fluorescent bead-based multiplex immunoassay. Two and 3 years after wP priming as well as 2 and 5 years after the aP booster at the age of 4, low plasma IgG levels to the pertussis proteins were found. At the same time, however pertussis protein-specific memory B-cells could be detected and their number increased with age. The number of tetanus-specific memory B-cells was similar in all age groups, whereas IgG-tetanus levels were high 2 years after tetanus booster compared to pre- and 5 years post-booster levels. This study shows the presence of long-term pertussis protein-specific memory B-cells in children despite waning antibody levels after vaccination, which suggests that memory B-cells in addition to antibodies may contribute to protection against pertussis. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Identification and evaluation of nextgeneration PTM-specific antibodies

    DEFF Research Database (Denmark)

    Persson, Nina Emilia

    -chain fragment variable (scFv)clones. Two different analyses are performed on the same microarray. There is no need for anypurification or enrichment before screening. In the first analysis, the ability of the individualscFv clone to bind to the soluble form of the antigens is evaluated. Favouring selection....... Including antibodies target several different categories of antigens suchas proteins, glycoproteins and glycolipids. Glycoproteins have become highlighted in cancerresearch since they are frequently involved in the initiation and spreading of cancer. One form ofglycosylation of proteins is the O...... for the patients. Severalmonoclonal antibodies have been generated against the Tn- and STn-antigens, but none has yetreached approval for therapeutic or diagnostic use. Indicating the need for a new generation ofantibodies against this type aberrant glycosylation.The two major techniques used for the production...

  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. 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. Antibody-Conjugated Nanoparticles for Biomedical Applications

    Directory of Open Access Journals (Sweden)

    Manuel Arruebo

    2009-01-01

    Full Text Available Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc., they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc. and be used in several diagnostic procedures, or even in therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example, they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated nanoparticles, offering the researcher an overview of the different applications and possibilities of these hybrid carriers.

  5. 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

  6. Simple dipstick assay for the detection of Salmonella typhi-specific IgM antibodies and the evolution of the immune response in patients with typhoid fever

    NARCIS (Netherlands)

    Hatta, Mochammad; Goris, Marga G. A.; Heerkens, Evy; Gooskens, Jairo; Smits, Henk L.

    2002-01-01

    Application of a dipstick assay for the detection of Salmonella typhi-specific IgM antibodies on samples collected from S. typhi or S. paratyphi culture-positive patients at the day of admission to the hospital revealed the presence of specific IgM antibodies in 43.5%, 92.9%, and 100% for samples

  7. Heterologous Coproduction of Enterocin A and Pediocin PA-1 by Lactococcus lactis: Detection by Specific Peptide-Directed Antibodies

    Science.gov (United States)

    Martínez, José M.; Kok, Jan; Sanders, Jan W.; Hernández, Pablo E.

    2000-01-01

    Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536–4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available. PMID:10919819

  8. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    N.D. Zegers (Netty)

    1995-01-01

    textabstractSynthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps

  9. 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...

  10. Occurrence of specific influenza antibodies in saliva and nasal secretion of monkeys (Macacus rhesus) after oral administration of influenza vaccine inactivated by gamma rays

    International Nuclear Information System (INIS)

    Tischner, H.; Huyuh, P.L.; Phan, P.N.; Bergmann, K.C.; Hoang, T.N.; Luther, P.; Nordheim, W.; Braeuniger, S.; Waldman, R.H.

    1984-01-01

    Antibodies in nasal secretion and saliva were measured in 10 Macacus rhesus wich had been immunized orally with a 60 Co-gamma-inactivated influenza vaccine. Prior to immunization monkeys had no detectable antibodies against hemagglutinin (HA) and neuraminidase, resp. in sera or secretions. Oral immunization using intraoesophageal tubing, induced the occurrence of both antiobodies in pilocarpine-stimulated secretions within 28 days but not in sera. 6 monkeys reacted with increasing HA antibodies in nasal secretions and 10 monkeys with increasing neuraminidase antibodies. Salivary HA antibodies occurred in 8 of 10 and neuraminidase antibodies in 9 of 10 animals. In most cases antibodies occurred in both secretions simultaneously. These results demonstrate the stimulation of antibodies specific to influenza in the respiratory tract of monkeys after oral immunization with an inactivated vaccine, for the first time. (author)

  11. 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...

  12. Structure of the extracellular domain of matrix protein 2 of influenza A virus in complex with a protective monoclonal antibody.

    Science.gov (United States)

    Cho, Ki Joon; Schepens, Bert; Seok, Jong Hyeon; Kim, Sella; Roose, Kenny; Lee, Ji-Hye; Gallardo, Rodrigo; Van Hamme, Evelien; Schymkowitz, Joost; Rousseau, Frederic; Fiers, Walter; Saelens, Xavier; Kim, Kyung Hyun

    2015-04-01

    The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e. M2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the

  14. Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

    Science.gov (United States)

    Mujib, Shariq; Liu, Jun; Rahman, A K M Nur-Ur; Schwartz, Jordan A; Bonner, Phil; Yue, Feng Yun; Ostrowski, Mario A

    2017-08-15

    Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS. IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and

  15. Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

    Science.gov (United States)

    Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

    2007-03-01

    Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

  16. 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.

  17. 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

  18. 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.

  19. Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

    Science.gov (United States)

    Tanaka, Kento; Yoshitomi, Tomomi; Hirahara, Kazuki

    2017-01-01

    Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

  20. 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.

  1. 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.

  2. Vulvovaginal-gingival Lichen Planus: Association with Lichen Planopilaris and Stratified Epithelium-specific Antinuclear Antibodies.

    Science.gov (United States)

    Olszewska, Malgorzata; Banka-Wrona, Agnieszka; Skrok, Anna; Rakowska, Adriana; Górska, Renata; Solomon, Lynn W; Rudnicka, Lidia

    2016-01-01

    Vulvovaginal-gingival lichen planus (VVG-LP) consists of a triad of symptoms: vulval, vaginal and gingival lichen planus lesions. The aim of this study was to analyse the prevalence of lesions in various anatomical locations in patients with VVG-LP. The study included 126 consecutive patients with lichen planus. Sixteen (12.7%) patients fulfilled the criteria of VVG-LP. In 12/16 (75%) patients with VVG-LP scalp lesions were also observed. Stratified epithelium-specific antinuclear antibodies (SES-ANA) and anti-ΔNp.3α antibodies were detected in 10/16 (75%) patients with VVG-LP and in 15/110 (13.6%) patients with other forms of lichen planus (p lichen planopilaris. The new entity may be termed "vulvovaginal-gingival-pilar lichen planus" and our study indicates that SES-ANA is a marker of this type of lichen planus with extensive, severe and refractory-to-therapy involvement of the mucous membranes, skin and scalp.

  3. A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera.

    Science.gov (United States)

    Sachse, Konrad; Rahman, Kh Shamsur; Schnee, Christiane; Müller, Elke; Peisker, Madlen; Schumacher, Thomas; Schubert, Evelyn; Ruettger, Anke; Kaltenboeck, Bernhard; Ehricht, Ralf

    2018-03-16

    Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

  4. 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

  5. Production of Monoclonal Antibodies Specific for Progesterone, Estradiole by Simultaneous Injection of Different Steroids

    OpenAIRE

    YÜCEL, Fatima ŞAHİNGÖZ

    2014-01-01

    We report here the development of hybrid cells producing monoclonal antibodies specific for two different steroid hormones with mixed immunization using hybridoma technology. BALB/c mice were immunized with a mixture of three steroid antigens: progesterone, estradiole and testosterone linked to bovine serum albumine. These mice were used for fusion. In the two fusion experiments, ELISA tests showed that among 645 wells only 2 hybrids reacted with progesterone (MAM 3C2, MAM 3E3) and one o...

  6. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

    DEFF Research Database (Denmark)

    Stryhn, A; Andersen, P S; Pedersen, L O

    1996-01-01

    Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide...... each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T...... cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody....

  7. Persistent humoral immune defect in highly active antiretroviral therapy-treated children with HIV-1 infection: loss of specific antibodies against attenuated vaccine strains and natural viral infection

    NARCIS (Netherlands)

    Bekker, Vincent; Scherpbier, Henriëtte; Pajkrt, Dasja; Jurriaans, Suzanne; Zaaijer, Hans; Kuijpers, Taco W.

    2006-01-01

    OBJECTIVE: In the pre-highly active antiretroviral therapy era, a loss of specific antibodies was seen. Our objective with this study was to describe the loss of specific antibodies during treatment with highly active antiretroviral therapy. METHODS: In a prospective, single-center, cohort study of

  8. 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.

  9. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  10. Use of Candida-specific chicken egg yolk antibodies to inhibit the adhering of Candida to denture base materials: prevention of denture stomatitis.

    Science.gov (United States)

    Kamikawa, Yoshiaki; Fujisaki, Junichi; Nagayama, Tomohiro; Kawasaki, Kiyotsugu; Hirabayashi, Daisuke; Hamada, Tomofumi; Sakamoto, Ryoich; Mukai, Hiroshi; Sugihara, Kazumasa

    2016-09-01

    Polyclonal anti-Candida chicken egg yolk antibodies (anti-IgY) were used to investigate the prevention of adherence of Candida species to denture base material in vitro. Candida is a potential virulence factor that can cause systemic infection and even death in immunocompromised individuals. Because long-term antifungal treatment may lead to the emergence of drug-resistant strains, it is necessary to develop novel preventive measures and treatments for candidiasis. Three types of chicken egg yolk antibodies were used in this study: non-specific antibody (control IgY), Candida albicans-specific antibody (anti-C.a.IgY) and Candida glabrata-specific antibody (anti-C.g.IgY). A mixture of different dilutions of each antibody with a suspension of Candida species and denture base material was incubated for 3 h, and then the colony-forming units of Candida on the denture base material were counted. Compared with control IgY, anti-C.a.IgY and anti-C.g.IgY significantly inhibited the adherence of C. albicans, but anti-C.a.IgY tended to be more potent than anti-C.g.IgY. The adherence of C. glabrata was also inhibited significantly by anti-C.a.IgY and anti-C.g.IgY with almost equivalent potency, indicating that their actions against C. glabrata were comparable. This study revealed the inhibitory effects of anti-C.a.IgY and anti-C.g.IgY against the adherence of C. albicans and C. glabrata to denture base material. This finding indicates the possibility of a beneficial effect of IgYs for the prevention of denture stomatitis and candidiasis in clinical settings. © 2014 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.

  11. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  12. Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment.

    Science.gov (United States)

    Stewart, Alex; Harrison, Joseph S; Regula, Lauren K; Lai, Jonathan R

    2013-04-08

    Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464-5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1-3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high

  13. Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk.

    Science.gov (United States)

    Lu, Ying; Wang, Weiming; Mao, Huiming; Hu, Hai; Wu, Yanling; Chen, Bing-Guan; Liu, Zhongmin

    2011-01-01

    Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37°C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Dietary cinnamaldehyde enhances acquisition of specific antibodies following helminth infection in pigs

    DEFF Research Database (Denmark)

    Williams, Andrew R.; Hansen, Tina V. A.; Krych, Lukasz

    2017-01-01

    immune responses during infection with an enteric pathogen. We examined the effect of dietary CA on plasma antibody levels in parasite-naïve pigs, and subsequently acquisition of humoral immune responses during infection with the parasitic nematode Ascaris suum. Parasite-naïve pigs fed diets supplemented...... with CA had higher levels of total IgA and IgG in plasma, and A. suum-infected pigs fed CA had higher levels of parasite-specific IgM and IgA in plasma 14days post-infection. Moreover, dietary CA increased expression of genes encoding the B-cell marker CD19, sodium/glucose co-transporter1 (SCA5L1...

  15. Specification of ROP flux shape

    Energy Technology Data Exchange (ETDEWEB)

    Min, Byung Joo [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of); Gray, A [Atomic Energy of Canada Ltd., Chalk River, ON (Canada)

    1997-06-01

    The CANDU 9 480/SEU core uses 0.9% SEU (Slightly Enriched Uranium) fuel. The use f SEU fuel enables the reactor to increase the radial power form factor from 0.865, which is typical in current natural uranium CANDU reactors, to 0.97 in the nominal CANDU 9 480/SEU core. The difference is a 12% increase in reactor power. An additional 5% increase can be achieved due to a reduced refuelling ripple. The channel power limits were also increased by 3% for a total reactor power increase of 20%. This report describes the calculation of neutron flux distributions in the CANDU 9 480/SEU core under conditions specified by the C and I engineers. The RFSP code was used to calculate of neutron flux shapes for ROP analysis. Detailed flux values at numerous potential detector sites were calculated for each flux shape. (author). 6 tabs., 70 figs., 4 refs.

  16. Specification of ROP flux shape

    International Nuclear Information System (INIS)

    Min, Byung Joo; Gray, A.

    1997-06-01

    The CANDU 9 480/SEU core uses 0.9% SEU (Slightly Enriched Uranium) fuel. The use f SEU fuel enables the reactor to increase the radial power form factor from 0.865, which is typical in current natural uranium CANDU reactors, to 0.97 in the nominal CANDU 9 480/SEU core. The difference is a 12% increase in reactor power. An additional 5% increase can be achieved due to a reduced refuelling ripple. The channel power limits were also increased by 3% for a total reactor power increase of 20%. This report describes the calculation of neutron flux distributions in the CANDU 9 480/SEU core under conditions specified by the C and I engineers. The RFSP code was used to calculate of neutron flux shapes for ROP analysis. Detailed flux values at numerous potential detector sites were calculated for each flux shape. (author). 6 tabs., 70 figs., 4 refs

  17. Donor-specific anti-HLA antibodies with antibody-mediated rejection and long-term outcomes following heart transplantation.

    Science.gov (United States)

    Clerkin, Kevin J; Farr, Maryjane A; Restaino, Susan W; Zorn, Emmanuel; Latif, Farhana; Vasilescu, Elena R; Marboe, Charles C; Colombo, Paolo C; Mancini, Donna M

    2017-05-01

    Donor-specific anti-HLA antibodies (DSA) are common after heart transplantation and are associated with rejection, cardiac allograft vasculopathy, and mortality. A noninvasive diagnostic test for pathologic antibody-mediated rejection (pAMR) does not exist. From January 1, 2010, through August 31, 2013, 221 consecutive adult patients underwent heart transplantation and were followed through October 1, 2015. The primary objective was to determine whether the presence of DSA could detect AMR at the time of pathologic diagnosis. Secondary analyses included association of DSA (stratified by major histocompatibility complex class and de novo status) during AMR with new graft dysfunction, graft loss (mortality or retransplantation), and development of cardiac allograft vasculopathy. During the study period, 69 patients (31.2%) had DSA (24% had de novo DSA), and there were 74 episodes of pAMR in 38 patients. Sensitivity of DSA at any mean fluorescence intensity to detect concurrent pAMR was only 54.3%. The presence of any DSA during pAMR increased the odds of graft dysfunction (odds ratio = 5.37; 95% confidence interval [CI], 1.34-21.47; p = 0.018), adjusting for age, sex, and timing of AMR. Circulating class II DSA after transplantation increased risk of future pAMR (hazard ratio = 2.97; 95% CI, 1.31-6.73; p = 0.009). Patients who developed de novo class II DSA had 151% increased risk of graft loss (contingent on 30-day survival) compared with patients who did not have DSA (95% CI, 1.11-5.69; p = 0.027). DSA were inadequate to diagnose pAMR. Class II DSA provided prognostic information regarding future pAMR, graft dysfunction with pAMR, and graft loss. Copyright © 2017 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

  18. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

    Directory of Open Access Journals (Sweden)

    George P Anderson

    Full Text Available Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

  19. Antimitochondrial antibodies and other antibodies in primary biliary cirrhosis: diagnostic and prognostic value.

    Science.gov (United States)

    Muratori, Luigi; Granito, Alessandro; Muratori, Paolo; Pappas, Georgios; Bianchi, Francesco B

    2008-05-01

    Antimitochondrial antibodies (AMA) are the serologic cornerstone in the diagnosis of primary biliary cirrhosis (PBC), even if they are not detectable in a proportion of patients, notwithstanding the most sensitive and sophisticated technologies used. To fill in the serologic gap in AMA-negative PBC, there is sound evidence to consider antinuclear antibody (ANA) patterns, such as anti-multiple nuclear dots and anti-membranous/rim-like, as PBC-specific surrogate hallmarks of the disease, and their detection can be considered virtually diagnostic. Furthermore, particular ANA specificities, such as anti-gp210, anti-p62, anticentromere antibodies, and anti-dsDNA, may provide additional diagnostic and prognostic information.

  20. Generalized Platform for Antibody Detection using the Antibody Catalyzed Water Oxidation Pathway

    OpenAIRE

    Welch, M. Elizabeth; Ritzert, Nicole L.; Chen, Hongjun; Smith, Norah L.; Tague, Michele E.; Xu, Youyong; Baird, Barbara A.; Abru?a, H?ctor D.; Ober, Christopher K.

    2014-01-01

    Infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. We report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. This biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the antibody ...

  1. Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase

    International Nuclear Information System (INIS)

    Berguido, F.J.; Bodjo, S.C.; Loitsch, A.; Diallo, A.

    2016-01-01

    Full text: Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The casual agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbilivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did not cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR. (author)

  2. Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60

    Directory of Open Access Journals (Sweden)

    O. Yu. Galkin

    2017-02-01

    Full Text Available The goal of this work was to study sensitivity and specificity of the developed ELISA set for the identification of IgG antibodies against Chlamydia trachomatis HSP-60 (using biotinylated tyramine-based signal amplification system. The study was conducted using a panel of characterized sera, as well as two reference ELISA sets of similar purpose. According to the results of ELISA informative value parameters, the ELISA we have developed showed the highest specificity and sensitivity parameters (no false negative or false positive results were registered. In 4 out of 15 intralaboratory panel serum samples initially identified as negative, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive. The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples. Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result. High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven. Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant ELISA-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents.

  3. Targeting Malignant Brain Tumors with Antibodies

    Directory of Open Access Journals (Sweden)

    Rok Razpotnik

    2017-09-01

    Full Text Available Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood–brain barrier (BBB makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs, and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A “Trojan horse” method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT. Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs and neural stem cells (NSCs show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts, are making their way into glioma treatment as another type of cell

  4. 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.

  5. 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.

  6. 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.

  7. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  8. Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry.

    Science.gov (United States)

    Kawahara, Akihiko; Taira, Tomoki; Abe, Hideyuki; Watari, Kosuke; Murakami, Yuichi; Fukumitsu, Chihiro; Takase, Yorihiko; Yamaguchi, Tomohiko; Azuma, Koichi; Akiba, Jun; Ono, Mayumi; Kage, Masayoshi

    2014-02-01

    Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. © 2013 American Cancer Society.

  9. Automatized spleen segmentation in non-contrast-enhanced MR volume data using subject-specific shape priors

    Science.gov (United States)

    Gloger, Oliver; Tönnies, Klaus; Bülow, Robin; Völzke, Henry

    2017-07-01

    To develop the first fully automated 3D spleen segmentation framework derived from T1-weighted magnetic resonance (MR) imaging data and to verify its performance for spleen delineation and volumetry. This approach considers the issue of low contrast between spleen and adjacent tissue in non-contrast-enhanced MR images. Native T1-weighted MR volume data was performed on a 1.5 T MR system in an epidemiological study. We analyzed random subsamples of MR examinations without pathologies to develop and verify the spleen segmentation framework. The framework is modularized to include different kinds of prior knowledge into the segmentation pipeline. Classification by support vector machines differentiates between five different shape types in computed foreground probability maps and recognizes characteristic spleen regions in axial slices of MR volume data. A spleen-shape space generated by training produces subject-specific prior shape knowledge that is then incorporated into a final 3D level set segmentation method. Individually adapted shape-driven forces as well as image-driven forces resulting from refined foreground probability maps steer the level set successfully to the segment the spleen. The framework achieves promising segmentation results with mean Dice coefficients of nearly 0.91 and low volumetric mean errors of 6.3%. The presented spleen segmentation approach can delineate spleen tissue in native MR volume data. Several kinds of prior shape knowledge including subject-specific 3D prior shape knowledge can be used to guide segmentation processes achieving promising results.

  10. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1982-01-01

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG 1 subclass. Affinities of antibodies for TSH were in the range 2 x 10 8 -5 x 10 10 M -1 . Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

  11. Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Paulius Lukas Tamošiūnas

    2014-01-01

    Full Text Available Porcine parvovirus (PPV is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs. Nine monoclonal antibodies (MAbs against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

  12. Detection of FMD virus type specific IgG1, IgG2 and IgA antibodies in milk and serum of buffaloes vaccinated with oil adjuvanted polyvalent FMD vaccine

    Directory of Open Access Journals (Sweden)

    R. Sharma

    2010-02-01

    Full Text Available The present investigation was carried out on 15 randomly selected milch buffaloes divided into three groups on the basis of lactation at an organized farm, to study the foot and mouth disease virus type specific antibodies in milk and serum following FMD vaccination. Milk and serum samples collected before vaccination i.e. 0 day and on 7, 14, 28, 42 and 56 days post vaccination, were analyzed for the detection of FMD virus specific IgG1, IgG2 and IgA antibody response by indirect double antibody sandwich ELISA. Significant FMD virus type specific antibody titres (IgG1, IgG2 and IgA were detected in milk and serum of buffaloes on different days post vaccination, though the levels of antibodies were lower in milk as compared to serum. FMD virus type specific IgG1 was found to be the predominant subclass as compared to IgG2 and IgA both in milk and serum of vaccinated buffaloes. Milk and serum IgG1, IgG2 and IgA antibody titres were positively correlated with values of regression coefficient (R as 0.506, 0.434 and 0.396, respectively.

  13. A point-of-care test for measles diagnosis: detection of measles-specific IgM antibodies and viral nucleic acid.

    Science.gov (United States)

    Warrener, Lenesha; Slibinskas, Rimantas; Chua, Kaw Bing; Nigatu, Wondatir; Brown, Kevin E; Sasnauskas, Kestutis; Samuel, Dhanraj; Brown, David

    2011-09-01

    To evaluate the performance of a newly developed point-of-care test (POCT) for the detection of measles-specific IgM antibodies in serum and oral fluid specimens and to assess if measles virus nucleic acid could be recovered from used POCT strips. The POCT was used to test 170 serum specimens collected through measles surveillance or vaccination programmes in Ethiopia, Malaysia and the Russian Federation: 69 were positive for measles immunoglobulin M (IgM) antibodies, 74 were positive for rubella IgM antibodies and 7 were positive for both. Also tested were 282 oral fluid specimens from the measles, mumps and rubella (MMR) surveillance programme of the United Kingdom of Great Britain and Northern Ireland. The Microimmune measles IgM capture enzyme immunoassay was the gold standard for comparison. A panel of 24 oral fluids was used to investigate if measles virus haemagglutinin (H) and nucleocapsid (N) genes could be amplified by polymerase chain reaction directly from used POCT strips. With serum POCT showed a sensitivity and specificity of 90.8% (69/76) and 93.6% (88/94), respectively; with oral fluids, sensitivity and specificity were 90.0% (63/70) and 96.2% (200/208), respectively. Both H and N genes were reliably detected in POCT strips and the N genes could be sequenced for genotyping. Measles virus genes could be recovered from POCT strips after storage for 5 weeks at 20-25 °C. The POCT has the sensitivity and specificity required of a field-based test for measles diagnosis. However, its role in global measles control programmes requires further evaluation.

  14. Antigen-specific H1N1 influenza antibody responses in acute respiratory tract infections and their relation to influenza infection and disease course.

    Science.gov (United States)

    Haran, John Patrick; Hoaglin, David C; Chen, Huaiqing; Boyer, Edward W; Lu, Shan

    2014-08-01

    Early antibody responses to influenza infection are important in both clearance of virus and fighting the disease. Acute influenza antibody titers directed toward H1-antigens and their relation to infection type and patient outcomes have not been well investigated. Using hemagglutination inhibition (HI) assays, we aimed to characterize the H1-specific antibody titers in patients with influenza infection or another respiratory infection before and after the H1N1-pandemic influenza outbreak. Among patients with acute influenza infection we related duration of illness, severity of symptoms, and need for hospitalization to antibody titers. There were 134 adult patients (average age 34.7) who presented to an urban academic emergency department (ED) from October through March during the 2008-2011 influenza seasons with symptoms of fever and a cough. Nasal aspirates were tested by viral culture, and peripheral blood serum was run in seven H1-subtype HI assays. Acutely infected influenza patients had markedly lower antibody titers for six of the seven pseudotype viruses. For the average over the seven titers (log units, base 2) their mean was 7.24 (95% CI 6.88, 7.61) compared with 8.60 (95% CI 8.27, 8.92) among patients who had a non-influenza respiratory illness, pinfection, titers of some antibodies correlated with severity of symptoms and with total duration of illness (pacute respiratory infections, lower concentrations of H1-influenza-specific antibodies were associated with influenza infection. Among influenza-infected patients, higher antibody titers were present in patients with a longer duration of illness and with higher severity-of-symptom scores. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. 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

  16. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  17. Cathepsin B Cleavage of vcMMAE-Based Antibody-Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific.

    Science.gov (United States)

    Gikanga, Benson; Adeniji, Nia S; Patapoff, Thomas W; Chih, Hung-Wei; Yi, Li

    2016-04-20

    Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the V(H), V(L), or C(H)2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in K(M) or k(cat) values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different

  18. PET-based compartmental modeling of {sup 124}I-A33 antibody: quantitative characterization of patient-specific tumor targeting in colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zanzonico, Pat; O' Donoghue, Joseph A.; Humm, John L. [Memorial Sloan Kettering Cancer Center, Department of Medical Physics, New York, NY (United States); Carrasquillo, Jorge A.; Pandit-Taskar, Neeta; Ruan, Shutian; Larson, Steven M. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Smith-Jones, Peter [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Stony Brook School of Medicine, Departments of Psychiatry and Radiology, Stony Brook, NY (United States); Divgi, Chaitanya [Columbia University Medical Center, New York, NY (United States); Scott, Andrew M. [La Trobe University, Olivia Newton-John Cancer Research Institute, Melbourne (Australia); Kemeny, Nancy E.; Wong, Douglas; Scheinberg, David [Memorial Sloan Kettering Cancer Center, Department of Medicine, New York, NY (United States); Fong, Yuman [Memorial Sloan Kettering Cancer Center, Department of Surgery, New York, NY (United States); City of Hope, Department of Surgery, Duarte, CA (United States); Ritter, Gerd; Jungbluth, Achem; Old, Lloyd J. [Memorial Sloan Kettering Cancer Center, Ludwig Institute for Cancer Research, New York, NY (United States)

    2015-10-15

    The molecular specificity of monoclonal antibodies (mAbs) directed against tumor antigens has proven effective for targeted therapy of human cancers, as shown by a growing list of successful antibody-based drug products. We describe a novel, nonlinear compartmental model using PET-derived data to determine the ''best-fit'' parameters and model-derived quantities for optimizing biodistribution of intravenously injected {sup 124}I-labeled antitumor antibodies. As an example of this paradigm, quantitative image and kinetic analyses of anti-A33 humanized mAb (also known as ''A33'') were performed in 11 colorectal cancer patients. Serial whole-body PET scans of {sup 124}I-labeled A33 and blood samples were acquired and the resulting tissue time-activity data for each patient were fit to a nonlinear compartmental model using the SAAM II computer code. Excellent agreement was observed between fitted and measured parameters of tumor uptake, ''off-target'' uptake in bowel mucosa, blood clearance, tumor antigen levels, and percent antigen occupancy. This approach should be generally applicable to antibody-antigen systems in human tumors for which the masses of antigen-expressing tumor and of normal tissues can be estimated and for which antibody kinetics can be measured with PET. Ultimately, based on each patient's resulting ''best-fit'' nonlinear model, a patient-specific optimum mAb dose (in micromoles, for example) may be derived. (orig.)

  19. 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.)

  20. 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.

  1. 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.)

  2. Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

    Science.gov (United States)

    Collazo, M. Thelma; Garza, John A.; Hayhurst, Andrew

    2010-01-01

    Background There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype. Methods and Findings A single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 µL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism. Conclusions Our panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively. PMID:20098614

  3. Evaluation of HPV-16 and HPV-18 specific antibody measurements in saliva collected in oral rinses and merocel® sponges.

    Science.gov (United States)

    Parker, Katherine H; Kemp, Troy J; Pan, Yuanji; Yang, Zhen; Giuliano, Anna R; Pinto, Ligia A

    2018-05-03

    Current Human papillomavirus (HPV) L1 VLP vaccines protect against HPV-16 and HPV-18-associated cancers, in females and males. Although correlates of protection have not been identified, HPV-specific antibodies at sites of infection are thought to be the main mechanism of protection afforded by vaccination. Oral sampling has gained increased attention as a potential alternative to serum in monitoring immunity to vaccination and understanding local immunity in oral cancers. Serum was collected via venipuncture, and saliva was collected via oral rinses and Merocel® sponges from healthy volunteers: 16 unvaccinated females, 6 females (ages 24-41) and 6 mid-adult aged males (ages 27-45) recipients of three doses of the HPV-16/18/6/11 vaccine (Gardasil®). Mid-adult male vaccine trial participants were compared to female participants. Samples were tested for anti-HPV-16 and anti-HPV-18 immunoglobulin G levels by an L1 virus-like particle-based enzyme-linked immunosorbent assay (ELISA). All vaccinated participants had detectable serum anti-HPV-16 and anti-HPV-18 antibodies. Optimal standard concentration range and sample serial dilutions for oral rinses were determined. The standard curve was not affected by the type of solution examined. Reproducibility of HPV-16 and HPV-18 antibody titers in mouthwash (overall CV  0.9) was observed for sera spiked controls in both solutions. HPV-16 and HPV-18 specific antibodies were detectable in saliva from vaccine recipients, both in mouthwash and in Merocel® sponges but levels were several logs lower than those in serum. This study confirms the application of HPV-16 and HPV-18 ELISAs currently used in sero-epidemiological studies of immunogenicity of HPV vaccines for use with oral samples. Oral samples may be a useful resource for the detection of HPV-16 and HPV-18-specific antibodies in saliva following vaccination. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Destructive arthritis in a patient with chikungunya virus infection with persistent specific IgM antibodies

    Directory of Open Access Journals (Sweden)

    Receveur Marie-Catherine

    2009-12-01

    Full Text Available Abstract Background Chikungunya fever is an emerging arboviral disease characterized by an algo-eruptive syndrome, inflammatory polyarthralgias, or tenosynovitis that can last for months to years. Up to now, the pathophysiology of the chronic stage is poorly understood. Case presentation We report the first case of CHIKV infection with chronic associated rheumatism in a patient who developed progressive erosive arthritis with expression of inflammatory mediators and persistence of specific IgM antibodies over 24 months following infection. Conclusions Understanding the specific features of chikungunya virus as well as how the virus interacts with its host are essential for the prevention, treatment or cure of chikungunya disease.

  5. 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

  6. Myasthenic Crisis Complicated with Myxedema, Positive for Both Anti-acetylcholine Receptor and Anti-muscle-specific Tyrosine Kinase Antibodies.

    Science.gov (United States)

    Horiuchi, Kazuhiro; Nagai, Azusa; Wakita, Masahiro; Ito, Shotaro; Takamura, Kei; Houzen, Hideki

    2018-01-15

    We herein report the case of myasthenic crisis occurring in a 51-year-old man. He had experienced ptosis, increased body weight with edema, and fatigue with dyspnea. He presented at our emergency department with disturbed consciousness. He was originally diagnosed with myxedema coma, and he required artificial respiration. Because his weakness persisted and he was positive for anti-acetylcholine receptor antibodies and anti-muscle-specific tyrosine kinase antibodies, we diagnosed myasthenic crisis after various examinations. His clinical response to treatment was good and he was discharged in an ambulatory status 3 months after admission. This case demonstrates that myasthenic crisis may occur in association with myxedema.

  7. [In vitro immunization for the production of antibodies to tetanus toxin and toxoid. 1. Systems for the detection of in vitro synthetized specific immunoglobulins. Strategies of test development].

    Science.gov (United States)

    Kiessig, S T; Jahn, S; Porstmann, T; von Baehr, R

    1987-01-01

    By means of semipurified tetanus toxin for solid phase coating in an enzyme immunoassay (ELISA) for detection of specific IgG and IgM antibodies a detection limit of 0.02 IU per litre was achieved. The addition of serum from animals like horses or goats as inert protein to the dilution medium was omitted to prevent a displacement of human antibodies by antitetanus antibodies present in the animals sera. The specificity of the ELISA was demonstrated by inhibition experiments with soluble antigen and in an ELISA for detection of anti-tetanus toxin antibodies from mice immunized with the toxoid from the different purification steps.

  8. Site-specific conjugation and labelling of prostate antibody 7E11C5.3 (CYT-351) with technetium-99m

    International Nuclear Information System (INIS)

    Stalteri, M.A.; Mather, S.J.; Belinka, B.A.; Coughlin, D.J.; Chengazi, V.U.; Britton, K.E.

    1997-01-01

    Attachment of chelating agents to the sugar residues of antibodies for subsequent radiolabelling is an attractive approach since it may have less effect on the immunoreactivity than attachment through lysine residues, which are distributed throughout the antibody and may be present near the antigen binding site. We have attached a new hydrazide-linked chelator CYT-395 (Cytogen Corp., Princeton, N.J.) to the sugar residues of the anti-prostate monoclonal antibody 7E11C5.3 and optimised the conditions for labelling the conjugate with technetium-99m in order to compare the conjugate to 7E11C5.3 antibody labelled directly with technetium using a mercaptoethanol reduction technique. Labelling yields of 70%-90% were obtained at specific activities up to 2000 MBq/mg antibody. The stability of the technetium-labelled conjugate in plasma or to a challenge with 0.1 or 1.0 mM cysteine was similar to that of direct-labelled antibody. In nine patients with prostate cancer, the plasma clearance of the labelled conjugate followed a two-compartment model, with an average β-phase half-life of 31.4±3.9 h. The average urinary clearance at 24 h was 15.3±5.0% of the injected dose. In this group of patients there was no significant difference between the blood and urine clearance of the labelled conjugate, and the clearances of the direct-labelled antibody. (orig.). With 5 figs

  9. 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

  10. Comparison of dot-ELISA and standard ELISA for detection of Neisseria meningitidis outer membrane complex-specific antibodies

    Directory of Open Access Journals (Sweden)

    Elza FT Belo

    Full Text Available Dot-ELISA using the outer membrane complex antigens of Neisseria meningitidis as a target was standardized for rapid detection of meningococcal-specific antibodies in human serum. We investigated the level of meningococcal-specific IgG, IgA, and IgM in serum using dot-ELISA with outer membrane antigens prepared from Neisseria meningitidis serotype B:4.19:P1.15,3,7,9 (a strain isolated from a Brazilian epidemic. The dot-ELISA is based on the same principles as the standard ELISA and is useful for detection of anti-N. meningitidis B antibodies in serum of patients with meningococcal infections. For the assay, outer membrane complexes (OMCs were absorbed by nitrocellulose membrane and blocked with a 5% skim milk solution. Serum samples were drawn upon hospital admission and during convalescence from patients with meningococcal septicemia, and single samples were drawn from uninfected controls. We retrospectively examined a total of 57 serum samples: 35 from patients infected with N. meningitidis B, 12 from patients infected with Haemophilus influenzae b, and 10 from health individuals. When performed at room temperature, dot-ELISA took approximately four hours to perform, and the optimum antigen concentration was 0.42 µg per dot. The specificity of IgG, IgM, and IgA demonstrates that dot-ELISA using OMCs from N. meningitidis B as a target is suitable for serologic verification of clinically suspected meningococcal disease in patients and for titer determination of antibodies produced during different phases of natural infection. Furthermore, the sensitivity of dot-ELISA was comparable to that of standard ELISA. Overall, dot-ELISA is simple to perform, rapid, and low cost. Further validation of the test as a screening tool is required.

  11. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    Science.gov (United States)

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Measurement of antibodies to tubulin by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Mead, G M; Cowin, P; Whitehouse, J M.A. [CRC Medical Oncology Unit, Southampton General Hospital, Southampton, U.K.

    1979-07-24

    A solid-phase double antibody radioimmunoassay capable of measuring antibody to tubulin, the principal component of microtubules, is described. This assay is simple, combining sensitivity with specificity and also allowing determination of antibody subclasses.

  13. Bispecific antibodies and their use in applied research

    Directory of Open Access Journals (Sweden)

    Harshit Verma

    Full Text Available Bispecific antibodies (BsAb can, by virtue of combining two binding specificities, improve the selectivity and efficacy of antibody-based treatment of human disease. Antibodies with two distinct binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo immunodiagnosis, therapy and for improving immunoassays. They have shown great promise for targeting cytotoxic effector cells, delivering radionuclides, toxins or cytotoxic drugs to specific targets, particularly tumour cells. The development of BsAb research goes through three main stages: chemical cross linking of murine-derived monoclonal antibody, hybrid hybridomas and engineered BsAb. This article is providing the potential applications of bispecific antibodies. [Vet World 2012; 5(12.000: 775-780

  14. Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

    International Nuclear Information System (INIS)

    Won, JaeSeon; Nam, PilWon; Lee, YongChan; Choe, MuHyeon

    2009-01-01

    Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G 4 S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38] 2 ) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.

  15. Cefditoren and ceftriaxone enhance complement-mediated immunity in the presence of specific antibodies against antibiotic-resistant pneumococcal strains.

    Directory of Open Access Journals (Sweden)

    Elisa Ramos-Sevillano

    Full Text Available BACKGROUND: Specific antibodies mediate humoral and cellular protection against invading pathogens such as Streptococcus pneumoniae by activating complement mediated immunity, promoting phagocytosis and stimulating bacterial clearance. The emergence of pneumococcal strains with high levels of antibiotic resistance is of great concern worldwide and a serious threat for public health. METHODOLOGY/PRINCIPAL FINDINGS: Flow cytometry was used to determine whether complement-mediated immunity against three antibiotic-resistant S. pneumoniae clinical isolates is enhanced in the presence of sub-inhibitory concentrations of cefditoren and ceftriaxone. The binding of acute phase proteins such as C-reactive protein and serum amyloid P component, and of complement component C1q, to pneumococci was enhanced in the presence of serum plus either of these antibiotics. Both antibiotics therefore trigger the activation of the classical complement pathway against S. pneumoniae. C3b deposition was also increased in the presence of specific anti-pneumococcal antibodies and sub-inhibitory concentrations of cefditoren and ceftriaxone confirming that the presence of these antibiotics enhances complement-mediated immunity to S. pneumoniae. CONCLUSIONS/SIGNIFICANCE: Using cefditoren and ceftriaxone to promote the binding of acute phase proteins and C1q to pneumococci, and to increase C3b deposition, when anti-pneumococcal antibodies are present, might help reduce the impact of antibiotic resistance in S. pneumoniae infections.

  16. Comparison of the specificity of antibodies to VAR2CSA in Cameroonian multigravidae with and without placental malaria

    DEFF Research Database (Denmark)

    Babakhanyan, Anna; Fang, Rui; Wey, Andrew

    2015-01-01

    BACKGROUND: Antibodies (Ab) to VAR2CSA prevent Plasmodium falciparum-infected erythrocytes from sequestrating in the placenta, i.e., prevent placental malaria (PM). The specificity of Ab to VAR2CSA associated with absence of PM is unknown. Accordingly, differences in the specificity of Ab to VAR2......CSA were compared between multigravidae with and without PM who had Ab to VAR2CSA. METHODS: In a retrospective case-control study, plasma collected from Cameroonian multigravidae with (n = 96) and without (n = 324) PM were screened in 21 assays that measured antibody levels to full length VAR2CSA (FV2......), individual VAR2CSA DBL domains, VAR2CSA domains from different genetic backgrounds (variants), as well as proportion of high avidity Ab to FV2. RESULTS: Multigravidae with and without PM had similar levels of Ab to FV2, the six VAR2CSA DBL domains and different variants, while the proportion of high avidity...

  17. THE USE OF THE ANTI-VENOM SPECIFIC ANTIBODIES ISOLATED FROM DUCK EGGS FOR INACTIVATION OF THE VIPER VENOM

    Directory of Open Access Journals (Sweden)

    ADRIANA CRISTE

    2008-05-01

    Full Text Available The activity of specific anti-venom can be demonstrated using protection test in laboratory mice. Our study aimed to emphasize the possibility of viper venom inactivation by the antibodies produced and isolated from duck eggs and also to the activation concentration of these antibodies. The venom used for inoculation was harvested from two viper species (Vipera ammodytes and Vipera berus. The immunoglobulin extract had a better activity on the venom from Vipera berus compared to the venom from Vipera ammodytes. This could be the result of a better immunological response, as consequence of the immunization with this type of venom, compared to the response recorded when the Vipera ammodytes venom was used. Besides the advantages of low cost, high productivity and reduced risk of anaphylactic shock, the duck eggs also have high activity up to dilutions of 1/16, 1/32, respectively, with specific activity and 100 surviving in individuals which received 3 x DL50.

  18. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Bradley

    2011-03-31

    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  19. Detection of anti-lactoferrin antibodies and anti-myeloperoxidase antibodies in autoimmune hepatitis: a retrospective study.

    Science.gov (United States)

    Tan, Liming; Zhang, Yuhong; Peng, Weihua; Chen, Juanjuan; Li, Hua; Ming, Feng

    2014-01-01

    Anti-lactoferrin antibodies (ALA) and anti-myeloperoxidase antibodies (AMPA) are specific serological markers for autoimmune hepatitis (AIH). The project aimed to detect ALA and AMPA and explore their clinical significances in AIH patients. 59 AIH patients, 217 non AIH patients, and 50 healthy controls were enrolled in this study. ALA and AMPA were detected by ELISA. Antineutropil cytoplasmic antibodies (ANCA) and anti-smooth muscle antibodies (ASMA) were examined by indirect immunofluorescence. Antimitochondrial antibody M2 subtype (AMA-M2), anti-liver kidney microsomal antibody Type 1 (LKM1), anti-liver cytosol antibody Type 1 (LC1), and anti-soluble liver antigen/liver-pancreas antibodies (SLA/LP) were tested by immunoblot. The positivity for ALA was 18.6% in AIH group, only one patient in non-AIH group was positive for ALA; the positivity for AMPA was 59.3% in AIH group, with significant differences (P < 0.01) compared with other groups. The specificities for ALA and AMPA were 99.63% and 97.75%; the sensitivities were 18.64% and 59.32%; and the accuracy rates were 84.97% and 90.80%, respectively. A certain correlation was observed between ALA and SLA/LP, AMPA and ANCA, ASMA in AIH group. ALA and AMPA were associated with AIH, and had high clinical diagnostic value. Co-detection with other relative autoantibodies could play an important role in differential diagnosis of AIH.

  20. Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species

    DEFF Research Database (Denmark)

    Koutaniemi, Sanna; Guillon, Fabienne; Tranquet, Olivier

    2012-01-01

    Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituen...

  1. 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.

  2. Rapid screening of monoclonal antibodies: new 'microstick' radioimmunoassay

    International Nuclear Information System (INIS)

    Scheinberg, D.A.; Strand, M.; Wilsnack, R.

    1983-01-01

    A new system for assaying monoclonal antibodies consisting of an 8 x 12 array of sticks which fits into a 96-well microtiter plate is described. Tests using virus specific monoclonal antibodies and virus proteins demonstrated sensitivity equivalent to the conventional microtiter plate assay. Antibody production, antigen specific antibody, and immunoglobulin isotypes could be measured under sterile conditions directly in the original fusion mixture wells and much greater rapidity than with the microtiter plate assay. (Auth.)

  3. Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl β-1,6 glucosamine specific antibody production using biofilm-embedded bacteria

    Science.gov (United States)

    Pérez, M. M.; Prenafeta, A.; Valle, J.; Penadés, J.; Rota, C.; Solano, C.; Marco, J.; Grilló, M.J.; Lasa, I.; Irache, J.M.; Maira-Litran, T.; Jiménez-Barbero, J.; Costa, L.; Pier, G.B.; de Andrés, D.; Amorena, B.

    2010-01-01

    Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-β-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis. PMID:19428854

  4. Monoclonal antibodies: potential role in radiation therapy and oncology

    International Nuclear Information System (INIS)

    Order, S.E.

    1982-01-01

    Specificity, which is a hallmark of the immune system, will be used in radiation oncology in both diagnosis and therapy through the application of radiolabelled monoclonal and polyclonal antibodies. Antigenic specificities, antibody preparations, and the tumor as a target for radiolabelled antibody is reviewed. Several clinical situations, i.e. single tumor cell suspensions, intraperitoneal single cells and masses, and solid tumors are reviewed in regard to both immune antibody targeting and specific differences between tumors in these regions. The concentration of tumor associated antigens is introductory to radiolabelled antibodies in diagnosis. In the radiation therapy of solid tumors, data regarding tumor dose, tumor effective half-life, varied antibody preparations, and the use of radiolabelled antibody as a method of tumor implantation is discussed using antiferritin 131 I-IgG as a model in hepatoma. The theoretical applications of monoclonal antibody integrated in cancer therapy are then presented as a new goal for future development

  5. Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression

    DEFF Research Database (Denmark)

    Tsimikas, Sotirios; Miyanohara, Atsushi; Hartvigsen, Karsten

    2011-01-01

    We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibod...... antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis....

  6. Expression of recombinant Antibodies

    Directory of Open Access Journals (Sweden)

    André eFrenzel

    2013-07-01

    Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  7. 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.

  8. 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.

  9. Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

    Czech Academy of Sciences Publication Activity Database

    Ohradanova-Repic, A.; Nogueira, E.; Hartl, I.; Gomes, A.C.; Preto, A.; Steinhuber, E.; Muehlgrabner, V.; Repic, M.; Kuttke, M.; Zwirzitz, A.; Prouza, M.; Suchánek, M.; Wozniak-Knopp, G.; Hořejší, Václav; Schabbauer, G.; Cavaco-Paulo, A.; Stockinger, H.

    2018-01-01

    Roč. 14, č. 1 (2018), s. 123-130 ISSN 1549-9634 Institutional support: RVO:68378050 Keywords : Active targeting * Liposome functionalization * Immunoliposome * Antibody engineering * Recombinant Fab antibody fragment Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 5.720, year: 2016

  10. Staphylococcal superantigen-specific IgE antibodies: degree of sensitization and association with severity of asthma

    OpenAIRE

    Elabras Filho, José; Mello, Fernanda Carvalho de Queiroz; Lupi, Omar; Bica, Blanca Elena Rios Gomes; Papi, José Angelo de Souza; França, Alfeu Tavares

    2016-01-01

    ABSTRACT Objective: To determine the presence of staphylococcal superantigen-specific IgE antibodies and degree of IgE-mediated sensitization, as well as whether or not those are associated with the severity of asthma in adult patients. Methods: This was a cross-sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of Rio de Janeiro, Brazil. Consecutive patients were divided into two groups according to the severity of asthma ba...

  11. 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)

  12. Absence of Association between Cord Specific Antibody Levels and Severe Respiratory Syncytial Virus (RSV) Disease in Early Infants: A Case Control Study from Coastal Kenya.

    Science.gov (United States)

    Nyiro, Joyce Uchi; Sande, Charles Jumba; Mutunga, Martin; Kiyuka, Patience Kerubo; Munywoki, Patrick Kioo; Scott, John Anthony G; Nokes, David James

    2016-01-01

    The target group for severe respiratory syncytial virus (RSV) disease prevention is infants under 6 months of age. Vaccine boosting of antibody titres in pregnant mothers could protect these young infants from severe respiratory syncytial virus (RSV) associated disease. Quantifying protective levels of RSV-specific maternal antibody at birth would inform vaccine development. A case control study nested in a birth cohort (2002-07) was conducted in Kilifi, Kenya; where 30 hospitalised cases of RSV-associated severe disease were matched to 60 controls. Participants had a cord blood and 2 subsequent 3-monthly blood samples assayed for RSV-specific neutralising antibody by the plaque reduction neutralisation test (PRNT). Two sample paired t test and conditional logistic regression were used in analyses of log2PRNT titres. The mean RSV log2PRNT titre at birth for cases and controls were not significantly different (P = 0.4) and remained so on age-stratification. Cord blood PRNT titres showed considerable overlap between cases and controls. The odds of RSV disease decreased with increase in log2PRNT cord blood titre. There was a 30% reduction in RSV disease per unit increase in log2PRNT titre (disease. Cord antibody levels show wide variation with considerable overlap between cases and controls. It is likely that, there are additional factors to specific PRNT antibody levels which determine susceptibility to severe RSV disease. In addition, higher levels of neutralizing antibody beyond the normal range may be required for protection; which it is hoped can be achieved by a maternal RSV vaccine.

  13. Antibody induction versus placebo, no induction, or another type of antibody induction for liver transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Wettergren, André; Wilson, Colin H

    2014-01-01

    . All 19 trials were with high risk of bias. Of the 19 trials, 16 trials were two-arm trials, and three trials were three-arm trials. Hence, we found 25 trial comparisons with antibody induction agents: interleukin-2 receptor antagonist (IL-2 RA) versus no induction (10 trials with 1454 participants....... Furthermore, serum creatinine was statistically significantly higher when T-cell specific antibody induction was compared with no induction (MD 3.77 μmol/L, 95% CI 0.33 to 7.21; low-quality evidence), as well as when polyclonal T-cell specific antibody induction was compared with no induction, but this small...... T-cell specific antibody induction, drug-related adverse events were less common among participants treated with interleukin-2 receptor antagonists (RR 0.23, 95% CI 0.09 to 0.63; low-quality evidence), but this was caused by the results from one trial, and trial sequential analysis could not exclude...

  14. Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis.

    Science.gov (United States)

    Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

    2017-06-12

    The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

  15. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

    DEFF Research Database (Denmark)

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual...

  16. Anti-Neospora caninum and anti-Sarcocystis spp. specific antibodies cross-react with Besnoitia besnoiti and influence the serological diagnosis of bovine besnoitiosis.

    Science.gov (United States)

    García-Lunar, P; Moré, G; Campero, L; Ortega-Mora, L M; Álvarez-García, G

    2015-11-30

    Bovine besnoitiosis control remains a challenge because the disease continues to spread and control relies solely on accurate diagnosis coupled to management measures. However, recent studies have reported that routinely used ELISAs may raise a high number of false-positive results. Herein, cross-reactions between Besnoitia besnoiti antigens and anti-Neospora caninum and/or anti-Sarcocystis spp.-specific antibodies were studied in an in house ELISA since N. caninum and Sarcocystis spp. are closely related parasites, and both infections are highly prevalent in cattle worldwide. The serum panel was composed of the following categories: sera from B. besnoiti-seronegative (n=75) and -seropositive cattle (n=66), B. besnoiti-based-ELISA false-positive reactors (n=96) together with N. caninum (n=36) and Sarcocystis spp. (n=42) -seropositive reference cattle sera. B. besnoiti tachyzoite based western blot (WB) results classified animals as seropositive or seronegative. Sera were analyzed for the detection of anti-N. caninum by WB and ELISA and anti-Sarcocystis spp.-specific antibodies by WB and IFAT. Those samples recognizing a Sarcocystis spp. 18-20 kDa antigenic region and N. caninum 17-18 kDa immunodominant antigen were considered to be Sarcocystis spp. and N. caninum seropositive, respectively. The category of B. besnoiti based-ELISA false-positive reactors showed the highest number of sera with specific anti-Sarcocystis spp. and anti-N. caninum antibodies (74%; 71/96), followed by the N. caninum-seropositive cattle category (52.8%; 19/36). In contrast, few B. besnoiti-seronegative and -seropositive cattle showed antibodies against Sarcocystis spp. and N. caninum (10.7%; 8/75 and 1.5%; 1/66), respectively). This study revealed that B. besnoiti false-positive ELISA results were associated not only with the presence of anti-N. caninum and anti-Sarcocystis spp. antibodies (χ(2): 78.36; pbovine besnoitiosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Thrombus imaging in a primate model with antibodies specific for an external membrane protein of activated platelets

    International Nuclear Information System (INIS)

    Palabrica, T.M.; Furie, B.C.; Konstam, M.A.; Aronovitz, M.J.; Connolly, R.; Brockway, B.A.; Ramberg, K.L.; Furie, B.

    1989-01-01

    The activated platelet is a potential target for the localization of thrombi in vivo since, after stimulation and secretion of granule contents, activated platelets are concentrated at sites of blood clot formation. In this study, we used antibodies specific for a membrane protein of activated platelets to detect experimental thrombi in an animal model. PADGEM (platelet activation-dependent granule-external membrane protein), a platelet alpha-granule membrane protein, is translocated to the plasma membrane during platelet activation and granule secretion. Since PADGEM is internal in unstimulated platelets, polyclonal anti-PADGEM and monoclonal KC4 antibodies do not bind to circulating resting platelets but do interact with activated platelets. Dacron graft material incubated with radiolabeled KC4 or anti-PADGEM antibodies in the presence of thrombin-activated platelet-rich plasma bound most of the antibody. Imaging experiments with 123I-labeled anti-PADGEM in baboons with an external arterial-venous Dacron shunt revealed rapid uptake in the thrombus induced by the Dacron graft; control experiments with 123I-labeled nonimmune IgG exhibited minimal uptake. Deep venous thrombi, formed by using percutaneous balloon catheters to stop blood flow in the femoral vein of baboons, were visualized with 123I-labeled anti-PADGEM. Thrombi were discernible against blood pool background activity without subtraction techniques within 1 hr. No target enhancement was seen with 123I-labeled nonimmune IgG. 123I-labeled anti-PADGEM cleared the blood pool with an initial half-disappearance time of 6 min and did not interfere with hemostasis. These results indicate that radioimmunoscintigraphy with anti-PADGEM antibodies can visualize thrombi in baboon models and is a promising technique for clinical thrombus detection in humans

  18. Kinetics of Epstein-Barr Virus (EBV) Neutralizing and Virus-Specific Antibodies after Primary Infection with EBV

    Science.gov (United States)

    Bu, Wei; Hayes, Gregory M.; Liu, Hui; Gemmell, Lorraine; Schmeling, David O.; Radecki, Pierce; Aguilar, Fiona; Burbelo, Peter D.; Woo, Jennifer; Balfour, Henry H.

    2016-01-01

    Prospective studies of antibodies to multiple Epstein-Barr virus (EBV) proteins and EBV neutralizing antibodies in the same individuals before, during, and after primary EBV infection have not been reported. We studied antibody responses to EBV in college students who acquired primary EBV infection during prospective surveillance and correlated the kinetics of antibody response with the severity of disease. Neutralizing antibodies and enzyme-linked immunosorbent assay (ELISA) antibodies to gp350, the major target of neutralizing antibody, reached peak levels at medians of 179 and 333 days after the onset of symptoms of infectious mononucleosis, respectively. No clear correlation was found between the severity of the symptoms of infectious mononucleosis and the peak levels of antibody to individual viral proteins or to neutralizing antibody. In summary, we found that titers of neutralizing antibody and antibodies to multiple EBV proteins increase over many months after primary infection with EBV. PMID:26888186

  19. Monoclonal antibody-based immunoassays.

    Science.gov (United States)

    Appleby, P; Reischl, U

    1998-01-01

    An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the application and use of immunoassays. Polyclonal reagents, with their associated problems of specificity and quality control, have now been largely replaced by readily available MAbs of potential immortality and well-defined specificity and affinity. This has resulted, in the last two decades, in a great expansion in the range of immunoassays available and also a significant improvement in their reproducibility and reliability.

  20. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  1. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    International Nuclear Information System (INIS)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-01-01

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV F , we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy

  2. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  3. [Possibilities of differentiation of antinuclear antibodies].

    Science.gov (United States)

    Müller, W; Rosenthal, M; Stojan, B

    1975-10-15

    Antinuclear antibodies can give diagnostic informations according to their titre values, the belonging to different classes of immune globulins and on the basis of different patterns of immunofluorescence connection. The determination of granulocyte-specific antibodies which frequently appear in progressive chronic polyarthritis further contributes to the differential-diagnostic classification of diseases of the connective tissue. An antibody against extractable nuclear antigen is specific for the so-called mixed connective tissue disease, an antimitochondrial antibody for the pseudo-LE-syndrome. Moreover, the own examinations resulted in a particularly high and frequent ability of complement fixation of the antinuclear factors in systematic lupus erythematosus and sclerodermy. In contrast to this in the progressive chronic polyarthritis the complement fixation was clearly more insignificant.

  4. 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.

  5. 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...

  6. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  7. Production and characterization of monoclonal antibodies specific to the strobilurin pesticide pyraclostrobin.

    Science.gov (United States)

    Mercader, Josep V; Suárez-Pantaleón, Celia; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2008-09-10

    Strobilurin fungicides are nowadays among the most important fungicides in the market of active agrochemicals. Pyraclostrobin, which belongs to the last generation of this family of molecules, shows a broader antifungal activity spectrum and higher efficiency and security profiles than previous fungicides. This paper describes the synthesis of functionalized haptens, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays (ELISA) for the detection of pyraclostrobin. A conformational analysis of hapten structure was performed, which provided relevant data concerning the length of the spacer arm. A very useful strategy has been followed for the screening of hybridomas, leading to the selection of a panel of high-affinity monoclonal antibodies to pyraclostrobin. Moreover, different immunoassays have been characterized using the conjugate-coated indirect ELISA format, and limits of detection below 0.1 microg/L have been obtained. Also, a simplified one-step procedure has been carried out with two indirect assays. Finally, these results have been compared with the performance of the same antibodies in the antibody-coated direct ELISA format.

  8. 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.

  9. Tumor detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Powe, J.; Hammond, N.D.

    1987-01-01

    Radioisotope conjugated to monoclonal antibody products has been used for imaging tumors targeted by the antibody. As imaging progresses, new sets of procedural and technical questions arise. In this chapter, we discuss several current problems in imaging tumor with radiolabeled monoclonal antibody. These include (1) methods for selection of specific antibody and, once the particular antibody is selected, which fragment form is to be used; (2) imaging procedures: what are the optimum imaging parameters, such as optimum time for imaging after administration of tracer and considerations regarding background subtraction; and (3) noninvasive quantitative techniques: quantitation of localization of antibody indirectly from quantitative information in the images.100 references

  10. Stability of rhenium-188 labeled antibody

    International Nuclear Information System (INIS)

    Lim, B. K.; Jung, J. M.; Jung, J. K.; Lee, D. S.; Lee, M. C.

    1999-01-01

    For clinical application of beta-emitter labeled antibody, high specific activity is important. Carrier-free Re-188 from W-188/Re-188 generator is an ideal radionuclide for this purpose. However, low stability of Re-188 labeled antibody, especially in high specific activity, due to radiolytic decomposition by high energy (2.1 MeV) beta ray was problem. We studied the stability of Re-188 labeled antibody, and stabilizing effect of several nontoxic radical-quenching agents. Pre-reduced monoclonal antibody (CEA79.4) was labeled with Re-188 by incubating with generator-eluted Re-188-perrhenate in the presence of stannous tartrate for 2 hr at room temperature. Radiochemical purity of each preparation was determined by chromatography (ITLC-SG/acetone, ITLC-SG/Umezawa, Whatman No.1/saline). Human serum albumin was added to the labeled antibodies(2%). Stability of Re-188-CEA79.4 was investigated in the presence of vitamin C, ethanol, or Tween 80 as radical-quenching agents. Specific activities of 4.29∼5.11 MBq/μg were obtained. Labeling efficiencies were 88±4%(n=12). Very low stability after removal of stannous tartrate from the preparation was observed. If stored after purging with N 2 , all the preparations were stable for 10 hr. However, if contacted with air, stability decreased. Perrhenate and Re-188-tartrate was major impurity in declined preparation (12∼47 and 9∼38% each, after 10 hr). Colloid-formation was not a significant problem in all cases. Addition of vitamin C stabilized the labeled antibodies either under N 2 or under air by reducing the formation of perrhenate. High specific activity Re-188 labeled antibody is unstable, especially, in the presence of oxygen. Addition of vitamin C increased the stability

  11. Monoclonal antibodies specific for the organophosphate pesticide azinphos-methyl

    NARCIS (Netherlands)

    Jones, WT; Harvey, D; Jones, SD; Ryan, GB; Wynberg, H; TenHoeve, W; Reynolds, PHS

    1995-01-01

    2-(2-Mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl,2-sulphide) methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos-methyl were prepared from mice immunized with the

  12. Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

    Science.gov (United States)

    Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

    2017-01-01

    The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z Bpa -Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z Bpa -Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z Bpa -Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL -1 , is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL -1 ). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Red Blood Cell Antibody Identification

    Science.gov (United States)

    ... antibodies may or may not be associated with adverse reactions, and identification of the specific type of RBC ... the only things that can cause a transfusion reaction. The recipient's immune ... or to drugs that the donor may have taken. Rarely, antibodies in the plasma ...

  14. A new immunization procedure for the obtention of anti-leucine enkephalin antibodies. Part I. Immunization procedure and physicochemical characteristics of antibodies.

    Science.gov (United States)

    Cupo, A; Vion-Dury, J; Jarry, T

    1986-10-01

    We described here a new immunization procedure to obtain high titre and high specific antibodies against Leu-enkephalin (LE). The immunogen form is composed of one part of LE conjugate and one part of LE-Arg6 conjugate. We have observed an increase of titre, affinity and specificity of the antibodies in the coimmunization procedure compared to those obtained by conventional immunization involving only the LE conjugate. The Leu-enkephalin antibodies exhibit a high affinity (KD 8 X 10(-12) M) and we are able to detect the Leu-enkephalin at the 10(-15) mole level. These LE antibodies are highly specific of the C part of LE peptide and cross-react weakly with Met-enkephalin (1%).

  15. Validation of celiac disease diagnoses recorded in the Danish National Patient Register using duodenal biopsies, celiac disease-specific antibodies, and human leukocyte-antigen genotypes

    DEFF Research Database (Denmark)

    Dydensborg Sander, Stine; Størdal, Ketil; Plato Hansen, Tine

    2016-01-01

    PURPOSE: The purpose of this study was to validate the celiac disease diagnoses recorded in the Danish National Patient Register. To validate the diagnoses, we used information on duodenal biopsies from a national register of pathology reports (the Patobank) and information on celiac disease......-specific antibodies and human leukocyte antigen (HLA) genotypes obtained from patient medical records. PATIENTS AND METHODS: We included all the children who were born from 1995 to 2012 and who were registered as having celiac disease in the Danish National Patient Register. We reviewed all the pathology reports...... on duodenal biopsies in the Patobank and the information in the medical records on celiac disease-specific antibodies (ie, anti-tissue transglutaminase 2 IgA and IgG, endomysial antibodies IgA, and anti-deamidated gliadin peptide IgG) and HLA genotypes. RESULTS: We identified 2,247 children who were...

  16. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer

    Science.gov (United States)

    Willett, Christopher G; Boucher, Yves; di Tomaso, Emmanuelle; Duda, Dan G; Munn, Lance L; Tong, Ricky T; Chung, Daniel C; Sahani, Dushyant V; Kalva, Sanjeeva P; Kozin, Sergey V; Mino, Mari; Cohen, Kenneth S; Scadden, David T; Hartford, Alan C; Fischman, Alan J; Clark, Jeffrey W; Ryan, David P; Zhu, Andrew X; Blaszkowsky, Lawrence S; Chen, Helen X; Shellito, Paul C; Lauwers, Gregory Y; Jain, Rakesh K

    2009-01-01

    The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors. PMID:14745444

  17. Immunoassay of 5-methyltetrahydrofolate: use of 125I-labeled protein A as the tracer molecule for specific antibody

    International Nuclear Information System (INIS)

    Langone, J.J.

    1980-01-01

    A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. 125 I-Labeled staphylococcal Protein A ( 125 I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of 125 I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific; 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15 to 20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits

  18. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

    Science.gov (United States)

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A; Schechtman, Deborah

    2016-02-25

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

  19. Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT.

    Science.gov (United States)

    Yoshihara, S; Maruya, E; Taniguchi, K; Kaida, K; Kato, R; Inoue, T; Fujioka, T; Tamaki, H; Ikegame, K; Okada, M; Soma, T; Hayashi, K; Fujii, N; Onuma, T; Kusunoki, Y; Saji, H; Ogawa, H

    2012-04-01

    A role of donor-specific HLA antibodies (DSA) in graft failure after SCT has been suggested, but the relevance of DSA in unmanipulated haploidentical SCT (haplo-SCT) remains unknown. We prospectively examined HLA antibodies using the Luminex-based single Ag assay for 79 adult patients undergoing unmanipulated haplo-SCT. Among them, 16 (20.2%) were HLA Ab-positive, including five patients with antibodies not corresponding to donor HLA Ags and 11 DSA-positive patients. Of the 11 DSA-positive patients, five received treatments to decrease DSA levels, including two, who received plasma exchange and rituximab, two who received platelet transfusions from healthy-related donors having DSA-corresponding HLA Ags and one who received bortezomib. Platelet transfusion was the most simple and effective treatment option for class I DSA. The cumulative incidence of neutrophil recovery was significantly lower in pretransplant (post-treatment) DSA-positive patients than in DSA-negative patients (61.9 vs 94.4%, P=0.026). Notably, three of five patients with high levels of DSA had graft failure. Donors should be selected on the basis of an evaluation of HLA antibodies. If haplo-SCT from donors with HLA Ags that correspond to high levels of DSA must be performed, then recipients should be treated for DSA to improve the chances of successful donor engraftment.

  20. Determination of Aspergillus pathogens in agricultural products by a specific nanobody-polyclonal antibody sandwich ELISA.

    Science.gov (United States)

    Wang, Ting; Li, Peiwu; Zhang, Qi; Zhang, Wen; Zhang, Zhaowei; Wang, Tong; He, Ting

    2017-06-28

    Aspergillus and its poisonous mycotoxins are distributed worldwide throughout the environment and are of particular interest in agriculture and food safety. In order to develop a specific method for rapid detection of Aspergillus flavus to forecast diseases and control aflatoxins, a nanobody, PO8-VHH, highly reactive to A. flavus was isolated from an immunized alpaca nanobody library by phage display. The nanobody was verified to bind to the components of extracellular and intracellular antigen from both A. flavus and A. parasiticus. To construct a sandwich format immunoassay, polyclonal antibodies against Aspergillus were raised with rabbits. Finally, a highly selective nanobody-polyclonal antibody sandwich enzyme-linked immunosorbent assay was optimized and developed. The results revealed that the detection limits of the two fungi were as low as 1 μg mL -1 , and that it is able to detect fungal concentrations below to 2 μg mg -1 of peanut and maize grains in both artificially and naturally contaminated samples. Therefore, we here provided a rapid and simple method for monitoring Aspergillus spp. contamination in agricultural products.

  1. Rapid analysis of charge variants of monoclonal antibodies using non-linear salt gradient in cation-exchange high performance liquid chromatography.

    Science.gov (United States)

    Joshi, Varsha; Kumar, Vijesh; Rathore, Anurag S

    2015-08-07

    A method is proposed for rapid development of a short, analytical cation exchange high performance liquid chromatography method for analysis of charge heterogeneity in monoclonal antibody products. The parameters investigated and optimized include pH, shape of elution gradient and length of the column. It is found that the most important parameter for development of a shorter method is the choice of the shape of elution gradient. In this paper, we propose a step by step approach to develop a non-linear sigmoidal shape gradient for analysis of charge heterogeneity for two different monoclonal antibody products. The use of this gradient not only decreases the run time of the method to 4min against the conventional method that takes more than 40min but also the resolution is retained. Superiority of the phosphate gradient over sodium chloride gradient for elution of mAbs is also observed. The method has been successfully evaluated for specificity, sensitivity, linearity, limit of detection, and limit of quantification. Application of this method as a potential at-line process analytical technology tool has been suggested. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Evaluation of multiplex assay platforms for detection of influenza hemagglutinin subtype specific antibody responses.

    Science.gov (United States)

    Li, Zhu-Nan; Weber, Kimberly M; Limmer, Rebecca A; Horne, Bobbi J; Stevens, James; Schwerzmann, Joy; Wrammert, Jens; McCausland, Megan; Phipps, Andrew J; Hancock, Kathy; Jernigan, Daniel B; Levine, Min; Katz, Jacqueline M; Miller, Joseph D

    2017-05-01

    Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex ® , and ForteBio ® platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera. Compared to the traditional HI, assay sensitivity ranged from 87% to 92% and assay specificity in sera collected from unexposed persons ranged from 65% to 100% across the platforms. High assay specificity (86-100%) for A(H5N1) rHA was achieved for sera from exposed or unexposed to hetorosubtype influenza HAs. In contrast, assay specificity for A(H1N1)pdm09 rHA using sera collected from A/Vietnam/1204/2004 (H5N1) vaccinees in 2008 was low (22-30%) in all platforms. Although cross-reactivity against rHA subtype proteins was observed in each assay platform, the correct subtype specific responses were identified 78%-94% of the time when paired samples were available for analysis. These results show that high throughput and portable multiplex assays that incorporate rHA can be used to identify influenza subtype specific infections. Published by Elsevier B.V.

  3. Lack of antibodies to NMDAR or VGKC-complex in GAD and cardiolipin antibody-positive refractory epilepsy.

    Science.gov (United States)

    Liimatainen, Suvi; Peltola, Jukka; Hietaharju, Aki; Sabater, Lidia; Lang, Bethan

    2014-03-01

    Over the last few years autoantibodies against neuronal proteins have been identified in several forms of autoimmune encephalitis and epilepsy. NMDA receptor (NMDAR) and voltage gated potassium channel (VGKC) complex antibodies are mainly associated with limbic encephalitis (LE) whereas glutamic acid decarboxylase antibodies (GADA) and anticardiolipin (ACL) antibodies are more commonly detected in patients with chronic epilepsy. Clinical features vary between these antibodies suggesting the specificity of different neuronal antibodies in seizures. Serum samples of 14 GADA positive and 24 ACL positive patients with refractory epilepsy were analyzed for the presence of VGKC or NMDAR antibodies. No positive VGKC or NMDAR antibodies were found in these patients. The results confirm the different significance of these neuronal antibodies in seizure disorders. Different autoantibodies have different significance in seizures and probably have different pathophysiological mechanisms of actions. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Determination of IgE antibodies to the benzylpenicilloyl determinant: a comparison of the sensitivity and specificity of three radio allergo sorbent test methods.

    Science.gov (United States)

    Garcia, J J; Blanca, M; Moreno, F; Vega, J M; Mayorga, C; Fernandez, J; Juarez, C; Romano, A; de Ramon, E

    1997-01-01

    The quantitation of in vitro IgE antibodies to the benzylpenicilloyl determinant (BPO) is a useful tool for evaluating suspected penicillin allergic subjects. Although many different methods have been employed, few studies have compared their diagnostic specificity and sensitivity. In this study, the sensitivity and specificity of three different radio allergo sorbent test (RAST) methods for quantitating specific IgE antibodies to the BPO determinant were compared. Thirty positive control sera (serum samples from penicillin allergic subjects with a positive clinical history and a positive penicillin skin test) and 30 negative control sera (sera from subjects with no history of penicillin allergy and negative skin tests) were tested for BPO-specific IgE antibodies by RAST using three different conjugates coupled to the solid phase: benzylpenicillin conjugated to polylysine (BPO-PLL), benzylpenicillin conjugated to human serum albumin (BPO-HSA), and benzylpenicillin conjugated to an aminospacer (BPO-SP). Receiver operator control curves (ROC analysis) were carried out by determining different cut-off points between positive and negative values. Contingence tables were constructed and sensitivity, specificity, negative predictive values (PV-), and positive predictive values (PV+) were calculated. Pearson correlation coefficients (r) and intraclass correlation coefficients (ICC) were determined and the differences between methods were compared by chi 2 analysis. Analysis of the areas defined by the ROC curves showed statistical differences among the three methods. When cut-off points for optimal sensitivity and specificity were chosen, the BPO-HSA assay was less sensitive and less specific and had a lower PV- and PV+ than the BPO-PLL and BPO-SP assays. Assessment of r and ICC indicated that the correlation was very high, but the concordance between the PLL and SP methods was higher than between the PLL and HSA or SP and HSA methods. We conclude that for quantitating Ig

  5. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

    Directory of Open Access Journals (Sweden)

    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  6. 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....

  7. Reduced 99mTc labelled NCA-95/CEA-antibody uptake in liver due to gentle antibody reconstitution

    International Nuclear Information System (INIS)

    Reske, S.N.; Buell, U.

    1990-01-01

    The influence of reconstituting a murine monoclonal IgG 1 antibody kit with pertechnetate Tc99m on antibody distribution in the liver, spleen and sternal bone marrow of patients was examined. The 99m Tc-labelled antibody used is directed against non-specific cross-reacting antigen (NCA-95) and carcinoembryonic antigen (CEA) and has been successfully applied for imaging tissue inflammation and bone marrow scanning. Radioactivity uptake was determined in the liver, spleen, bone marrow and a precordial background region in a consecutive series of 25 patients, examined with an antibody preparation, routinely radiolabelled according to the manufacturer's recommendations and in 14 patients, in whom the antibody was reconstituted with special care, avoiding bubble formation and dropping of buffer into the antibody-containing vial. Gentle compared with routine antibody reconstitution caused a highly significant reduction of the antibody uptake in the liver, as determined by count densities, normalized to injected dose and acquisition time (13.2±5.5 vs 20.1±6.0 cpm per pixel, anti x±SD, P=0.008). The liver to background ratio was reduced from 3.4±1.4 to 1.9±0.5 (P<0.001). Spleen, sternal bone marrow and precordial background count rates were not significantly affected. These results clearly demonstrate that gentle antibody reconstitution can decrease non-specific antibody uptake in the liver by 34%±6.4% (anti x±SEM). Thus, scan quality is improved, and the potential deleterious camouflage of underlying structures is avoided. (orig.)