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Sample records for cysticercosis antigens antibodies

  1. Serodiagnosis of bovine cysticercosis by detecting live Taenia saginata cysts using a monoclonal antibody-based antigen-ELISA

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    W. Wanzala

    2002-07-01

    Full Text Available An ante mortem antigen-ELISA-based diagnosis of Taenia saginata cysticercosis was studied in artificially (n = 24 and naturally (n = 25 infected cattle with the objective of further validating the assay as a field diagnostic test. Based on total dissection as the definitive method of validity, the assay minimally detected 14 live cysticerci in artificially infected calves and 2 in naturally infected steers. In natural infections, the minimum number of live cysticerci consistently detected by Ag-ELISA was 5 while in artificial infections it was above 14. However, other animals with 12 and 17 live cysticerci in artificially infected calves, and 1 and 2 live cysticerci in naturally infected steers, escaped detection for unknown reasons. Animals harbouring dead cysticerci gave negative reactions in the assay as was the case in non-infected experimental control calves. There was a statistically significant positive linear correlation between Ag-ELISA optical density values and burdens of live cysticerci as obtained by total dissection of both artificially infected calves (r = 0.798, n = 24 ; P < 0.05 and naturally infected steers (r = 0.631, n = 25 ; P < 0.05. These results clearly show the potential effectiveness of ante mortem monoclonal antibody-based antigen detection ELISA in the diagnosis of bovine cysticercosis in cattle. Its value lies in the diagnosis of infection in cattle as a screening test in a herd, rather than as a diagnostic test at the individual level, due to false positive and negative reactions. In a herd of heavily infected cattle, the assay may, however, provide for individual diagnosis. Nevertheless, more work is recommended to increase its sensitivity so as to be able to diagnose light infections consistently in the field.

  2. Cysticercosis

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    Moosa, A.; Dawood, A.A. (Natal Univ., Durban (South Africa). Dept. of Pediatrics and Child Health)

    1983-12-01

    Cysticercosis is the most common parasitic disease in the world affecting the central nervous system. The article discusses the pathogenesis of cysticercosis as well as the pathological types of cysticercosis such as subcutaneous nodules, lingual nodules and orbital nodules. Cerebral cysticercosis is discussed in greater depth together with its clinical features. Attention is also given to the diagnosis of cerebral cysticercosis and diagnostic aids such as computerized tomography that can be used.

  3. Expression and immunolocalisation of TpFABP as a candidate antigen for the serodiagnosis of rabbit Taenia pisiformis cysticercosis.

    Science.gov (United States)

    Yang, Deying; Chen, Lin; Xie, Yue; Wu, Xuhang; Nong, Xiang; Peng, Xi; Lai, Weimin; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2013-01-01

    The larval stage of Taenia pisiformis, also known as Cysticercus pisiformis, is the causative agent of cysticercosis and the cause of severe health problems in rabbits that negatively impacts on husbandry production. To date, there is no fast detection method to identify early infections in rabbits. In the present study, a new dot-ELISA-based on an endogenous antigen fatty acid-binding protein (FABP) was developed for the detection of cysticercosis, and its potential was then evaluated using test serum samples. Immunolocalisation showed that T. pisiformis FABP (TpFABP) localised to the parenchyma of the bladder wall of the cysticercus and perinuclear cytoplasm of parenchyma of the adult parasite. After cloning and expression, recombinant TpFABP (rTpFABP) protein was used for serodiagnosis of T. pisiformis infection in rabbits by dot-ELISA. The antibody was detected 14 days post-infection in rabbits experimentally infected with T. pisiformis. Based on the necropsy results, the sensitivity and specificity of 169 serum samples tested by rTpFABP dot-ELISA were found to be 98.2% (54/55) and 92.1% (105/114), respectively. These data suggest that the dot-ELISA developed in this study has potential for detection of T. pisiformis infection in rabbits. © D. Yang et al., published by EDP Sciences, 2013.

  4. Expression and immunolocalisation of TpFABP as a candidate antigen for the serodiagnosis of rabbit Taenia pisiformis cysticercosis

    OpenAIRE

    Yang, Deying; Chen, Lin; Xie, Yue; Wu, Xuhang; Nong, Xiang; Peng, Xi; Lai, Weimin; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2013-01-01

    The larval stage of Taenia pisiformis, also known as Cysticercus pisiformis, is the causative agent of cysticercosis and the cause of severe health problems in rabbits that negatively impacts on husbandry production. To date, there is no fast detection method to identify early infections in rabbits. In the present study, a new dot-ELISA-based on an endogenous antigen fatty acid-binding protein (FABP) was developed for the detection of cysticercosis, and its potential was then evaluated using ...

  5. Antigen antibody interactions

    CERN Document Server

    DeLisi, Charles

    1976-01-01

    1. 1 Organization of the Immune System One of the most important survival mechanisms of vertebrates is their ability to recognize and respond to the onslaught of pathogenic microbes to which they are conti- ously exposed. The collection of host cells and molecules involved in this recognition­ 12 response function constitutes its immune system. In man, it comprises about 10 cells 20 (lymphocytes) and 10 molecules (immunoglobulins). Its ontogenic development is c- strained by the requirement that it be capable of responding to an almost limitless variety of molecular configurations on foreign substances, while simultaneously remaining inert to those on self components. It has thus evolved to discriminate, with exquisite precision, between molecular patterns. The foreign substances which induce a response, called antigens, are typically large molecules such as proteins and polysaccharides. The portions of these with which immunoglobulins interact are called epitopes or determinants. A typical protein epitope m...

  6. Antigen recognition by IgG4 antibodies in human trichinellosis

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    Pinelli E.

    2001-06-01

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

  7. Factors associated with the prevalence of circulating antigens to porcine cysticercosis in three villages of burkina faso.

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    Rasmané Ganaba

    2011-01-01

    Full Text Available little is known about porcine cysticercosis in Burkina Faso. We conducted a pilot study to estimate the prevalence of antigens of Taenia solium cysticercosis and to identify associated factors in pigs of three villages in Burkina Faso, selected to represent different pig management practices: one village where pigs are allowed to roam freely (Batondo, one village where pigs are penned part of the time (Pabré and one village with limited pig farming (Nyonyogo.a clustered random sampling design was used. Data on socio-demographic characteristics (source of drinking water, presence of latrines in the household, type and number of breeding animals and pig management practices were collected using a standardized questionnaire. Blood samples were collected from one pig per household to determine the presence of antigens of the larval stages of T. solium by the B158/B60 Ag-ELISA. The associations between seropositivity and socio-demographic and pig management practices were estimated using logistic regression. Proportions of 32.5% (95% CI 25.4-40.3, 39.6% (31.9-47.8, and 0% of pigs, were found positive for the presence of circulating antigens of T. solium in Batondo, Pabré, and Nyonyogo, respectively. The results of the logistic regression analyses suggested that people acquire knowledge on porcine cysticercosis following the contamination of their animals. The presence of antigens in the pigs' sera was not associated with the absence of latrines in the household, the source of drinking water or the status of infection in humans but was associated with pig rearing practices during the rainy season.the results suggest that education of pig farmers is urgently needed to reduce the prevalence of this infection.

  8. Factors associated with the prevalence of circulating antigens to porcine cysticercosis in three villages of burkina faso.

    Science.gov (United States)

    Ganaba, Rasmané; Praet, Nicolas; Carabin, Hélène; Millogo, Athanase; Tarnagda, Zékiba; Dorny, Pierre; Hounton, Sennen; Sow, Adama; Nitiéma, Pascal; Cowan, Linda D

    2011-01-04

    little is known about porcine cysticercosis in Burkina Faso. We conducted a pilot study to estimate the prevalence of antigens of Taenia solium cysticercosis and to identify associated factors in pigs of three villages in Burkina Faso, selected to represent different pig management practices: one village where pigs are allowed to roam freely (Batondo), one village where pigs are penned part of the time (Pabré) and one village with limited pig farming (Nyonyogo). a clustered random sampling design was used. Data on socio-demographic characteristics (source of drinking water, presence of latrines in the household, type and number of breeding animals) and pig management practices were collected using a standardized questionnaire. Blood samples were collected from one pig per household to determine the presence of antigens of the larval stages of T. solium by the B158/B60 Ag-ELISA. The associations between seropositivity and socio-demographic and pig management practices were estimated using logistic regression. Proportions of 32.5% (95% CI 25.4-40.3), 39.6% (31.9-47.8), and 0% of pigs, were found positive for the presence of circulating antigens of T. solium in Batondo, Pabré, and Nyonyogo, respectively. The results of the logistic regression analyses suggested that people acquire knowledge on porcine cysticercosis following the contamination of their animals. The presence of antigens in the pigs' sera was not associated with the absence of latrines in the household, the source of drinking water or the status of infection in humans but was associated with pig rearing practices during the rainy season. the results suggest that education of pig farmers is urgently needed to reduce the prevalence of this infection.

  9. Taenia saginata: production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens.

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    Vicentini-Oliveira, Josy Campanhã; Golim, Marjorie A; de Cássia Paulan, Silvana; Biondi, Germano Francisco; Rossi-Ferreira, Rosana; Deffune, Elenice; Nunes, Cáris Maroni

    2010-12-01

    Cysticercosis is a major cause of economic loss in bovine production due to meat condemnation. Chemotherapy is being used in Brazilian cattle and a diagnostic test to improve the treatment program is desired. We produced monoclonal antibodies against crude (TAEB) and cyst fluid (TAEF) Taenia saginata metacestode antigens using immunized BALB/c mice. After cell fusion, 10 TAEB and nine TAEF hybrids were selected and cloned resulting in 18 IgG(1) and 32 IgM TAEB clones, and 9 IgG(1) and 9 IgM TAEF clones. Ascites was produced and Western blot testing was performed resulting in reactivity to protein fractions of low molecular weight (<18kDa), 43, 55, 66 and 100kDa. The indirect immunofluorescence test, with one monoclonal antibody against crude and one against cyst fluid antigens, recognized antigenic fractions of both the scolex and the bladder wall of metacestodes from naturally infected bovine.

  10. Serological diagnosis of Taenia solium in pigs: No measurable circulating antigens and antibody response following exposure to Taenia saginata oncospheres.

    Science.gov (United States)

    Dorny, P; Dermauw, V; Van Hul, A; Trevisan, C; Gabriël, S

    2017-10-15

    Taenia solium taeniasis/cysticercosis is a zoonosis included in the WHO's list of neglected tropical diseases. Accurate diagnostic tools for humans and pigs are needed to monitor intervention outcomes. Currently used diagnostic tools for porcine cysticercosis all have drawbacks. Serological tests are mainly confronted with problems of specificity. More specifically, circulating antigen detecting tests cross-react with Taenia hydatigena and the possibility of transient antigens as a result of aborted infections is suspected. Furthermore, the hypothesis has been raised that hatched ingested eggs of other Taenia species may lead to a transient antibody response or to the presence of circulating antigen detectable by serological tests used for porcine cysticercosis. Here we describe the results of a study that consisted of oral administration of Taenia saginata eggs to five piglets followed by serological testing during five weeks and necropsy aiming at studying possible cross reactions in serological tests used for porcine cysticercosis. The infectivity of the eggs was verified by in vitro hatching and by experimental infection of a calf. One piglet developed acute respiratory disease and died on day 6 post infection. The remaining four piglets did not show any clinical signs until euthanasia. None of the serum samples from four piglets collected between days 0 and 35 post infection gave a positive reaction in the B158/B60 Ag-ELISA and in a commercial Western blot for antibody detection. In conclusion, this study showed that experimental exposure of four pigs to T. saginata eggs did not result in positive serologies for T. solium. These results may help interpreting serological results in monitoring of T. solium control programmes. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Serological studies of neurologic helminthic infections in rural areas of southwest cameroon: toxocariasis, cysticercosis and paragonimiasis.

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    Nkouawa, Agathe; Sako, Yasuhito; Itoh, Sonoyo; Kouojip-Mabou, Alida; Nganou, Christ Nadège; Saijo, Yasuaki; Knapp, Jenny; Yamasaki, Hiroshi; Nakao, Minoru; Nakaya, Kazuhiro; Moyou-Somo, Roger; Ito, Akira

    2010-07-06

    Both epilepsy and paragonimiasis had been known to be endemic in Southwest Cameroon. A total of 188 people (168 and 20 with and without symptoms confirmed by clinicians, respectively, 84.6% under 20 years old) were selected on a voluntary basis. Among 14 people (8.3%) with history of epilepsy, only one suffered from paragonimiasis. Therefore, we challenged to check antibody responses to highly specific diagnostic recombinant antigens for two other helminthic diseases, cysticercosis and toxocariasis, expected to be involved in neurological diseases. Soil-transmitted helminthic infections were also examined. Fecal samples were collected exclusively from the 168 people. Eggs of Ascaris lumbricoides, Trichuris trichiura and hookworms were found from 56 (33.3%), 72 (42.8%), and 19 (11.3%) persons, respectively. Serology revealed that 61 (36.3%), 25 (14.9%) and 2 (1.2%) of 168 persons showed specific antibody responses to toxocariasis, paragonimiasis and cysticercosis, respectively. By contrast, 20 people without any symptoms as well as additional 20 people from Japan showed no antibody responses. Among the 14 persons with epilepsy, 5 persons were seropositive to the antigen specific to Toxocara, and one of them was simultaneously positive to the antigens of Paragonimus. The fact that 2 children with no history of epilepsy were serologically confirmed to have cysticercosis strongly suggests that serological survey for cysticercosis in children is expected to be useful for early detection of asymptomatic cysticercosis in endemic areas. Among persons surveyed, toxocariasis was more common than paragonimiasis, but cysticercosis was very rare. However, the fact that 2 children were serologically confirmed to have cysticercosis was very important, since it strongly suggests that serology for cysticercosis is useful and feasible for detection of asymptomatic cysticercotic children in endemic areas for the early treatment.

  12. Serological studies of neurologic helminthic infections in rural areas of southwest cameroon: toxocariasis, cysticercosis and paragonimiasis.

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    Agathe Nkouawa

    2010-07-01

    Full Text Available Both epilepsy and paragonimiasis had been known to be endemic in Southwest Cameroon. A total of 188 people (168 and 20 with and without symptoms confirmed by clinicians, respectively, 84.6% under 20 years old were selected on a voluntary basis. Among 14 people (8.3% with history of epilepsy, only one suffered from paragonimiasis. Therefore, we challenged to check antibody responses to highly specific diagnostic recombinant antigens for two other helminthic diseases, cysticercosis and toxocariasis, expected to be involved in neurological diseases. Soil-transmitted helminthic infections were also examined.Fecal samples were collected exclusively from the 168 people. Eggs of Ascaris lumbricoides, Trichuris trichiura and hookworms were found from 56 (33.3%, 72 (42.8%, and 19 (11.3% persons, respectively. Serology revealed that 61 (36.3%, 25 (14.9% and 2 (1.2% of 168 persons showed specific antibody responses to toxocariasis, paragonimiasis and cysticercosis, respectively. By contrast, 20 people without any symptoms as well as additional 20 people from Japan showed no antibody responses. Among the 14 persons with epilepsy, 5 persons were seropositive to the antigen specific to Toxocara, and one of them was simultaneously positive to the antigens of Paragonimus. The fact that 2 children with no history of epilepsy were serologically confirmed to have cysticercosis strongly suggests that serological survey for cysticercosis in children is expected to be useful for early detection of asymptomatic cysticercosis in endemic areas.Among persons surveyed, toxocariasis was more common than paragonimiasis, but cysticercosis was very rare. However, the fact that 2 children were serologically confirmed to have cysticercosis was very important, since it strongly suggests that serology for cysticercosis is useful and feasible for detection of asymptomatic cysticercotic children in endemic areas for the early treatment.

  13. Evaluation of an Antigen-Antibody

    African Journals Online (AJOL)

    GB

    detect samples with high viral loads have a significant implication for this assay designed to detect both antigen and antibody proteins. Arguably, HCV as a member of Flaviviridae family has an icosahedral capsid of T3 or T4 symmetry with 180 or 240 core protein subunits. Since one RNA genome contains 240 subunits of.

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

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    Edith S Málaga-Machaca

    2017-11-01

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

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

    NARCIS (Netherlands)

    Duarte, Joao Nuno Silva

    2017-01-01

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

  16. Prevalence of porcine cysticercosis and associated risk factors in Homa Bay District, Kenya

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    Eshitera Eric E

    2012-12-01

    Full Text Available Abstract Background Taenia solium is an important zoonosis in many developing countries. Cysticercosis poses a serious public health risk and leads to economic losses to the pig production industry. Due to scarcity of data on the epidemiology of porcine cysticercosis in Kenya, the present study was conducted to determine the prevalence and risk factors for porcine cysticercosis within Homa Bay district. A cross-sectional survey was carried out in 2010, and a total of 392 pigs were recruited in a household survey, with all being tested by ante-mortem lingual palpation (together with questionnaire data on pig production, occurrence and transmission of porcine cysticercosis, risk factors and awareness of porcine cysticercosis collected from the households from which pigs were sampled. Sufficient serum was collected from 232 of the pigs to be tested for the presence of circulating parasite antigen using a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (Ag-ELISA. Results Seventy six pigs were found positive by the Ag-ELISA (32.8%, 95% C.I. 26.8-39.2%, while by tongue inspection cysticerci were detected in 22/ 392 pigs (5.6% 95% C.I. 3.6-8.4%. The most important risk factor for porcine cysticercosis in the Homa Bay area was for pigs to belong to a farm where latrine use by members of the household was not evident (OR = 1.9, 95% CI = 1.13–2.37. Conclusion The present findings indicate that porcine cysticercosis is endemic in Homa Bay District, and that latrine provision, in conjunction with free-range pig keeping contributes significantly to porcine cysticercosis transmission.

  17. Lingual cysticercosis

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    A P Pichare

    2014-01-01

    Full Text Available Cysticercosis is a disease caused by larval form of tapeworm. Cysticercus cellulose primarily develops in tissues of pigs. Infection of human tissues is unusual and affliction of the oral cavity is rare. We, herein, present a case of lingual cysticercosis without involvement of any other site. A 5-year-old male, non-vegetarian child presented with a painless, pearly white, solitary nodular swelling on posterior dorsal side of tongue since 3 months. Excision biopsy was done. Histopathology revealed cysticercous cellulose in tongue muscles.

  18. 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

  19. Antibody-antigen interactions: contact analysis and binding site topography.

    Science.gov (United States)

    MacCallum, R M; Martin, A C; Thornton, J M

    1996-10-11

    We have analysed antigen-contacting residues and combining site shape in the antibody Fv and Fab crystal structures now available from the Protein Data Bank. Antigen-contacting propensities are presented for each antibody residue, allowing a new definition for the complementarity determining regions (CDRs) to be proposed based on observed antigen contacts. Contacts are more common at CDR residues which are located centrally within the combining site; some less central CDR residues are only contacted by large antigens. Non-contacting residues within the CDRs coincide with residues identified by Chothia and co-workers as important in defining "canonical" conformations. An objective means of classifying protein surfaces by gross topography has been developed and applied to the antibody combining site surfaces. The surfaces have been clustered into four topographic classes: concave and moderately concave (mostly hapten binders), ridged (mostly peptide binders) and planar (mostly protein binders). We have determined the topographic classes for ten pairs of complexed and uncomplexed antibody-antigen crystal structures; four change topographic class on complexation. The results will be of use in antibody engineering, antigen docking and in clinical immunology. To demonstrate one application, we show how the data can be used to locate the antigen binding pocket on antibody structures.

  20. Antígeno intradérmico para diagnóstico de cisticercose An intradermal antigen for cysticercosis diagnosis

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    José M. Marlet

    1978-03-01

    Full Text Available É descrito novo antígeno para teste cutâneo no diagnóstico da cisticercose. São relatados os resultados obtidos em experimentação realizada em 64 pessoas não parasitadas, em 18 portadores de ténia, em 34 de cisticercose cerebral e em 51 de Hymenolepis nana. Após considerações sobre a especificidade, sensibilidade, facilidade de aplicação e leitura e inocuidade do antígeno proposto, é indicado como teste de triagem em estudos epidemiológicos.A new antigen for a cutaneous test used for cyticercosis diagnosis is described. The author refers to technical details of antigen preparation and the results obtained in tests performed on 64 controls, on 18 subjects infected with Taenia, on 34 subjects with cerebral cysticercosis and on 51 subjects with Hymenolepis nana. The antigen is considered specific, sensible, innocuous and very easy to apply. The test is pointed out as being the test of choice in epidemiologic studies.

  1. Recognition of Coccidioides immitis Antigens with Monoclonal Antibodies.

    Science.gov (United States)

    1986-09-30

    AD-A114 322 RECOGNITION OF COCCIDIOIDES IMMITIS ANTIGENS NITH 1/1 MONOCLONAL ANT ISODIESMU CALIFORNIA UNIV OAKLAND NAVAL SIOSCIENCES LAB S J KRAEGER...031 NR204-123_ 11 (1lEWnld SeuiyCasification) ([L ECOGNITIB’N OF COCCIDIQIDES IMMITIS ANTIGENS WITH MONOCLONAL ANTIBODIES 12 PERSONAL A THOR(S...specificity and suitability for diagnostic use of seven 1gM monoclonal antibodies (MAbs) prepared in 1984 with c. immitis Silveira spherules and

  2. Bullous pemphigoid : Serum antibody titre and antigen specificity

    NARCIS (Netherlands)

    Pas, H H; de Jong, M C; Jonkman, M F; Heeres, K; Slijper-Pal, I J; van der Meer, J B

    1995-01-01

    2 antigens have been identified as possible targets for autoantibody depositions in bullous pemphigoid: a 230-kD protein (BP230) and a 180-kD protein (BP180). We studied the relationship of these 2 antigens with the immunofluorescence determined serum antibody titre: 2 groups of bullous pemphigoid

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

    OpenAIRE

    Jiménez, T; Díaz, A M; Zlotnik, H

    1990-01-01

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

  4. Generation of monoclonal antibodies against highly conserved antigens.

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    Hongzhe Zhou

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

  5. Prevalence of hepatitis B antigen and C antibody among blood ...

    African Journals Online (AJOL)

    The prevalence of hepatitis B surface antigen (HBsAg) and hepatitis C (HCV) antibody were determined in 560 blood donor sera using ELISA kits (DIALAB Austria). Out of these 48(8.57%) were positive to hepatitis B virus infection, while 33(5.89%) were positive to hepatitis C virus antibodies. The sex distribution of ...

  6. Serum Antibody Repertoire Profiling Using In Silico Antigen Screen.

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    Xinyue Liu

    Full Text Available Serum antibodies are valuable source of information on the health state of an organism. The profiles of serum antibody reactivity can be generated by using a high throughput sequencing of peptide-coding DNA from combinatorial random peptide phage display libraries selected for binding to serum antibodies. Here we demonstrate that the targets of immune response, which are recognized by serum antibodies directed against sequential epitopes, can be identified using the serum antibody repertoire profiles generated by high throughput sequencing. We developed an algorithm to filter the results of the protein database BLAST search for selected peptides to distinguish real antigens recognized by serum antibodies from irrelevant proteins retrieved randomly. When we used this algorithm to analyze serum antibodies from mice immunized with human protein, we were able to identify the protein used for immunizations among the top candidate antigens. When we analyzed human serum sample from the metastatic melanoma patient, the recombinant protein, corresponding to the top candidate from the list generated using the algorithm, was recognized by antibodies from metastatic melanoma serum on the western blot, thus confirming that the method can identify autoantigens recognized by serum antibodies. We demonstrated also that our unbiased method of looking at the repertoire of serum antibodies reveals quantitative information on the epitope composition of the targets of immune response. A method for deciphering information contained in the serum antibody repertoire profiles may help to identify autoantibodies that can be used for diagnosing and monitoring autoimmune diseases or malignancies.

  7. Pathogen specific carbohydrate antigen microarrays: a chip for detection of Salmonella O-antigen specific antibodies.

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    Blixt, Ola; Hoffmann, Julia; Svenson, Stefan; Norberg, Thomas

    2008-01-01

    A Salmonella O-antigen microarray was developed by covalent coupling of oligosaccharide antigens specific for serogroups Salmonella enterica sv. Paratyphi (group A), Typhimurium (group B) and Enteritidis (group D). Antibodies were correctly detected in sera from patients with culture verified salmonellosis. High serogroup-specificity was seen with the disaccharide antigens. With the larger antigens, containing the backbone sequence Manalpha1-2Rhaalpha1-2Gal (MRG), common backbone-specific antibodies (O-antigen 12) were also detected. This is "proof of principle" that pathogen-specific carbohydrate antigen microarrays constitute a novel technology for rapid and specific serological diagnosis in either individual patients or larger sero-epidemiological and vaccine studies.

  8. Diagnosis of cysticercosis in endemic regions

    OpenAIRE

    Garcia, H. H.; Martinez, M.; Gilman, R.; Herrera, G.; Tsang, V. C. W.; Pilcher, J. B.; Diaz, F; Verastegui, M.; Gallo, C.; Porras, M.; Alvarado, M.; Naranjo, J.; Miranda, E.

    1991-01-01

    Taenia solium cysticercosis is a frequent cause of neurological disease in developing countries. Specific diagnosis of cysticercosis is difficult. We obtained serum and/or CSF samples from 204 consecutive patients admitted to a neurological ward in Lima, Peru, and looked for antibodies specific for T solium with the enzyme-linked immunoelectrotransfer blot (EITB) assay. 21 (12%) of 173 serum samples from these patients were EITB-positive. In contrast only 2 (1·5%) of 135 patients attending a ...

  9. Reaktivitas Antibodi Poliklonal SSV terhadap Antigen Homolog dan Heterolog

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    Sri Sulandari

    1998-07-01

    Full Text Available Polyclonal antibodies for Soybean Stunt Virus (SSV were produced in white rabbit through the following procedures: approximately 100 mg of purified virions emulsified in Complete Freund’s Adjuvant (CFA were injected intramuscularly first. In the second and third injection 150 mg of purified virions in Incomplete Freund’s Adjuvant (IFA per injection were injected intramuscularly. Finally, about 300 mg of purified virions were injected intravenously as a booster. The injection were done at 2 weeks interval. Antiserum was collected 5 days after the final injection. Antisera was purified by precipitation in saturated ammonium sulfate. Purified antibody was tested for the titer and reactivity of antibodies against the homologous and heterologous antigen. The studies were conducted with non-precoated I-ELISA test. This research was able to obtain about 25 ml of crude antisera for SSV, the concentration of purified polyclonal antibodies was about 9 mg/ml. the titer of polyclonal antibodies was 10.000 in I-ELISA. Without absorbtion with sap of healthy plant, the antibodies could not be use to identify the infected and healthy plant samples. In the following test, the absorbed antibody was used. Using antibodies to SSV at a dilution of 1:1000 and 1:10.000 against sap extracts sample of healthy and infected plant at a dilution of 1:10 by non-precoated I-ELISA test, indicated that the antibody could be used to identify the healthy and infected samples. By the same test, the antibody could be reacted to both homologous antigen (SSV and heterologous antigen (CMV isolated from banana. Key words: SSV, polyclonal antibodies, ELISA

  10. Cysticercosis in experimentally and naturally infected pigs: parasitological and immunological diagnosis

    Directory of Open Access Journals (Sweden)

    Márcia R.M. da Silva

    2012-04-01

    Full Text Available Our objective was to evaluate the diagnosis of swine cysticercosis by examining "ante mortem" (inspection of the tongue, "post mortem" (inspection and detailed necropsy and ELISA for research in serum of antibodies (Ab-ELISA and antigens (Ag-ELISA. Seven (7 pigs were experimentally infected orally with eggs of Taenia solium and another 10 were naturally infected. In the pigs experimentally infected, inspection of the tongue was negative in all animals, in the routine inspection detailed necropsy and cysticercis were identified in all of them. In pigs with heavy natural infection, inspection of the tongue identified cysticerci in two (20%, while at inspection with necropsy the parasites were identified in large quantities in all animals. In ELISA for antibody search (Ab-ELISA TS-14 recombinant protein was used, and in search for antigen (Ag-ELISA a monoclonal antibody against this protein. In animals experimentally infected, blood was collected weekly for 140 days. The Ab-ELISA identified an increase in titers of antibody to cysticerci 21 days after infection, and at the end of the experimental period six animals (86% were positive to the test. The search for circulating antigens (Ag-ELISA was positive in two pigs 28 to 91 days after infection. All naturally infected pigs were positive for Ag-ELISA and Ab-ELISA. The search for antibodies and antigens by ELISA in serum from 30 pigs of a local farm and without history of cysticercosis was negative. Thus, the use of TS-14 antigen in ELISA test (Ab-ELISA can be useful for the diagnosis of cysticercosis in pigs with low infection.

  11. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Science.gov (United States)

    2010-04-01

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

  12. 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...

  13. Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cássia dos Coqueiros, SP determined by ELISA and immunoblotting using Taenia crassiceps antigens

    Directory of Open Access Journals (Sweden)

    BRAGAZZA Lúcia M.

    2002-01-01

    Full Text Available Considering the impact of cysticercosis on public health, especially the neurologic form of the disease, neurocysticercosis (NC, we studied the frequency of positivity of anti-Taenia solium cysticercus antibodies in serum samples from 1,863 inhabitants of Cássia dos Coqueiros, SP, a municipal district located 80 km from Ribeirão Preto, an area considered endemic for cysticercosis. The 1,863 samples were tested by enzyme linked immunosorbent assay (ELISA using an antigenic extract from Taenia crassiceps vesicular fluid (Tcra. The reactive and inconclusive ELISA samples were tested by immunoblotting. Of the 459 samples submitted to immunoblotting, 40 were strongly immunoreactive to the immunodominant 18 and 14 kD peptides. Considering the use of immunoblotting as confirmatory due to its high specificity, the anti-cysticercus serum prevalence in this population was 2.1%.

  14. Structural analysis of hepatitis B surface antigen by monoclonal antibodies.

    Science.gov (United States)

    Ben-Porath, E; Wands, J R; Marciniak, R A; Wong, M A; Hornstein, L; Ryder, R; Canlas, M; Lingao, A; Isselbacher, K J

    1985-10-01

    A method has been developed for the analysis of hepatitis B surface antigen (HBsAg) antigenic structure at the molecular level that creates "fingerprints" or "signatures" of various hepatitis B viral (HBV) strains. This technique employs high affinity IgM and IgG monoclonal antibodies (anti-HBs) directed against distinct and separate determinants on HBsAg. In performing this antigenic structural analysis, separate binding curves for different monoclonal anti-HBs are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum by radioimmunoassay. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples are aligned by an iterative least-squares procedure to generate the numerical signature characteristic of the viral strain. The numerical signatures are then displayed on a computer-graphic plot. The signature profiles of HBsAg subtypes are a true reflection of their antigenic structure, and in vertical and horizontal transmission studies the molecular characteristics of the viral epitopes are conserved. By signature analysis we found substantial antigenic heterogeneity among the ayw3 strain both in the U.S. and France, as well as in populations of the Far East and Africa. Populations in Ethiopia, Gambia, and the Philippines were infected with two antigenically distinct HBV strains. In some newly identified HBV strains, it was found that epitopes identified by some monoclonal antibodies were absent or substantially reduced, which suggested that a genetic mutation may have occurred. Thus this study suggests that there is far more antigenic heterogeneity in HBV than previously recognized. These variants are antigenically distinct from each other at the epitope level, and were heretofore unrecognized by polyvalent anti-HBsAg antibodies.

  15. TSOL18 Vaccine Antigen of Taenia solium: Development of Monoclonal Antibodies and Field Testing of the Vaccine in Cameroon

    Directory of Open Access Journals (Sweden)

    Assana, E.

    2010-01-01

    Full Text Available Chapter 1 reviews the literature about the immunological aspects of taeniid cestode infections and the existing vaccines against Taenia solium cysticercosis in pigs. One of the most promising vaccines is TSOL18, a protein that has been identified in the oncosphere of Taenia solium and expressed as a recombinant molecule in E. coli. Repeated experimental trials have shown that this vaccine is able to protect up to 100% of the immunised pigs against a challenge infection with T. solium. Antibodies raised by the vaccine are capable of killing the parasite in in vitro cultures and it is believed that antibody and complement mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. The identification of the villages with a high risk of T. solium infection, which could subsequently be used in the vaccine trial, is reported in chapter 2. A survey was conducted in 150 households owning 1756 pigs in the rural areas of Mayo-Danay division in the far north region of Cameroon. A questionnaire survey was carried out to collect information on the pig farming system and to identify potential risk factors for T. solium cysticercosis infection in pigs. Blood samples were collected from 398 pigs with the aim of estimating the sero-prevalence of Taenia solium cysticercosis. The results showed that 90.7% of the pigs were free roaming during the dry season and that 42.7% of households keeping pigs in the rural areas had no latrine facility. Seventy six percent of the interviewed pig owners affirmed that the members of the household used open field defecation. ELISA for antigen and antibody detection showed an apparent prevalence of porcine cysticercosis of 24.6% and 32.2%, respectively. A Bayesian approach using the conditional dependence between the two diagnostic tests indicated that the true sero-prevalence of cysticercosis in Mayo-Danay was 26.6%. Binary logistic regression analysis indicated that the

  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. Urine antibody against human cancer antigen NY-ESO-1

    OpenAIRE

    Jäger, Dirk; Stockert, Elisabeth; Karbach, Julia; Herrlinger, Kristina; Atmaca, Akin; Arand, Michael; Chen, Yao-Tseng; Gnjatic, Sacha; Old, Lloyd J; Knuth, Alexander; Jäger, Elke

    2002-01-01

    NY-ESO-1 is one of the most immunogenic tumor antigens known to date. Spontaneous humoral and cellular immune responses against NY-ESO-1 are detected in a substantial proportion of patients with NY-ESO-1 positive cancers. NY-ESO-1 serum antibody is dependent on the presence of NY-ESO-1+ cancer cells, and antibody titers correlate with the clinical development of the disease. NY-ESO-1 serum antibody is associated with detectable NY-ESO-1-specific CD8+ T cell reactivity. High titers of NY-ESO-1...

  18. Focused antibody response to influenza linked to antigenic drift.

    Science.gov (United States)

    Huang, Kuan-Ying A; Rijal, Pramila; Schimanski, Lisa; Powell, Timothy J; Lin, Tzou-Yien; McCauley, John W; Daniels, Rodney S; Townsend, Alain R

    2015-07-01

    The selective pressure that drives antigenic changes in influenza viruses is thought to originate from the human immune response. Here, we have characterized the B cell repertoire from a previously vaccinated donor whose serum had reduced neutralizing activity against the recently evolved clade 6B H1N1pdm09 viruses. While the response was markedly polyclonal, 88% of clones failed to recognize clade 6B viruses; however, the ability to neutralize A/USSR/90/1977 influenza, to which the donor would have been exposed in childhood, was retained. In vitro selection of virus variants with representative monoclonal antibodies revealed that a single amino acid replacement at residue K163 in the Sa antigenic site, which is characteristic of the clade 6B viruses, was responsible for resistance to neutralization by multiple monoclonal antibodies and the donor serum. The K163 residue lies in a part of a conserved surface that is common to the hemagglutinins of the 1977 and 2009 H1N1 viruses. Vaccination with the 2009 hemagglutinin induced an antibody response tightly focused on this common surface that is capable of selecting current antigenic drift variants in H1N1pdm09 influenza viruses. Moreover, amino acid replacement at K163 was not highlighted by standard ferret antisera. Human monoclonal antibodies may be a useful adjunct to ferret antisera for detecting antigenic drift in influenza viruses.

  19. Diagnosis of cysticercosis in endemic regions

    Science.gov (United States)

    Garcia, H. H.; Martinez, M.; Gilman, R.; Herrera, G.; Tsang, V. C. W.; Pilcher, J. B.; Diaz, F.; Verastegui, M.; Gallo, C.; Porras, M.; Alvarado, M.; Naranjo, J.; Miranda, E.

    2010-01-01

    Taenia solium cysticercosis is a frequent cause of neurological disease in developing countries. Specific diagnosis of cysticercosis is difficult. We obtained serum and/or CSF samples from 204 consecutive patients admitted to a neurological ward in Lima, Peru, and looked for antibodies specific for T solium with the enzyme-linked immunoelectrotransfer blot (EITB) assay. 21 (12%) of 173 serum samples from these patients were EITB-positive. In contrast only 2 (1·5%) of 135 patients attending a public endoscopy clinic and 1 (1%) of 88 patients attending a private endoscopy clinic were seropositive. 1 (1%) of 98 pregnant women living in a Lima shanty town was EITB-positive. 15 (58%) of 26 neurology patients diagnosed clinically as having cysticercosis were seronegative. Routine screening by EITB of all patients with neurological symptoms from areas of endemic cysticercosis would avoid misdiagnosis of this common and treatable disease. PMID:1678809

  20. Bovine cysticercosis: Preliminary observations on the immunohistochemical detection of Taenia saginata antigens in lymph nodes of an experimentally infected calf

    Science.gov (United States)

    2004-01-01

    Abstract A newly developed immunohistochemical test was used for the first time to demonstrate the presence of Taenia saginata (Cysticercus bovis) antigens in the lymph nodes of a heifer calf experimentally inoculated with Taenia saginata eggs. The new test should aid in the differential diagnosis of eosinophilic lymphadenitis in cattle. PMID:15532887

  1. Experimental Immunization Based on Plasmodium Antigens Isolated by Antibody Affinity

    Directory of Open Access Journals (Sweden)

    Ali N. Kamali

    2015-01-01

    Full Text Available Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethal Plasmodium yoelii yoelii 17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses of P. yoelii yoelii 17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites.

  2. Antibody profiling with protein antigen microarrays in early stage cancer.

    Science.gov (United States)

    Liu, Brian C-S; Dijohnson, Daniel A; O'Rourke, Dennis J

    2012-05-01

    Proteins not present in normal cells, that is, cancer cells, may elicit a host immune response that leads to the generation of antibodies that might react with these tumor-associated proteins. In recent years, a growing number of reports have showed that autoantibody profiling may provide an alternative approach for the detection of cancer. However, most studies of antigen-autoantibody reactivity have relied on recombinant proteins. Recombinant proteins lack the proper post-translational modifications present in native proteins. Because of this limitation, native or natural protein antigen microarrays are gaining popularity for profiling antibody responses. i) To illustrate some examples of autoantibodies as signatures for early stage cancer; ii) to briefly outline the various protein antigen microarray platforms; iii) to illustrate the use of native or natural protein microarrays in the discovery of potential biomarkers and iv) to discuss the advantages of native protein antigen microarrays over other approaches. The nature of protein microarray platforms is conducive to multiplexing, which amplifies the potential for uncovering effective biomarkers for many significant diseases. However, the major challenge will be in integrating microarray platforms into multiplexed clinical diagnostic tools, as the main drawback is the reproducibility and coefficient of variation of the results from array to array, and the transportability of the array platform to a more automatable platform.

  3. Recognition of multiple Mycoplasma bovis antigens by monoclonal antibodies.

    Science.gov (United States)

    Dénes, Béla; Tenk, Miklós; Tekes, Lajos; Varga, Ildikó; Ferenczné, Ildikó P; Stipkovits, László

    2003-02-01

    To produce monoclonal antibodies (MAbs), Balb/c AnN Crl BR mice were inoculated with the cell suspension of a Hungarian Mycoplasma bovis strain designated 26034. Three days after the last immunization the spleen of the immunized mouse was removed aseptically. The fusion of spleen cells with Sp2/0-Ag14 murine myeloma cells was performed in the presence of polyethylene glycol. The obtained hybrid cells were selected with hipoxantine, aminopterine and thymidine (HAT) medium. Two weeks after the fusion, the supernatants of the grown cells were tested by a self-developed indirect enzyme-linked immunosorbent assay (ELISA). The results showed that 63 antibody-producing hybridomas had been obtained. For accurate determination of the molecular weight of antigen determinants, the supernatants giving positive reaction in the ELISA were tested by Western blotting. According to the results, the obtained MAbs recognize the antigen determinants of the following molecular weights: 1B11: 63 kDa, 1C7: 63 kDa, 2C5: 22, 25 and 27 kDa, 2C9: 69 kDa, 3G12: 67, 69 and 72 kDa, 4H9: 63 kDa, 5B8: 22, 25 and 27 kDa, 5D3: 22, 25 and 27 kDa, 5C11: 69 kDa, 5E5: 22, 25 and 27 kDa, 6F11: 63 kDa, and 6H10: 22, 25 and 27 kDa. The 12 cell groups selected on the basis of the Western blotting were cloned twice by end-point dilution method. The cloned cells were propagated, and with 5 cell lines antibodies were produced in the CELLine bioreactor (Integra Biosciences, Zurich, Switzerland). Cell line 3G12 showed the highest productivity with an average daily output of 1.5 mg immunoglobulin. Cell line 5E5 produced 1.1 mg, 6H10 0.8 mg, 2C9 0.47 mg and 6F11 0.4 mg antibody per day. The isotype of the antibodies was determined by ELISA. The antibodies produced by the 12 cell lines tested were assigned to the IgG(1) subclass according to the heavy chain. Ten cell lines produced kappa and two produced lambda light-chain antibody. Possible cross-reactions of the produced monoclonal anti-M. bovis antibodies with

  4. Cysticercosis of masseter

    Directory of Open Access Journals (Sweden)

    B Dilip Kumar

    2011-01-01

    Full Text Available Cysticercosis is a parasitic infestation caused by the larval stage of Taenia solium, a cestodic paratise. It is a common disease in developing countries where it is also endemic. The most commonly infested body organs include subcutaneous tissues, brain and skeletal muscles. It is interesting to note that oral lesion of cysticercosis is a rare event. Here we report an isolated lesion of cysticercosis in the masseter muscle.

  5. Effect of pH on antigen binding by clonotypic antibodies with different isoelectric points

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Y.; Miyai, K.; Hata, N.; Iijima, Y.

    1987-02-01

    Polyclonal rabbit antibodies to thyroxine, human myoglobin, human growth hormone, human thyrotropin, human alpha-fetoprotein, and human thyroglobulin were fractionated into clonotypic antibodies with different isoelectric points by agarose isoelectric focusing or chromatofocusing. The effect of pH on the binding of these antigens by their respective clonotypic antibodies was assessed by radioimmunoassay. The profiles of the pH effect differed both for different antigens and for different pI's of the antibodies used. The pH optima in the radioimmunoassays for protein antigens were found to be expressed as a function of pI and molecular weight of both antigen and antibody molecules.

  6. A monoclonal antibody defining human B cell differentiation antigen (HLB-1 antigen).

    Science.gov (United States)

    Kasai, K; Koshiba, H; Ishii, Y; Kikuchi, K

    1983-01-01

    A new monoclonal antibody specific for human B cell differentiation antigen (HLB-1) is produced by a hybridoma established by fusion of splenocytes of mice immunized with the Epstein-Barr virus (EBV)-transformed peripheral B cell line, RPMI-8057. This monoclonal, antibody designated anti-HLB-1 monoclonal antibody (anti-HLB-1), reacted with surface immunoglobulin (sIg)-positive B cells of normal peripheral blood and lymphoid tissues and sIg-positive leukemic cells. The cells of T cell leukemia, non-T non-B acute lymphoblastic leukemia (ALL) and nonlymphoid leukemia were HLB-1 negative. These data were further confirmed by studying a panel of cultured human hematopoietic cell lines. Anti-HLB-1 reacted with B cell lines derived from pre-B, Burkitt's lymphoma, B cell type ALL and EBV-transformed peripheral B cells. Anti-HLB-1 was reactive with only one of three human myeloma cell lines, and with none of the T cell, myeloid and non-T non-B ALL cell lines. This newly defined HLB-1 antigen is different from other conventional human B cell markers such as sIg, Ia antigens, and receptors for the Fc portion of Ig and complement C3.

  7. Cysticercosis in the Democratic Republic Of Congo

    Directory of Open Access Journals (Sweden)

    P. Dorny

    2012-06-01

    Full Text Available Cysticercosis, caused by Taenia solium eggs, is a zoonotic disease whose consequences can be severe especially in the cerebral localisation (neuorcysticercosis. Indeed, neurocysticercosis is the first cause of epilepsy amongst the infectious etiology group. Following the increase of epilepsy cases in Kinshasa and Bas-Congo, it was important to assess the fraction attributable to neurocysticercosis especially as data on cysticercosis in Democratic Republic Of Congo (DRC dating from 1970.A joint study between veterinary and human doctors was conducted in the provinces of Bas-Congo and Kinshasa between 2008 and 2010. Blood samples were collected from the general population, patients with epilepsy and pigs. These samples were analysed using ELISA antigen in the laboratory of the Institute of Tropical Medicine in Antwerp. Patients positive to ELISA antigen took the CT scan exam for the confirmation of neurocysticercosis. In the province of Kinshasa, of 530 epileptic patients, 6.3% were identified as neurocysticercosis cases. Out of a total of 498 pigs, 38.9% were positive for cysticercosis. In the province of Bas- Congo, of 943 inhabitants from Malanga village, 21.6% were positive with predominance in males (26.4% versus 17.5%. A total of 145 pigs from 5 villages were examined and 41.2% found positive.We can conclude that cysticercosis in the DRC has been neglected for a long time and cysticercosis could be a real major public health problem. Prospective studies addressing the consequences of cysticercosis in communities are needed in order to prevent epilepsy due to neurocysticercosis.

  8. Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop (CLAW).

    Science.gov (United States)

    Cobbold, S; Metcalfe, S

    1994-03-01

    A panel of 127 monoclonal antibodies against canine leukocyte antigens, including controls, was distributed to 29 laboratories that performed a variety of experiments to identify groups of antibodies against the canine equivalents of some of the human CD antigens. Cluster analysis was performed centrally, using the submitted antibody binding data from immunofluorescence, ELISA and immuno-histology experiments. Immunoprecipitation for molecular weight determination was also performed centrally with T-cell blasts and a B-cell line as the sources of antigen. Clusters of three or more antibodies were found that defined the canine equivalents of the CD5, CD4, CD8 and Thy-1 antigens, and these could be used to label T-cell subsets from the peripheral blood. Other groups of monoclonal antibodies recognized the canine homologues of the CD11/18 group of antigens, CD44 and the CD45/CD45R antigen family: these should be useful in isolating functional subsets of CD4+ helper T cells. There was a cluster of four antibodies that bound strongly to platelets (probably CD41 antigen), three antibodies that were specific to B cells (including CD21) and two antibodies against a granulocyte antigen (possibly CD15). A number of reagents were found against canine MHC-II and immunoglobulin, with some of the latter able to distinguish between Ig subclasses. Properties of each of the canine antigens defined by these monoclonal antibodies are discussed and compared with other species. The availability of such a panel of reagents should allow rapid improvements in the immunological diagnosis of canine disease, and there might now be a potential for testing novel therapeutic strategies in a clinical veterinary setting.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  10. Structural Consequences of Mutations in Interfacial Tyr Residues of a Protein Antigen-Antibody Complex

    National Research Council Canada - National Science Library

    Mitsunori Shiroishi; Kouhei Tsumoto; Yoshikazu Tanaka; Akiko Yokota; Takeshi Nakanishi; Hidemasa Kondo; Izumi Kumagai

    2007-01-01

    Tyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies...

  11. Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen

    Directory of Open Access Journals (Sweden)

    Anizah Rahumatullah

    2017-11-01

    Full Text Available Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv clones against a specific filarial antigen (BmR1. The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications.

  12. Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

    Science.gov (United States)

    Zhang, Shaohua; Luo, Xuenong; Guo, Aijiang; Zhu, Xueliang; Cai, Xuepeng

    2016-07-01

    The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

    Science.gov (United States)

    Devanaboyina, Siva Charan; Lynch, Sandra M; Ober, Raimund J; Ram, Sripad; Kim, Dongyoung; Puig-Canto, Alberto; Breen, Shannon; Kasturirangan, Srinath; Fowler, Susan; Peng, Li; Zhong, Haihong; Jermutus, Lutz; Wu, Herren; Webster, Carl; Ward, E Sally; Gao, Changshou

    2013-01-01

    A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a “buffering” effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0–7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination. PMID:24492341

  14. Acute HIV-1 Infection in Antigen/Antibody-negative Blood Donors in ...

    African Journals Online (AJOL)

    Acute HIV-1 Infection in Antigen/Antibody-negative Blood Donors in Dar es Salaam, Tanzania. ... Fourth generation human immunodeficiency virus (HIV) antigen (Ag)/antibody (Ab) enzyme-linked immunosorbent assay (ELISA) test used in the current screening of blood donors at the National Blood Transfusion Service ...

  15. Chimeric antigen receptors and bispecific antibodies to retarget T cells in pediatric oncology.

    Science.gov (United States)

    Suzuki, Maya; Curran, Kevin J; Cheung, Nai-Kong V

    2015-08-01

    Cancer immunotherapy using antigen-specific T cells has broad therapeutic potential. Chimeric antigen receptors and bispecific antibodies can redirect T cells to kill tumors without human leukocyte antigens (HLA) restriction. Key determinants of clinical potential include the choice of target antigen, antibody specificity, antibody affinity, tumor accessibility, T cell persistence, and tumor immune evasion. For pediatric cancers, additional constraints include their propensity for bulky metastatic disease and the concern for late toxicities from treatment. Nonetheless, the recent preclinical and clinical developments of these T cell based therapies are highly encouraging. © 2015 Wiley Periodicals, Inc.

  16. Evaluation of the siemens HIV antigen-antibody immunoassay.

    Science.gov (United States)

    Vallefuoco, Luca; Aden Abdi, Fatima; Sorrentino, Rosanna; Spalletti-Cernia, Daniela; Mazzarella, Claudia; Barbato, Sara; Perna, Enzo; Buffolano, Wilma; Di Nicuolo, Giuseppe; Portella, Giuseppe

    2014-01-01

    Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibodies are available on the international market and are currently used for blood donor screening and for HIV diagnosis. In this study we evaluated the performance of the novel automated fourth-generation ADVIA Centaur® HIV Ag/Ab Combo assay. The assay detected seroconversion at the same bleed or at least one bleed earlier in panels with respect to other assays and showed a detection efficacy equal to those of other assays in a low-titer panel. Samples obtained from blood donors (n = 2,778) or from HIV-positive patients (HIV-1 B subtype, n = 82; non-B subtype, n = 71) were also tested, showing a good correlation with other fourth-generation assays. We assessed the performance of 3 fourth-generation assays for detecting in utero transmitted anti-HIV antibodies and found a more specific detection efficiency with the ADVIA Centaur HIV Ag/Ab Combo assay compared to the other fourth-generation assays.

  17. Improvements and Variants of the Multiple Antigen Blot Assay-MABA: An Immunoenzymatic Technique for Simultaneous Antigen and Antibody Screening.

    Science.gov (United States)

    Noya, Oscar; Losada, Sandra; Toledo, Marilyan; Gauna, Adriana; Lorenzo, María Angelita; Bermúdez, Henry; de Noya, Belkisyolé Alarcón

    2015-01-01

    This simple, versatile, reliable, reproducible, multipurpose, and inexpensive technique is based on the adhesion of different antigens to a single nitrocellulose strip using, as template, an acrylic device containing 28 parallel channels. The inclusion of channels containing normal human serum improves the quality control of this assay. Antigen-sensitized nitrocellulose strips are cut perpendicularly to the antigen-rows, exposed to immune sera followed by the appropriate conjugate. Positive signals are recorded using chemiluminescent or precipitable colorimetric substrates. This assay allows the simultaneous qualitative demonstration of antigenicity and immunogenicity of antigens obtained as synthetic peptides, recombinant molecules, or crude preparations, with high sensitivity and specificity. Its major value is based on the rapid and simultaneous comparative evaluation of various antigenic preparations allowing the diagnosis of a variety of infectious, allergic, and autoimmune diseases. It can in general be used to detect any type of antibody or circulating antigen. Some improvements and variants of the original technique are included.

  18. From Monoclonal Antibodies to Chimeric Antigen Receptors for the Treatment of Human Malignancies

    OpenAIRE

    Caruana, Ignazio; Diaconu, Iulia; Dotti, Gianpietro

    2014-01-01

    Monoclonal antibodies (mAbs) and their directly derived cell-based application known as chimeric antigen receptors (CARs) ensue from the need to develop novel therapeutic strategies that retain high anti-tumor activity, but carry reduced toxicity compared to conventional chemo- and radio-therapies. In this concise review article we will summarize the application of antibodies designed to target antigens expressed by tumor cells, and the transition from these antibodies to the generation of CARs.

  19. A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens

    OpenAIRE

    Tan, Joshua; Pieper, Kathrin; Piccoli, Luca; Abdi, Abdirahman; Perez, Mathilde Foglierini; Geiger, Roger; Tully, Claire Maria; Jarrossay, David; Maina Ndungu, Francis; Wambua, Juliana; Bejon, Philip; Fregni, Chiara Silacci; Fernandez-Rodriguez, Blanca; Barbieri, Sonia; Bianchi, Siro

    2015-01-01

    Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies 1?4 . Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here, we report the isolation of human monoclonal antibodies that recogn...

  20. Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination

    Science.gov (United States)

    Kang, Myungsun; Pelet, Jeisa M.; Ruda, Vera M.; Foley, Maria H.; Hu, Joyce K.; Kumari, Sudha; Crampton, Jordan; Baldeon, Alexis D.; Sanders, Rogier W.; Moore, John P.; Crotty, Shane; Langer, Robert; Anderson, Daniel G.; Chakraborty, Arup K.; Irvine, Darrell J.

    2016-01-01

    Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1–2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency. PMID:27702895

  1. New immune strategies for the treatment of acute lymphoblastic leukemia: antibodies and chimeric antigen receptors.

    Science.gov (United States)

    Advani, Anjali S

    2013-01-01

    The prognosis of adult acute lymphoblastic leukemia (ALL) remains poor and novel treatment strategies are needed. Antibody-based therapies represent such an approach. ALL cells express various surface antigens that are targets for monoclonal antibodies. This review focuses on 4 major classes of antibody therapy: (1) naked antibodies, (2) T-cell-engaging bispecific single-chain antibodies, (3) immunoconjugates/immunotoxins, and (4) chimeric antigen receptors. Preclinical and clinical data are reviewed. This area of research represents an exciting new approach to help improve the outcome of this disease. Several clinical trials are currently incorporating this therapy in the treatment of newly diagnosed and relapsed adult ALL patients.

  2. [Evaluation of Abbott Fourth Generation HIV Antigen and Antibody Assays.].

    Science.gov (United States)

    Kang, Hee Jung; Yoo, Kyeong Ha; Kim, Han Sung; Cho, Hyoun Chan

    2006-02-01

    In order to reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and serological diagnosis, new fourth generation screening assays which detect HIV p24 antigen and specific antibody simultaneously have been developed. In this study, we evaluated the performance of a new fourth generation assay. We compared a new fourth generation assay, Architect HIV Ag/Ab combo, with another fourth generation assay AxSYM HIV Ag/Ab combo and a third generation assay, AxSYM HIV 1/2 gO for their performance. The assays were evaluated using 3 HIV seroconversion panels, 305 sera of healthy subjects and 100 sera of patients with HBsAg or anti-HCV antibodies. Within-run and total coefficient variations of the three screening assays were analyzed for the evaluation of precision. Architect HIV Ag/Ab combo shortened the window period by 8.7+/-2.1 days relative to AxSYM HIV 1/2 gO and 2.0+/-2.0 days relative to AxSYM HIV Ag/Ab combo in seroconversion panels. Architect HIV Ag/Ab combo presented the best performance in precision among the three reagents; total CV for positive control was 3.6%, 9.6% and 4.6% for Architect HIV Ag/Ab combo, AxSYM HIV Ag/Ab combo and AxSYM HIV 1/2 gO, respectively. Specificities of three assays were not different in this study. HIV Ag/Ab combined assays reduced the diagnostic window as compared to the third generation screening assays, enabling an earlier diagnosis of HIV infection. A new fourth generation assay, Architect HIV Ag/Ab combo presents a better performance than AxSYM HIV Ag/Ab combo, showing improved seroconversion sensitivity and precision.

  3. Anti-cytotoxic T lymphocyte antigen-4 antibodies in melanoma

    Directory of Open Access Journals (Sweden)

    Tosti G

    2013-10-01

    Full Text Available Giulio Tosti, Emilia Cocorocchio, Elisabetta PennacchioliDivisione Melanomi e Sarcomi, Istituto Europeo di Oncologia, Milano, ItalyAbstract: Approaches aimed at enhancement of the tumor specific response have provided proof for the rationale of immunotherapy in cancer, both in animal models and in humans. Ipilimumab, an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4 antibody, is a new generation immunotherapeutic agent that has shown activity in terms of disease free and overall survival in metastatic melanoma patients. Its use was approved by the US Food and Drug Administration in March 2011 to treat patients with late stage melanoma that has spread or that cannot be removed by surgery. The mechanism of action of CTLA-4 antibodies in the activation of an antitumor immune response and selected clinical studies of ipilimumab in advanced melanoma patients are discussed. Ipilimumab treatment has been associated with immune related adverse events due to T-cell activation and proliferation. Most of these serious adverse effects are associated with the gastrointestinal tract and include severe diarrhea and colitis. The relationship between immune related adverse events and antitumor activity associated with ipilimumab was explored in clinical studies. Potential biomarkers predictive for clinical response and survival in patients treated with anti-CTLA-4 therapy are presently under investigation. Besides the conventional patterns of response and stable disease as defined by standard Response Evaluation Criteria in Solid Tumors criteria, in subsets of patients, ipilimumab has shown patterns of delayed clinical activity which were associated with an improved overall survival. For this reason a new set of response criteria for tumor immunotherapy has been proposed, which was termed immune related response criteria. These new criteria are presently used to better analyze clinical activity of immunotherapeutic regimens. Ipilimumab is currently under

  4. MRI of intraspinal cysticercosis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seung Cheol; Chang, Kee Hyun; Han, Moon Hee; Han, Gi Seok [Seoul Natioal University College of Medicine, Seoul (Korea, Republic of); Hwang, Hee Young [Chung Ang Gil Hospital, Incheon (Korea, Republic of)

    1995-01-15

    To describe the MR features of intraspinal cysticercosis. Medical records and MR images of four cases of intraspinal cysticercosis were retrospectively reviewed. The MR findings were described with regard to the location and signal intensity of the lesions, contrast enhancement, presence or absence of associated intracranial cysticerci, and other findings. There were three cases of subarachnoidal form and one case of intramedullary form. Cysticerci of subarachnoidal form in three cases were located in retromedullary space at C2 level, anterior to cord at C1-C6 levels, and lumbosacral area, respectively. The signal intensities of the lesions were same as those of CSF. Localized arachnoidal enhancement was found in all three cases. In one case there was a large area of high signal intensity within the spinal cord on T2-weighted image suggesting either ischemia secondary to vascular compromise or inflammatory edema. All of these three cases accompanied intracranial cysticercosis. Intramedullary cysticercosis in one case was shown as a single 1 cm cystic lesion at C2 level, which showed hypointense signal on T1-weighted image, hyperintense signal on T2-weighted image, and signet-ring-like enhancement. This lesion did not accompany intracranial cysticerci. Intraspinal cysticercosis manifested as single or multiple cysts within either spinal cord or subarachnoid space, and were frequently associated with arachnoiditis.

  5. Antigen-specific human monoclonal antibodies from transgenic mice.

    Science.gov (United States)

    Mompó, Susana Magadán; González-Fernández, Africa

    2014-01-01

    Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germ line configuration. The engineered mouse genome can undergo productive rearrangement in the B cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animals expressing human Ig genes. This chapter describes the type of transgenic knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs.

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

    OpenAIRE

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire res...

  7. Hepatitis B Virus DNA in Blood Samples Positive for Antibodies to Core Antigen and Negative for Surface Antigen

    Science.gov (United States)

    Gutiérrez, C.; León, G.; Loureiro, C. L.; Uzcátegui, N.; Liprandi, F.; Pujol, F. H.

    1999-01-01

    Anti-hepatitis B core antigen (HBcAg)-positive hepatitis B surface antigen (HBsAg)-negative plasma samples from blood donors were tested by nested PCR. DNA positivity was more significantly associated with high levels of anti-HBcAg than with low levels of anti-HBsAg antibodies. Analysis of a dilution of anti-HBcAg antibodies might result in a more rational exclusion of anti-HBcAg-positive HBsAg-negative samples, reducing the number of donations discarded and enabling more countries to incorporate anti-HBcAg testing. PMID:10473534

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Yi Liu

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

  10. Cysticercosis of the masseter

    Directory of Open Access Journals (Sweden)

    Prabhath Ramakrishnan

    2012-01-01

    Full Text Available Cysticercosis of the oral cavity is a very rare soft tissue lesion and very few cases have been reported worldwide. Here we report a case of a cysticercous cellulosae within the masseter muscle which was diagnosed with the help of high resolution ultrasonography (USG and ultrasound guided fine needle aspiration cytology (FNAC and managed conservatively using oral antiparasitic medication. Cysticercosis is not commonly considered in the diagnosis of swellings of the head and neck and this is the reason why they are of utmost interest to the practitioner and have to be studied.

  11. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  12. Studies on associations of antinuclear antibodies with antibodies to an uveitogenic peptide of retinal S antigen in children with uveitis.

    Science.gov (United States)

    Rosenberg, A M; Hauta, S A; Prokopchuk, P A; Romanchuk, K G

    1996-02-01

    To determine if, in children with uveitis, antinuclear antibodies (ANA) are associated with antibodies to an uveitogenic peptide of a soluble retinal antigen and to the homologous nuclear antigen, histone 3 (H3). ANA occur in most children with juvenile rheumatoid arthritis (JRA) and associated uveitis. An uveitogenic segment of retinal soluble antigen (S antigen peptide) is homologous with a similarly uveitogenic peptide of H3. We investigated a possible association between ANA positivity, antibodies to H3, and antibodies to the uveitogenic S antigen peptide. The sera of 31 children with uveitis (20 of whom had associated JRA) were tested for the presence of ANA by indirect immunofluorescence. Antibodies to H3 and to an uveitogenic peptide of S antigen (an 18 mer segment having the amino acid sequence DTNLASSTIIKEGIDKTV) were measured by enzyme immunoassay. 19 of 20 children (95%) with JRA and associated uveitis and none of 11 with uveitis not associated with JRA had positive tests for ANA (X2 = 14.97; p < 0.00001). 16 of 19 ANA positive sera from subjects with JRA (84%) displayed reactivity with the chromosomal regions of metaphase cells. 9 of 20 patients with JRA with uveitis (45%) and 2 of 11 patients (18%) with uveitis not associated with JRA had antibodies to H3. Two uveitic patients with JRA (10%) and 2 non-JRA patients with uveitis (18%) reacted with S antigen peptide. Antibodies to H3 occurred significantly more frequently in children with uveitis than in all adult control subjects (X2 = 12.98; p = 0.003) and in adults with uveitis (X2 = 5.62; p = 0.022). Humoral immune responses to the uveitogenic peptide of S antigen and the homologous H3 antigen appear not to be uniquely important in the immunopathology of uveitis associated with JRA. Antibodies to isolated H3 do not exclusively account for ANA positivity in the uveitic patient with JRA. A unique immunopathogenic mechanism for the development of uveitis associated with JRA is suggested by the

  13. Differing rates of antibody acquisition to merozoite antigens in malaria: implications for immunity and surveillance.

    Science.gov (United States)

    McCallum, Fiona J; Persson, Kristina E M; Fowkes, Freya J I; Reiling, Linda; Mugyenyi, Cleopatra K; Richards, Jack S; Simpson, Julie A; Williams, Thomas N; Gilson, Paul R; Hodder, Anthony N; Sanders, Paul R; Anders, Robin F; Narum, David L; Chitnis, Chetan; Crabb, Brendan S; Marsh, Kevin; Beeson, James G

    2017-04-01

    Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance. © Society for Leukocyte Biology.

  14. Differential antibody response of Gambian donors to soluble Plasmodium falciparum antigens

    DEFF Research Database (Denmark)

    Jakobsen, P H; Riley, E M; Allen, S J

    1991-01-01

    A seroepidemiological and clinical study was performed in an area of West Africa (The Gambia) where Plasmodium falciparum is endemic with seasonal transmission. Plasma samples were tested by intermediate gel immunoelectrophoresis for antibodies against 7 soluble P. falciparum antigens. There were...... who had had a documented attack of clinical malaria or parasitaemia. There was no difference in antibody profiles to soluble antigens between children with sickle cell trait and children with normal haemoglobin....

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

    Science.gov (United States)

    Samuelsen, Simone V.; Solov'Yov, Ilia A.; Balboni, Imelda M.; Mellins, Elizabeth; Nielsen, Christoffer Tandrup; Heegaard, Niels H. H.; Astakhova, Kira

    2016-10-01

    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 oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies.

  16. Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody

    Directory of Open Access Journals (Sweden)

    Judith Abelha

    1981-06-01

    Full Text Available Trypanomastigote forms of Trypanosoma cruzi were derived from tissue culture and incubated with immune and non-immune human sera. All immune sera showed high titers of specific humoral antibodies of the IgM or the IgG type. Agglutination and swelling of parasites were observed after incubation at 37ºC, but many trypomastigotes remained free-swimming in the sera for two to three days. The quantitiy of immune serum capable of lysing a maximum of 10 x 10 [raised to the power of 6] sensitized red cells was not capable of lysing 4 x 10 [raised to the power of 3] tripomastigotes. Typically, the parasites underwent cyclical changes with the formation of clumps of amastigotes and the appearance of epimastigote forms. Multiplication of the parasites was observed in immune sera. Further, the infectivity of the parasites to susceptible mice was not lost. All sera used produced similar general effects on the growth of the parasite. The antibody bound to T. cruzi appeard to enter cells by antigen-receptor mediated endocytosis. The ferritin-conjugated antibody was internalized and delivered to phagolysosomes where they might be completely degraded to amino-acids. This seemed to be a coupled process by which the immunoglobulin is first bound to specific parasite surface receptor and then rapidly endocytosed by the cell.Formas tripomastigotas de Trypanosoma cruzi derivadas de cultura de tecido foram encubadas com soros humanos imunes e não-imunes.Todos os soros humanos usados tinham títulos elevados de anticorpos das classes IgM ou IgG. Aglutinação e entumescimento dos parasitos eram observados apos encubação a 37ºC mas muitos tripomastigotas permaneceram circulando livremente nos soros por dois a três dias. A quantidade de soro imune capaz de lisar um máximo de 10 x 10 [elevado a 6] hemácias sensibilizadas não foi capaz de lisar 4 x 10 [elevado a 3] tripomastigotas. Tipicamente, os parasitos apresentavam alterações cíclicas com formação de

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

    Indian Academy of Sciences (India)

    WINTEC

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

  18. 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 ...

  19. Specific and common antigens of Trichomonas vaginalis detected by monoclonal antibodies.

    Science.gov (United States)

    Torian, B E; Connelly, R J; Stephens, R S; Stibbs, H H

    1984-01-01

    Monoclonal antibodies to Trichomonas vaginalis were prepared by immunizing mice with a cloned isolate of T. vaginalis. Eight antibodies reacted with the same four isolates or strains but did not react with the other T. vaginalis strains or isolates tested. All eight antibodies reacted uniformly with both the body and flagella of T. vaginalis in the immunofluorescence assay but were unreactive by immunoblotting. The antigen(s) recognized by these antibodies was determined to be present on the surface membrane by indirect immunofluorescence assay of live organisms. The antigen(s) was found to be sensitive to periodate oxidation but resistant to pronase digestion. In addition, one monoclonal antibody was derived which reacted with all T. vaginalis isolates or strains tested, as well as with Trichomonas gallinae, Tritrichomonas foetus, and Giardia lamblia. This antibody reacted with the body but not the flagella of Formalin-fixed protozoa in the immunofluorescence assay but failed to react with live organisms. The antigen was found to be periodate resistant but pronase labile. In the immunoblot assay, this antibody detected a single T. vaginalis polypeptide with a molecular weight of 62,000. Images PMID:6360900

  20. Comparison of five assays for antibody to varicella-zoster virus and the fluorescent-antibody-to-membrane-antigen test.

    OpenAIRE

    Larussa, P; Steinberg, S; Waithe, E; Hanna, B; Holzman, R

    1987-01-01

    Three commercially available assays (the Varicelisa Test Kit [Whittaker M.A. Bioproducts, Walkersville, Md.], the VZV Indirect Fluorescent-Antibody Test [Electro-Nucleonics, Inc., Columbia, Md.], and the Litton VZV Bio-EnzaBead Screen Kit [Litton Bionetics, Inc., Charleston, S.C.]) and two enzyme-linked immunosorbent assays used in our laboratory, one using a membrane-associated antigen and the other using a soluble antigen dotted on nitrocellulose paper, were compared with a varicella-zoster...

  1. An evaluation of a recombinant multiepitope based antigen for detection of Toxoplasma gondii specific antibodies.

    Science.gov (United States)

    Hajissa, Khalid; Zakaria, Robaiza; Suppian, Rapeah; Mohamed, Zeehaida

    2017-12-29

    The inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents. Recently, epitope-based antigens have emerged as an alternative diagnostic marker for the achievement of highly sensitive and specific capture antigens. In this study, the diagnostic utility of a recombinant multiepitope antigen (USM.TOXO1) for the serodiagnosis of human toxoplasmosis was evaluated. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis. The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody. This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.

  2. Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    NARCIS (Netherlands)

    Wilson, R. (Robert); J. Cohen (Jonathan); Reglinski, M. (Mark); R.J. Jose; Chan, W.Y. (Win Yan); Marshall, H. (Helina); C.P. de Vogel (Corné); S.B. Gordon (Stephen); Goldblatt, D. (David); Petersen, F.C. (Fernanda C.); H. Baxendale (Helen); Brown, J.S. (Jeremy S.)

    2017-01-01

    textabstractNaturally acquired immunity against invasive pneumococcal disease (IPD) is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous

  3. Antibody response against gastrointestinal antigens in demyelinating diseases of the central nervous system

    DEFF Research Database (Denmark)

    Banati, M; Csecsei, P; Koszegi, E

    2013-01-01

    BACKGROUND: Antibodies against gastrointestinal antigens may indicate altered microbiota and immune responses in the gut. Recent experimental data suggest a connection between gastrointestinal immune responses and CNS autoimmunity. METHODS: Antibodies against gliadin, tissue transglutaminase (t...... (MS), and 48 healthy controls (HC). RESULTS: Thirty-seven percentages of patients with AQP4-seropositive NMO/NMO-SD and 28% of patients with MS had at least one particular antibody in contrast to 8% of HC (P ...

  4. From monoclonal antibodies to chimeric antigen receptors for the treatment of human malignancies.

    Science.gov (United States)

    Caruana, Ignazio; Diaconu, Iulia; Dotti, Gianpietro

    2014-10-01

    Monoclonal antibodies (mAbs) and their directly derived cell-based application known as chimeric antigen receptors (CARs) ensue from the need to develop novel therapeutic strategies that retain high anti-tumor activity, but carry reduced toxicity compared to conventional chemo- and radiotherapies. In this concise review article, we will summarize the application of antibodies designed to target antigens expressed by tumor cells, and the transition from these antibodies to the generation of CARs. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. 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.

  6. Cysticercosis of the eye in south India - A case series

    Directory of Open Access Journals (Sweden)

    Kaliaperumal S

    2005-01-01

    Full Text Available Purpose: To study the clinical presentation and treatment outcome of patients with ocular cysticercosis in southern India. Methods: This study included 10 patients who were diagnosed to have ocular or adnexal cysticercosis over a period of one year in Pondicherry, India. The clinical presentation, results of investigation and treatment outcome of the cases were analysed retrospectively. Results: Age of these patients ranged from 12 to 55 years. Four presented with loss of vision, two with a swelling in the eyelid, one with proptosis, one with diplopia and two with conjunctival involvement. ELISA for cysticercus antibodies in serum was positive in all cases. Albendazole and prednisolone were given for the treatment of these cases. Two patients responded well to treatment and were completely cured of the disease. There was partial improvement in 6 cases. Surgery in the form of excision was performed in two cases following a course of medical therapy. There was no significant change in visual acuity in eyes with intraocular cysticercosis following treatment. Conclusion: Ultrasonography B scan and ELISA for anticysticercal antibodies help to establish the diagnosis of ocular cysticercosis. A combination of oral albendazole and corticosteroids is found to be effective in confirmed cases. Intraocular cysticercosis is associated with a poor prognosis for vision.

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

    KAUST Repository

    Di Palma, Francesco

    2017-08-03

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

  8. Failure of a novel, rapid antigen and antibody combination test to detect antigen-positive HIV infection in African adults with early HIV infection.

    Directory of Open Access Journals (Sweden)

    William Kilembe

    Full Text Available BACKGROUND: Acute HIV infection (prior to antibody seroconversion represents a high-risk window for HIV transmission. Development of a test to detect acute infection at the point-of-care is urgent. METHODS: Volunteers enrolled in a prospective study of HIV incidence in four African cities, Kigali in Rwanda and Ndola, Kitwe and Lusaka in Zambia, were tested regularly for HIV by rapid antibody test and p24 antigen ELISA. Five subgroups of samples were also tested by the Determine Ag/Ab Combo test 1 Antigen positive, antibody negative (acute infection; 2 Antigen positive, antibody positive; 3 Antigen negative, antibody positive; 4 Antigen negative, antibody negative; and 5 Antigen false positive, antibody negative (HIV uninfected. A sixth group included serial dilutions from a p24 antigen-positive control sample. Combo test results were reported as antigen positive, antibody positive, or both. RESULTS: Of 34 group 1 samples with VL between 5x105 and >1.5x107 copies/mL (median 3.5x106, 1 (2.9% was detected by the Combo antigen component, 7 (20.6% others were positive by the Combo antibody component. No group 2 samples were antigen positive by the Combo test (0/18. Sensitivity of the Combo antigen test was therefore 1.9% (1/52, 95% CI 0.0, 9.9. One false positive Combo antibody result (1/30, 3.3% was observed in group 4. No false-positive Combo antigen results were observed. The Combo antigen test was positive in group 6 at concentrations of 80 pg/mL, faintly positive at 40 and 20 pg/mL, and negative thereafter. The p24 ELISA antigen test remained positive at 5 pg/mL. CONCLUSIONS: Although the antibody component of the Combo test detected antibodies to HIV earlier than the comparison antibody tests used, less than 2% of the cases of antigen-positive HIV infection were detected by the Combo antigen component. The development of a rapid point-of-care test to diagnose acute HIV infection remains an urgent goal.

  9. Failure of a novel, rapid antigen and antibody combination test to detect antigen-positive HIV infection in African adults with early HIV infection.

    Science.gov (United States)

    Kilembe, William; Keeling, Michelle; Karita, Etienne; Lakhi, Shabir; Chetty, Paramesh; Price, Matt A; Makkan, Heeran; Latka, Mary; Likoti, Morongwe; Ilukui, Kenneth; Hurlston, Mackenzie; Allen, Susan; Stevens, Gwynn; Hunter, Eric

    2012-01-01

    Acute HIV infection (prior to antibody seroconversion) represents a high-risk window for HIV transmission. Development of a test to detect acute infection at the point-of-care is urgent. Volunteers enrolled in a prospective study of HIV incidence in four African cities, Kigali in Rwanda and Ndola, Kitwe and Lusaka in Zambia, were tested regularly for HIV by rapid antibody test and p24 antigen ELISA. Five subgroups of samples were also tested by the Determine Ag/Ab Combo test 1) Antigen positive, antibody negative (acute infection); 2) Antigen positive, antibody positive; 3) Antigen negative, antibody positive; 4) Antigen negative, antibody negative; and 5) Antigen false positive, antibody negative (HIV uninfected). A sixth group included serial dilutions from a p24 antigen-positive control sample. Combo test results were reported as antigen positive, antibody positive, or both. Of 34 group 1 samples with VL between 5x105 and >1.5x107 copies/mL (median 3.5x106), 1 (2.9%) was detected by the Combo antigen component, 7 (20.6%) others were positive by the Combo antibody component. No group 2 samples were antigen positive by the Combo test (0/18). Sensitivity of the Combo antigen test was therefore 1.9% (1/52, 95% CI 0.0, 9.9). One false positive Combo antibody result (1/30, 3.3%) was observed in group 4. No false-positive Combo antigen results were observed. The Combo antigen test was positive in group 6 at concentrations of 80 pg/mL, faintly positive at 40 and 20 pg/mL, and negative thereafter. The p24 ELISA antigen test remained positive at 5 pg/mL. Although the antibody component of the Combo test detected antibodies to HIV earlier than the comparison antibody tests used, less than 2% of the cases of antigen-positive HIV infection were detected by the Combo antigen component. The development of a rapid point-of-care test to diagnose acute HIV infection remains an urgent goal.

  10. Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes

    Science.gov (United States)

    Hjelm, Barbara; Forsström, Björn; Löfblom, John; Rockberg, Johan; Uhlén, Mathias

    2012-01-01

    A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar. PMID:23284606

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

    DEFF Research Database (Denmark)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-01-01

    A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene...... against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore......, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use....

  12. Characteristics of 26 kDa antigen of H. Pylori by Monoclonal Antibody.

    Science.gov (United States)

    Ghahremani, Hossein; Farshad, Shohreh; Amini Najafabadi, Hossein; Kashanian, Susan; Momeni Moghaddam, Mohammad Amin; Moradi, Nariman; Paknejad, Maliheh

    2015-02-01

    Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen) is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs) against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated.

  13. Human sperm antigens and antisperm antibodies I. Studies on vasectomy patients.

    Science.gov (United States)

    Tung, K S

    1975-01-01

    This study documents the types and incidence of antisperm antibody, detectable by indirect immunofluorescence, in 114 patients before vasectomy, 112 at 2 months and 71 patients at 6-9 months after vasectomy. Indirect immunofluorescence techniques revealed antibodies to seven distinct sperm antigens. Five of these antigens were readily accessible to antibody in vitro, and the remaining two were accessible only after treatment of spermatozoa with dithiothreitol and trypsin. Antisperm antibodies were detected in 61% of patients before vasectomy. The incidence rose to 77% at 2 months and 90% at 6-9 months after vasectomy. These antibodies were distinguishable into two groups based on their incidence before vasectomy. The first group included antibodies to antigens in the acrosome with a diffuse distribution, the equatorial region, the postacrosomal region and the midpiece of the tail. Its incidence was 61% before vasectomy; increased to 73% at 2 months and 80% at 6-9 months after vasectomy. The second group included antibodies to the sperm nucleus, the tail and to discrete antigens over the acrosome. They were found rarely (3%) in patients before vasectomy; increased in incidence to 25% at 2 months and 55% at 6-9 months after vasectomy. Antisperm antibodies of both groups existed as IgG and IgM classes; an exception being antibodies to sperm nucleus which were almost exclusively IgG. Of the antibodies, 14% were found to fix complement in vitro. Other autoantibodies, including antinuclear, antimitochondrial and antismooth muscle antibodies, did not develop following vasectomy. Images FIG. 1 PMID:1106922

  14. Mutated epitopes of hepatitis B surface antigen fused to the core antigen of the virus induce antibodies that react with the native surface antigen.

    Science.gov (United States)

    Shiau, A L; Murray, K

    1997-03-01

    Fusion of peptide epitopes to the core antigen (HBcAg) of hepatitis B virus (HBV) enhances their immunogenicity, both quantitatively and qualitatively. In a number of vaccine-induced mutants of HBV, glycine145 of the surface antigen S polypeptide (HBsAg) has been replaced by arginine, resulting in loss of cross-reactivity with antibodies to normal (wild-type) HBsAg. HBcAg fusion proteins carrying the immunodominant epitope of HBsAg, in which glycine145 was replaced by arginine, glutamic acid, or lysine, were produced in Escherichia coli and formed particles that displayed HBc antigenicity and immunogenicity similar to that of HBcAg itself. The fusion proteins also elicited T-cell proliferative responsiveness to HBcAg and HBsAg. Fusions carrying either wild-type or mutated epitopes of HBsAG showed HBs antigenicity in immunoblot analysis and antigen-capture immunoradiometric assay, but both mutant and wild-type derivatives induced antibodies that cross-reacted with wild-type HBsAG. The results emphasise the potential for HBcAg fusion proteins in vaccines by broadening the antibody response in a way that could confer protection against both wild-type and variant form of HBV.

  15. Human antibody response to Lethocerus salivary antigens as a ...

    African Journals Online (AJOL)

    Interestingly in addition to a few immunogenic salivary proteins (85, 64, 37 and 33 kDa bands), a 28 kDa protein derived from salivary glands homogenate of aquatic insects was able to bind to Mycobacterium ulcerans and to be recognized by IgG antibodies of healthy subjects in endemic areas. The antibody responses to ...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification...

  17. Monoclonal antibodies against genetically manipulated hepatitis B core antigen.

    Science.gov (United States)

    Hlozánek, I; Korec, E; Dostálová, V; Stará, J; König, J; Bichko, V V; Seichertová, A; Gren, E J

    1987-01-01

    Four different hybridoma clones secreting anti-HBcAg antibodies were constructed by fusing cells of the mouse myeloma line SP2/0 with lymphocytes from mice immunized with bacterially produced HBcAg. The monoclonal antibodies were immunologically characterized and used for HBcAg detection by ELISA. This monoclonal-antibody-based assay was compared with ELISA based on polyclonal human anti-HBcAg IgG for sensitivity and specificity. The monoclonal antibody reacted specifically both with the bacterially produced HBcAg and HBcAg isolated from human liver, but did not react with HBeAg. The human polyclonal antibody reacted with HBcAg, but also with HBeAg.

  18. Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens.

    Directory of Open Access Journals (Sweden)

    Robert Wilson

    2017-01-01

    Full Text Available Naturally acquired immunity against invasive pneumococcal disease (IPD is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous immunoglobulin preparation (IVIG, representing natural IgG responses to S. pneumoniae, to identify the classes of antigens that are functionally relevant for immunity to IPD. IgG in IVIG recognised capsular antigen and multiple S. pneumoniae protein antigens, with highly conserved patterns between different geographical sources of pooled human IgG. Incubation of S. pneumoniae in IVIG resulted in IgG binding to the bacteria, formation of bacterial aggregates, and enhanced phagocytosis even for unencapsulated S. pneumoniae strains, demonstrating the capsule was unlikely to be the dominant protective antigen. IgG binding to S. pneumoniae incubated in IVIG was reduced after partial chemical or genetic removal of bacterial surface proteins, and increased against a Streptococcus mitis strain expressing the S. pneumoniae protein PspC. In contrast, depletion of type-specific capsular antibody from IVIG did not affect IgG binding, opsonophagocytosis, or protection by passive vaccination against IPD in murine models. These results demonstrate that naturally acquired protection against IPD largely depends on antibody to protein antigens rather than the capsule.

  19. 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.

  20. 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

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  2. Rapid Isolation of Monoclonal Antibodies Specific for Cell Surface Differentiation Antigens

    Science.gov (United States)

    Barclay, Stephen L.; Smith, Alan M.

    1986-06-01

    Two immunization procedures were compared for their ability to yield monoclonal antibodies that react with plasma membrane-bound differentiation antigens of Dictyostelium. In the first method, hybridomas prepared from BALB/c mice immunized with aggregating amoebae produced monoclonal antibodies that recognized antigens present on both growing and aggregating Dictyostelium amoebae. None of the monoclonal antibodies reacted with only the injected aggregation-stage cell type. In contrast, monoclonal antibodies that reacted with differentiation antigens were easily obtained by primary immunization of BALB/c mice with living aggregation-stage cells, followed by secondary immunization with a preparation of plasma membrane from aggregating cells or intact aggregating cells mixed with polyclonal BALB/c antiserum raised against undifferentiated cells. By this method, approximately 20% of all anti-Dictyostelium monoclonal antibodies obtained in a fusion are specific for differentiation antigens. The properties and developmental regulation of several of these antigens are described. The possible uses of this immunological method to detect unique determinants on other kinds of cells and the likely immune mechanisms that make it successful are discussed.

  3. Monoclonal antibodies to hepatitis B surface antigen: production and characterization.

    Science.gov (United States)

    Hlozánek, I; Dostálová, V; Korec, E; Zelený, V; König, J; Nĕmecek, V

    1986-01-01

    Hybridomas secreting anti-HBsAg antibodies were produced by fusion of the mouse myeloma cell line SP2/0 with lymphocytes from mice immunized with purified HBsAg. All clones produced antibodies of the IgG1 idiotype that react with the subtype a determinant of HBsAg. An enzyme immunoassay for detection of HBsAg in human sera using monoclonal antibodies was developed and compared with commercial Sevatest ELISA HBsAg/micro I kit for detection of HBsAg in clinical serum samples.

  4. The chicken erythrocyte-specific MHC antigen. Characterization and purification of the B-G antigen by monoclonal antibodies

    DEFF Research Database (Denmark)

    Salomonsen, J; Skjødt, K; Crone, M

    1987-01-01

    -labeled chicken erythrocyte membranes (CEM) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The B-G antigen had an approximate molecular mass of 46-48 kd in reduced samples, depending on the haplotype, and in unreduced samples contained either dimers (85 kd......-G to be synthesized as a monomer, with dimerization taking place after 20-30 min. No change in the monomer's molecular mass due to posttranslational modifications was revealed. The antigen was purified from detergent extract of CEM by affinity chromatography with a monoclonal antibody, and then reduced and alkylated...... from the affinity chromatography step was 3-4 micrograms B-G/ml blood, calculated from Coomassie-stained SDS-PAGE of B-G using ovalbumin standards. The monoclonal antibodies were also used to identify the B-G (class IV) precipitation arc in crossed immunoelectrophoresis. No common precipitate...

  5. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

    DEFF Research Database (Denmark)

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

    1999-01-01

    BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously...

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Andrew R Williams

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

  8. Use of the Abbott Architect HIV antigen/antibody assay in a low incidence population.

    Science.gov (United States)

    Dubravac, Terry; Gahan, Thomas F; Pentella, Michael A

    2013-12-01

    With the availability of 4th generation HIV diagnostic tests which are capable of detecting acute infection, Iowa evaluated the 3rd and 4th generation HIV test and compared the performance of these products in a low incidence population. This study was conducted to evaluate the performance of an HIV antigen/antibody combination (4th generation) assay compared to an EIA 3rd generation assay. Over a 4 month period, 2037 specimens submitted for HIV screening were tested by Bio-Rad GS HIV-1/HIV-2 Plus O EIA and the Abbott Architect i1000SR HIV Ag/Ab Combo. The performance characteristics of sensitivity, specificity, positive predictive value and negative predictive value were determined. Of the 2037 specimens tested, there were 13 (0.64%) true positives detected. None of the positive specimens were from patients in the acute phase of infection. The Abbott antigen/antibody combo assay had a sensitivity, specificity, positive-predictive value and negative predictive value of 100%, 99.85%, 81.25%, and 100% respectively. The Bio-Rad EIA assay had a sensitivity, specificity, positive-predictive value and negative predictive value of 100%, 99.80%, 76.47% and 100%, respectively. The EIA had four false positive results which tested negative by the antigen/antibody assay and western blot. In a low-incidence state where early infections are less commonly encountered, the EIA assay and the antigen/antibody assay performed with near equivalency. The antigen/antibody assay had one less false positive result. While no patients were detected in the acute stage of infection, the use of the antigen/antibody assay presents the opportunity to detect an infected patient sooner and prevent transmission to others. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Hafezeh Alizadeh

    2012-12-01

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

  10. Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

    Science.gov (United States)

    Neumann-Schaal, Meina; Messerschmidt, Katrin; Grenz, Nicole; Heilmann, Katja

    2013-03-01

    Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Defining Influenza A Virus Hemagglutinin Antigenic Drift by Sequential Monoclonal Antibody Selection

    OpenAIRE

    Das, Suman R.; Hensley, Scott E.; Ince, William L.; Brooke, Christopher B.; Subba, Anju; Delboy, Mark G.; Russ, Gustav; Gibbs, James S.; Bennink, Jack R.; Yewdell, Jonathan W.

    2013-01-01

    Human influenza A virus (IAV) vaccination is limited by “antigenic drift,” rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined ...

  12. Influenza virus antigenic variation, host antibody production and new approach to control epidemics

    Directory of Open Access Journals (Sweden)

    Deng Yi-Mo

    2009-03-01

    Full Text Available Abstract Influenza is an infectious disease and can lead to life-threatening complications like pneumonia. The disease is caused by three types of RNA viruses called influenza types A, B and C, each consisting of eight negative single-stranded RNA-segments encoding 11 proteins. Current annual vaccines contain two type A strains and one type B strain and are capable of inducing strong antibody responses to both the surface glycoprotein hemagglutinin and the neuraminidase. While these vaccines are protective against vaccine viruses they are not effective against newly emerging viruses that contain antigenic variations known as antigenic drift and shift. In nature, environmental selection pressure generally plays a key role in selecting antigenic changes in the antigen determining spots of hemagglutinin, resulting in changes in the antigenicity of the virus. Recently, a new technology has been developed where influenza-specific IgG+ antibody-secreting plasma cells can be isolated and cloned directly from vaccinated humans and high affinity monoclonal antibodies can be produced within several weeks after vaccination. The new technology holds great promise for the development of effective passive antibody therapy to limit the spread of influenza viruses in a timely manner.

  13. Streptococci and Actinomyces induce antibodies which cross react with epithelial antigens in periodontitis

    Science.gov (United States)

    Ye, P; Harty, D W S; Chapple, C C; Nadkarni, M A; Carlo, A A D E; Hunter, N

    2003-01-01

    Perturbation of epithelial structure is a prominent but poorly understood feature of the immunopathological response to bacterial antigens which characterizes the destructive lesion of periodontitis. Western analysis of sera from 22 patients with periodontitis detected multiple antigens in extracts of epithelial cells whereas sera from 12 periodontally healthy subjects displayed only trace reaction with epithelial antigens. To investigate a possible relationship between the bacterial flora adjacent to diseased sites and the presence of antibodies reactive with epithelium, subgingival plaque samples were taken from deep periodontal pockets and cultured anaerobically. Gram positive bacteria containing antigens cross-reactive with epithelial cells were reproducibly isolated by probing membrane colony-lifts with affinity-isolated (epithelium-specific) antibodies and identified by 16S rDNA sequence homology as streptococci (S. mitis, S. constellatus and two S. intermedius strains) and Actinomyces (A. georgiae, and A. sp. oral clone). Conversely, when serum from patients with periodontitis was absorbed with the captured bacterial species the number of epithelial antigens recognized was specifically reduced. It was concluded that development of cross-reactive antibodies related to these organisms may contribute to perturbation of the epithelial attachment to the tooth and the progression of periodontitis. These autoreactive antibodies could also be a contributing factor in other diseases affecting epithelia. PMID:12605700

  14. [Biodynamics of the antigen-antibody reaction in vivo].

    Science.gov (United States)

    Scherrmann, J M

    2000-01-01

    The success of the toxin neutralization by a specific antibody in a living system depends on multiple factors. First, the type of the actual available antibody structures (immunoglobulin G, Fab2 or Fab fragments) must be selected according to the toxin molecular weight: Fab is adapted to the neutralization of haptens, IgG and Fab2 to macromolecular toxins. Other factors involved in the success of immunotherapy are issued from the pharmacokinetic properties of the toxin: non reversible binding to the receptor, intracellular distribution of a macromolecular toxin are disadvantageous. Finally, the selection of the antibody dose according to its association constant value for the toxin allows to administer the optimal capacity of immunoneutralization. The control of all these factors inserted in the biodynamics of the living system contributes to the success of immunotherapy.

  15. Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens

    DEFF Research Database (Denmark)

    Sparholt, S H; Barington, T; Schou, C

    1991-01-01

    An enzyme-linked immunospot assay (ELI-spot assay) has been established to count individual cells secreting antibodies to Dermatophagoides spp. allergens. Initial optimization of the assay was performed using Der p I-specific murine hybridoma cell lines. Inhibition with soluble purified allergen...... demonstrated that spot formation was specific. Addition of cycloheximide showed that spot formation was due to antibodies actively produced and secreted and not caused by preformed surface-associated Ig. An analogous method was used to count immunoglobulin-secreting cells (IgSC) of any specificity...... Alutard, showed that 1 week after injection, circulating allergen-specific antibody-secreting cells (AbSC) were detected with this assay. The ranges were between 1 and 13 IgM-, 2 and 20 IgG- and 20 and 55 IgA allergen-specific AbSC per 10(6) MNC. It is expected that this assay will help to understand more...

  16. Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic

    Energy Technology Data Exchange (ETDEWEB)

    Ayeb, M.E.; Bahraoui, E.M.; Granier, C.; Beress, L.; Rochat, H.

    1986-11-04

    Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp=9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and ..cap alpha..-NH/sub 2/ of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.

  17. Plant Virus Particles Carrying Tumour Antigen Activate TLR7 and Induce High Levels of Protective Antibody

    OpenAIRE

    Jantipa Jobsri; Alex Allen; Deepa Rajagopal; Michael Shipton; Kostya Kanyuka; Lomonossoff, George P.; Christian Ottensmeier; Diebold, Sandra S.; Stevenson, Freda K.; Natalia Savelyeva

    2015-01-01

    Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a n...

  18. Receptor-Carrier Based Models for Antigen-Antibody Interactions

    Science.gov (United States)

    1989-07-07

    antigen bearing cell and then looks for other antigens. The 4 Cell-Mediated Immune Response T-lymphocyte memory T-cell 00 antigeno cttxcTilhpeT-elsprso 0...f+s(Ksa)SVo, O (17) f=0 s=O n n-f AbO = a + Ksal + I I (s)dfs(Kfl)f+S(Ksa)sVoc f=0 s=O (18) n n-f LO = I + KsaA + I Z: (f+s)df,s(Kfl)f+S(Ksa)SVo, o f...conservation equation can be described in similar terms AbO = a + J(n)Vo,o + W(n) V 2 0 ,0 (33) J(n) and W(n) are defined as n/24 J(n) = I (ixi + (n-i)xn-i)S(i

  19. Detection of avian influenza antibodies and antigens in poultry and ...

    African Journals Online (AJOL)

    The detection of AI (NP) antibodies in wild birds but failure to detect the viruses showed that the exposure might not be recent. We recommend that poultry should be prevented from contact with wild water birds and a broad based surveillance for AI viruses in poultry and wild birds should be carried out in Kogi state, Nigeria.

  20. Detection of avian influenza antibodies and antigens in poultry and ...

    African Journals Online (AJOL)

    The global spread of HPAI (H5N1) between 2005 and 2006 was blamed on movement of migratory wild birds and trade in live poultry across continents from infected regions. A survey was carried out to detect the presence of avian influenza (AI) antibodies in wild birds and AI viruses in poultry and wild birds from Kogi state, ...

  1. Masking of antigenic epitopes by antibodies shapes the humoral immune response to influenza

    Science.gov (United States)

    Zarnitsyna, Veronika I.; Ellebedy, Ali H.; Davis, Carl; Jacob, Joshy; Ahmed, Rafi; Antia, Rustom

    2015-01-01

    The immune responses to influenza, a virus that exhibits strain variation, show complex dynamics where prior immunity shapes the response to the subsequent infecting strains. Original antigenic sin (OAS) describes the observation that antibodies to the first encountered influenza strain, specifically antibodies to the epitopes on the head of influenza's main surface glycoprotein, haemagglutinin (HA), dominate following infection with new drifted strains. OAS suggests that responses to the original strain are preferentially boosted. Recent studies also show limited boosting of the antibodies to conserved epitopes on the stem of HA, which are attractive targets for a ‘universal vaccine’. We develop multi-epitope models to explore how pre-existing immunity modulates the immune response to new strains following immunization. Our models suggest that the masking of antigenic epitopes by antibodies may play an important role in describing the complex dynamics of OAS and limited boosting of antibodies to the stem of HA. Analysis of recently published data confirms model predictions for how pre-existing antibodies to an epitope on HA decrease the magnitude of boosting of the antibody response to this epitope following immunization. We explore strategies for boosting of antibodies to conserved epitopes and generating broadly protective immunity to multiple strains. PMID:26194761

  2. Antibodies against food antigens in patients with autistic spectrum disorders.

    Science.gov (United States)

    de Magistris, Laura; Picardi, Annarita; Siniscalco, Dario; Riccio, Maria Pia; Sapone, Anna; Cariello, Rita; Abbadessa, Salvatore; Medici, Nicola; Lammers, Karen M; Schiraldi, Chiara; Iardino, Patrizia; Marotta, Rosa; Tolone, Carlo; Fasano, Alessio; Pascotto, Antonio; Bravaccio, Carmela

    2013-01-01

    Immune system of some autistic patients could be abnormally triggered by gluten/casein assumption. The prevalence of antibodies to gliadin and milk proteins in autistic children with paired/impaired intestinal permeability and under dietary regimen either regular or restricted is reported. 162 ASDs and 44 healthy children were investigated for intestinal permeability, tissue-transglutaminase (tTG), anti-endomysium antibodies (EMA)-IgA, and total mucosal IgA to exclude celiac disease; HLA-DQ2/-DQ8 haplotypes; total systemic antibodies (IgA, IgG, and IgE); specific systemic antibodies: α-gliadin (AGA-IgA and IgG), deamidated-gliadin-peptide (DGP-IgA and IgG), total specific gliadin IgG (all fractions: α, β, γ, and ω), β-lactoglobulin IgG, α-lactalbumin IgG, casein IgG; and milk IgE, casein IgE, gluten IgE,-lactoglobulin IgE, and α-lactalbumin IgE. AGA-IgG and DPG-IgG titers resulted to be higher in ASDs compared to controls and are only partially influenced by diet regimen. Casein IgG titers resulted to be more frequently and significantly higher in ASDs than in controls. Intestinal permeability was increased in 25.6% of ASDs compared to 2.3% of healthy children. Systemic antibodies production was not influenced by paired/impaired intestinal permeability. Immune system of a subgroup of ASDs is triggered by gluten and casein; this could be related either to AGA, DPG, and Casein IgG elevated production or to impaired intestinal barrier function.

  3. Antibodies against Food Antigens in Patients with Autistic Spectrum Disorders

    Directory of Open Access Journals (Sweden)

    Laura de Magistris

    2013-01-01

    Full Text Available Purpose. Immune system of some autistic patients could be abnormally triggered by gluten/casein assumption. The prevalence of antibodies to gliadin and milk proteins in autistic children with paired/impaired intestinal permeability and under dietary regimen either regular or restricted is reported. Methods. 162 ASDs and 44 healthy children were investigated for intestinal permeability, tissue-transglutaminase (tTG, anti-endomysium antibodies (EMA-IgA, and total mucosal IgA to exclude celiac disease; HLA-DQ2/-DQ8 haplotypes; total systemic antibodies (IgA, IgG, and IgE; specific systemic antibodies: α-gliadin (AGA-IgA and IgG, deamidated–gliadin-peptide (DGP-IgA and IgG, total specific gliadin IgG (all fractions: α, β, γ, and ω, β-lactoglobulin IgG, α-lactalbumin IgG, casein IgG; and milk IgE, casein IgE, gluten IgE, -lactoglobulin IgE, and α-lactalbumin IgE. Results. AGA-IgG and DPG-IgG titers resulted to be higher in ASDs compared to controls and are only partially influenced by diet regimen. Casein IgG titers resulted to be more frequently and significantly higher in ASDs than in controls. Intestinal permeability was increased in 25.6% of ASDs compared to 2.3% of healthy children. Systemic antibodies production was not influenced by paired/impaired intestinal permeability. Conclusions. Immune system of a subgroup of ASDs is triggered by gluten and casein; this could be related either to AGA, DPG, and Casein IgG elevated production or to impaired intestinal barrier function.

  4. Lymphocyte antigens targetable by monoclonal antibodies in non-systemic vasculitic neuropathy.

    Science.gov (United States)

    Schneider, Christian; Wunderlich, Gilbert; Bleistein, Johannes; Fink, Gereon R; Deckert, Martina; Brunn, Anna; Lehmann, Helmar Christoph

    2017-09-01

    To identify the most relevant antigens for monoclonal antibodies in lymphocytic infiltrates in non-systemic vasculitic neuropathy (NSVN). Current immunosuppressive treatment for NSVN is insufficient. Monoclonal antibodies might be a treatment option, but the expression profile for targetable antigens on lymphocytic infiltrates in NSVN is unknown. Sural nerve biopsies from a cohort of patients with NSVN were immunohistochemically studied for the expression of potential candidate antigens in perivascular and intramural lymphocytic infiltrates and correlated with neurological and electrophysiological parameters. 20 patients with treatment naïve NSVN and 5 patients with idiopathic axonal neuropathy were included. The CD52, BAFF and CD49d antigens were expressed in epineurial, perivascular or intramural lymphocytes of all (20/20) patients. CD52 was most prominently expressed in 21.49% of all inflammatory infiltrates. BAFF and CD49d were detected in 11.25% and 10.99% of these lymphocytes, respectively. The CD20, CD25 and CD126 antigens were found less frequently and at low levels only (CD20: 10/20 patients, 5.84% of lymphocytes; CD25: 17/20 patients, 5.22% of lymphocytes; CD126: 3/20 patients, 0.15% of lymphocytes). This is the first study in NSVN that identifies antigens expressed by pathogenic lymphocytes, which are potential targets for future monoclonal antibody treatment. Our data suggest that NSVN is amenable to monoclonal antibodies and, moreover, that targeting CD52 may be particularly promising. Our results strongly warrant future clinical trials in NSVN with monoclonal antibodies. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  5. Spatial clustering of porcine cysticercosis in Mbulu district, northern Tanzania.

    Directory of Open Access Journals (Sweden)

    Helena A Ngowi

    Full Text Available BACKGROUND: Porcine cysticercosis is caused by a zoonotic tapeworm, Taenia solium, which causes serious disease syndromes in human. Effective control of the parasite requires knowledge on the burden and pattern of the infections in order to properly direct limited resources. The objective of this study was to establish the spatial distribution of porcine cysticercosis in Mbulu district, northern Tanzania, to guide control strategies. METHODOLOGY/PRINCIPAL FINDINGS: This study is a secondary analysis of data collected during the baseline and follow-up periods of a randomized community trial aiming at reducing the incidence rate of porcine cysticercosis through an educational program. At baseline, 784 randomly selected pig-keeping households located in 42 villages in 14 wards were included. Lingual examination of indigenous pigs aged 2-12 (median 8 months, one randomly selected from each household, were conducted. Data from the control group of the randomized trial that included 21 of the 42 villages were used for the incidence study. A total of 295 pig-keeping households were provided with sentinel pigs (one each and reassessed for cysticercosis incidence once or twice for 2-9 (median 4 months using lingual examination and antigen ELISA. Prevalence of porcine cysticercosis was computed in Epi Info 3.5. The prevalence and incidence of porcine cysticercosis were mapped at household level using ArcView 3.2. K functions were computed in R software to assess general clustering of porcine cysticercosis. Spatial scan statistics were computed in SatScan to identify local clusters of the infection. The overall prevalence of porcine cysticercosis was 7.3% (95% CI: 5.6, 9.4; n = 784. The K functions revealed a significant overall clustering of porcine cysticercosis incidence for all distances between 600 m and 5 km from a randomly chosen case household based on Ag-ELISA. Lingual examination revealed clustering from 650 m to 6 km and between 7.5 and 10 km

  6. Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

    Directory of Open Access Journals (Sweden)

    Kristina M. Ilieva

    2017-09-01

    Full Text Available Monoclonal antibodies find broad application as therapy for various types of cancer by employing multiple mechanisms of action against tumors. Manipulating the Fc-mediated functions of antibodies that engage immune effector cells, such as NK cells, represents a strategy to influence effector cell activation and to enhance antibody potency and potentially efficacy. We developed a novel approach to generate and ascertain the functional attributes of Fc mutant monoclonal antibodies. This entailed coupling single expression vector (pVitro1 antibody cloning, using polymerase incomplete primer extension (PIPE polymerase chain reaction, together with simultaneous Fc region point mutagenesis and high yield transient expression in human mammalian cells. Employing this, we engineered wild type, low (N297Q, NQ, and high (S239D/I332E, DE FcR-binding Fc mutant monoclonal antibody panels recognizing two cancer antigens, HER2/neu and chondroitin sulfate proteoglycan 4. Antibodies were generated with universal mutagenic primers applicable to any IgG1 pVitro1 constructs, with high mutagenesis and transfection efficiency, in small culture volumes, at high yields and within 12 days from design to purified material. Antibody variants conserved their Fab-mediated recognition of target antigens and their direct anti-proliferative effects against cancer cells. Fc mutations had a significant impact on antibody interactions with Fc receptors (FcRs on human NK cells, and consequently on the potency of NK cell activation, quantified by immune complex-mediated calcium mobilization and by antibody-dependent cellular cytotoxicity (ADCC of tumor cells. This strategy for manipulation and testing of Fc region engagement with cognate FcRs can facilitate the design of antibodies with defined effector functions and potentially enhanced efficacy against tumor cells.

  7. Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens

    DEFF Research Database (Denmark)

    Ofori, Michael F; Dodoo, Daniel; Staalsoe, Trine

    2002-01-01

    In areas of intense Plasmodium falciparum transmission, protective immunity is acquired during childhood in parallel with acquisition of agglutinating antibodies to parasite-encoded variant surface antigens (VSA) expressed on parasitized red blood cells. In a semi-immune child in such an area......, clinical disease is caused mainly by parasites expressing VSA not recognized by preexisting VSA-specific antibodies in that child. Such malaria episodes are known to cause an increase in agglutinating antibodies specifically recognizing VSA expressed by the parasite isolate causing the illness, whereas...

  8. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  9. Monoclonal antibodies produced against antigenic determinants present in complex mixtures of proteins.

    Science.gov (United States)

    King, S W; Morrow, K J

    1988-10-01

    This overview provides information concerning the production of monoclonal antibodies (MAbs) against specific antigenic determinants present in complex mixtures of proteins. We review five specific techniques for the production of these antibodies (Abs): (a) So-called "shotgun," non-selective approach; (b) cascade procedure; (c) lymphocyte "panning"; (d) cyclophosphamide elimination of unwanted Ab producers; and finally (e) use of polyclonal antisera to extinguish unwanted antibody production. We discuss the relative advantages and disadvantages of these various procedures, and suggest alternative strategies by which specific MAbs might be generated.

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

    Science.gov (United States)

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Brasil, Tatiana de Arruda Campos; Foti, Leonardo; Souza, Wayner Vieira de; Silva, Edmilson Domingos; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Fred Luciano Neves Santos

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

  13. Evaluation of the impact of a control program against taeniasis-cysticercosis (Taenia solium.

    Directory of Open Access Journals (Sweden)

    Aline S de Aluja

    2014-05-01

    Full Text Available Objetive. The impact of a control program is evaluated to eventually eradicate taeniasis-cysticercosis (Taenia solium based on education and vaccination of pigs. Materials and methods. The prevalence of porcine cysticercosis was estimated using tongue inspection, ultrasound and determination of antibodies, before and three years after the application in three regions of the state of Guerrero. Results. A significant reduction in the prevalence of porcine cysticercosis of 7 to 0.5% and 3.6 to 0.3% estimated by tongue examination or ultrasound respectively (p menor que 0.01 and a no significant decrease in seroprevalence from 17.7 to 13.3% were observed. Conclusions. The reduction of the prevalence of taeniasis-cysticercosis establishes the program’s effectiveness in preventing infection. The sustained presence of antibodies, compatible with contact of Taenia solium or other related helminths, underlines the importance of maintaining interventionsto achieve eradication.

  14. Hepatitis B virus surface antigen and antibody markers in children at ...

    African Journals Online (AJOL)

    Hepatitis B virus surface antigen and antibody markers in children at a major paediatric hospital after the pentavalent DTP-HBV-Hib vaccination. ... Ghana Medical Journal ... Abstract. Objectives: The knowledge about outcomes of infant vaccination against HBV infections using the DPT-HepB-Hib vaccine in Ghana is limited.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-05-01

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

  16. Acute HIV-1 Infection in Antigen/Antibody-negative Blood Donors in ...

    African Journals Online (AJOL)

    The aim of this study was to determine the prevalence of acute HIV infection among HIV antigen/antibody negative blood donors. This was a cross-sectional blood donation based facility study conducted at Eastern Zone Blood Transfusion Services in Dar es Salaam from December 2009 to April 2010. Apparently healthy ...

  17. From therapeutic antibodies to chimeric antigen receptors (CARs): making better CARs based on antigen-binding domain.

    Science.gov (United States)

    Wu, Yanling; Jiang, Shibo; Ying, Tianlei

    2016-12-01

    A variety of approaches are being pursued to improve the safety and antitumor potency of chimeric antigen receptor (CAR) T-cell therapy. However, most engineering efforts have thus far been focused on its intracellular signaling domain, while its extracellular antigen-binding domain has received less attention. Areas covered: Herein, the authors summarize the current knowledge of CAR T-cell therapy. Accordingly, they focus on its antigen-binding domain, discuss key considerations for selecting an optimal single-chain variable fragment (scFv) when designing a CAR, and suggest potential directions aimed at developing the next-generation CARs. Expert opinion: The extracellular region of CARs can play a decisive role in their safety and efficacy. Instead of directly translating an available therapeutic mAb to a scFv-based CAR construct, the authors suggest that various CAR-displayed scFvs with different affinity, specificity and binding epitopes against an individual target molecule should be generated and evaluated side-by-side. Incorporating new antibody formats that possess characteristics superior to those of scFvs may be one way to engineer safer and more effective CARs. The authors expect that further CAR engineering will enable us to target more antigens involved in hematological and solid malignancies with minimal side effects to serve unmet clinical needs.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    a naturally occurring subfraction from human serum, to Galalpha containing neoglycoproteins and mouse laminin that were immobilized on microtiter plates. RESULTS: Galalpha reactive antibodies with similar monosaccharide specificity have distinct structural preference for sugar ligands. Laminin...... and neoglycoproteins were treated with alpha-galactosidase and subsequently incubated with antibodies and lectins. The enzyme treatment was more deleterious on antibody binding than on lectin binding. CONCLUSION: Antibodies and lectins may bind to different galactose determinants on the glycoproteins. Two anti......-Galalpha1 antibodies that both have been raised against glycans on rabbit red blood cells may recognize Galalpha-antigens with varying specificities. Binding results obtained after digestion with alpha-galactosidase indicate that some xenoreactive Galalpha groups are not directly accessible for removal...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-02-01

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

  20. Class specific antibody responses to newborn larva antigens during Trichinella spiralis human infection

    Directory of Open Access Journals (Sweden)

    Mendez-Loredo B.

    2001-06-01

    Full Text Available A follow-up study of the class antibody responses to newborn larva (NBL antigens in individuals involved in an outbreak of human trichinellosis was carried out by ELISA assays. The data showed that similar kinetics of antibody responses of different magnitude developed in trichinellosis patients; it was low by week 3, a peak raised by week 5 and decreased from week 7 up to the end of the study. The IgA-ELISA assay was the most sensitive and specific while the IgM was the least sensitive and specific. IgA antibodies to NBL antigens were detected in 80 % of patients while IgE, IgG and IgM responses were observed in 44, 31 and 19 % of the patients by week 3, respectively. From weeks 5 to 7, IgA antibodies were found in 89 to 100 % of the patients while lower percentages (0-82 % were found for the other isotypes. Reactivity of IgA, IgE, IgG and IgM to NBL antigens decreased from week 37 to 57 after infection (0-38 %. These results suggest that detection of IgA antibodies may be useful for early diagnosis and epidemiological studies in human trichinellosis.

  1. Opsonic and protective properties of antibodies raised to conjugate vaccines targeting six Staphylococcus aureus antigens.

    Directory of Open Access Journals (Sweden)

    Clarissa Pozzi

    Full Text Available Staphylococcus aureus is a major cause of nosocomial and community-acquired infections for which a vaccine is greatly desired. Antigens found on the S. aureus outer surface include the capsular polysaccharides (CP of serotype 5 (CP5 or 8 (CP8 and/or a second antigen, a β-(1→6-polymer of N-acetyl-D-glucosamine (PNAG. Antibodies specific for either CP or PNAG antigens have excellent in vitro opsonic killing activity (OPKA, but when mixed together have potent interference in OPKA and murine protection. To ascertain if this interference could be abrogated by using a synthetic non-acetylated oligosaccharide fragment of PNAG, 9GlcNH(2, in place of chemically partially deacetylated PNAG, three conjugate vaccines consisting of 9GlcNH(2 conjugated to a non-toxic mutant of alpha-hemolysin (Hla H35L, CP5 conjugated to clumping factor B (ClfB, or CP8 conjugated to iron-surface determinant B (IsdB were used separately to immunize rabbits. Opsonic antibodies mediating killing of multiple S. aureus strains were elicited for all three vaccines and showed carbohydrate antigen-specific reductions in the tissue bacterial burdens in animal models of S. aureus skin abscesses, pneumonia, and nasal colonization. Carrier-protein specific immunity was also shown to be effective in reducing bacterial levels in infected lungs and in nasal colonization. However, use of synthetic 9GlcNH(2 to induce antibody to PNAG did not overcome the interference in OPKA engendered when these were combined with antibody to either CP5 or CP8. Whereas each individual vaccine showed efficacy, combining antisera to CP antigens and PNAG still abrogated individual OPKA activities, indicating difficulty in achieving a multi-valent vaccine targeting both the CP and PNAG antigens.

  2. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Jr., Joseph J. (SAIC); (NCI)

    2012-10-16

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

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

    Science.gov (United States)

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

    2018-02-01

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

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

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    Ondigo Bartholomew N

    2012-12-01

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

  5. Anti-idiotypic antibody with potential use as an Eimeria tenella sporozoite antigen surrogate for vaccination of chickens against coccidiosis.

    Science.gov (United States)

    Bhogal, B S; Nollstadt, K H; Karkhanis, Y D; Schmatz, D M; Jacobson, E B

    1988-01-01

    Anti-idiotypic antibodies were raised in rabbits against four monoclonal antibodies with specificity for the surface antigenic determinants of Eimeria tenella sporozoites, the infective stage of the coccidial parasite. Two of the monoclonal antibodies (1073 and 15-1) transferred passive protection in chickens against E. tenella infection. The polyclonal anti-idiotype antibody preparations against protective monoclonal antibodies contained specificities for the paratope-associated idiotypes of these monoclonal antibodies, as assessed by the competitive inhibition of binding of the homologous idiotype-anti-idiotype by the sporozoite antigen. Competitive inhibition of binding of homologous idiotype-anti-idiotype by the parasite antigen was not observed when the anti-idiotype antibody preparations against monoclonal antibodies 1546 and 1096 were tested. The anti-idiotype 1073 and 15-1 antibodies functioned as surrogate antigens in vivo when used for vaccination of young chickens, as evidenced by the induction of partial protective immunity against subsequent challenge infection with virulent parasites and induction of antisporozoite antibodies. These data clearly support the view that anti-idiotypic antibodies raised against the paratope-associated idiotypes can mimic pathogen antigens and therefore can provide a possible alternative approach for the vaccination of chickens against coccidiosis. PMID:3258583

  6. Taenia solium Human Cysticercosis: A Systematic Review of Sero-epidemiological Data from Endemic Zones around the World.

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    Marco Coral-Almeida

    Full Text Available Taenia solium cysticercosis is a zoonotic neglected disease responsible for severe health disorders such as seizures and death. Understanding the epidemiology of human cysticercosis (HCC in endemic regions will help to expose critical information about the transmission of the disease, which could be used to design efficient control programs. This review gathered serological data on apparent prevalence of T. solium circulating antigens and/or seroprevalence of T. solium antibodies, apparent prevalence of human taeniasis and risk factors for HCC from endemic communities in order to understand the differences in exposure to the parasite and active infections with T. solium metacestodes in endemic areas around the world.Three databases were used to search sero-epidemiological data from community-based studies conducted between 1989 and 2014 in cysticercosis endemic communities worldwide. The search focused on data obtained from T. solium circulating antigen detection by monoclonal antibody-based sandwich ELISA and/or T. solium antibody seroprevalence determined by Enzyme-linked Immunoelectrotransfer Blot (EITB. A meta-analysis was performed per continent.A total of 39,271 participants from 19 countries, described in 37 articles were studied. The estimates for the prevalence of circulating T. solium antigens for Africa, Latin America and Asia were: 7.30% (95% CI [4.23-12.31], 4.08% (95% CI [2.77-5.95] and 3.98% (95% CI [2.81-5.61], respectively. Seroprevalence estimates of T. solium antibodies were 17.37% (95% CI [3.33-56.20], 13.03% (95% CI [9.95-16.88] and 15.68% (95% CI [10.25-23.24] respectively. Taeniasis reported prevalences ranged from 0 (95% CI [0.00-1.62] to 17.25% (95% CI [14.55-20.23].A significant variation in the sero-epidemiological data was observed within each continent, with African countries reporting the highest apparent prevalences of active infections. Intrinsic factors in the human host such as age and immunity were main

  7. Different types of monoclonal antibodies to Ogawa-specific and group-specific antigens of Vibrio cholerae O1.

    OpenAIRE

    Ito, T; Yokota, T

    1987-01-01

    Nine monoclonal antibodies to Ogawa-specific antigenic determinants of Vibrio cholerae O1 and seven monoclonal antibodies to the Ogawa-Inaba common antigenic determinants were obtained. Specificities and reactivities were examined by slide or microdilution agglutination methods, along with enzyme-linked immunosorbent assays and immunoblotting analysis. In both the Ogawa-specific and Ogawa-Inaba common groups, it was revealed that there were two types of antibodies. One type showed strong aggl...

  8. Systemic antibody responses of calves to low molecular weight Cooperia oncophora antigens.

    Science.gov (United States)

    Nieuwland, M G; Ploeger, H W; Kloosterman, A; Parmentier, H K

    1995-10-01

    The systemic antibody responses to adult Cooperia oncophora antigen were studied using sera obtained from calves during a 6-week period following a single oral infection with either 20,000 or 100,000 third-stage C. oncophora larvae. Dose dependent increasing titres of IgG binding complete adult Cooperia antigen were found in the sera of Cooperia-infected calves. SDS-gel electrophoresis under reducing conditions, followed by Western blotting, revealed that the increase of IgG binding Cooperia antigens could be attributed mainly to specific binding of IgG to a complex of 12-15 kDa protein fragments of Cooperia adult antigen. This protein may represent a Cooperia oncophora-specific component that can be used for serodiagnosis.

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

    NARCIS (Netherlands)

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

    1998-01-01

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

  10. Multiple transfusion fail to provoke antibodies against blood cell antigens in human infants.

    Science.gov (United States)

    Floss, A M; Strauss, R G; Goeken, N; Knox, L

    1986-01-01

    We conducted studies of both red cell (RBC) and leukocyte (WBC) antibody formation in infants following multiple transfusions given during the first weeks of life. Fifty-three infants received 683 RBC transfusions from 503 different donors, plus 62 platelet, 4 granulocyte, and 53 fresh-frozen plasma units during the first 4 months of life. Three hundred fifty serum samples were obtained before, during, and after the transfusions. None of the infants formed unexpected RBC antibodies when tested at 37 degrees C by a two-cell low-ionic-strength solution antibody screen that included an anti-globulin phase. Twenty posttransfusion serums were negative when tested at room temperature. Lymphocytotoxic and granulocytotoxic WBC antibodies were measured in posttransfusion serums from 13 infants, and none were found. Despite exposure to many RBC and WBC antigens, no infants produced alloantibodies against blood cell antigens. Thus, immunologically mediated transfusion reactions should be quite rare in young infants, and this study supports recommendations of the American Association of Blood Banks Standards to omit repeat RBC compatibility testing during the first 4 months of life in infants whose initial RBC antibody screens reveal no unexpected antibodies.

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

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    Spiropoulou Christina F

    2010-06-01

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

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

    Science.gov (United States)

    Chiang, Cheng-Feng; Lo, Michael K; Rota, Paul A; Spiropoulou, Christina F; Rollin, Pierre E

    2010-06-03

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

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

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    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.

  14. Antibodies anti granulocytic antigens in IBD: from microscopic morphology to antigenic specificity

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    A. Montanelli

    2011-09-01

    Full Text Available Objective: ANCA (p-ANCA and x-ANCA have been documented to occur in many inflammatory disorders. The specific ANCA antigens and the clinical correlation of a positive ANCA test in these disorders are still for the most part obscure. The aim of the present study was to investigate the prevalence of and the target antigens for ANCA in patients with IBD. Methods: 104 patients (67 age between 3-18 years, mean age 8+3 and 37 age between 25-70 years, mean age 48+15 clinically and hystopathologically diagnosed as: 67 ulcerative colitis, 16 Crohn’ disease, 21 other colitis (7 indeterminate colitis were enrolled in our study. ANCA were determined by ELISA and IIF methods. Results: We observed a good performance in terms of sensibility and specificity of ANCA, and a good correlation between the two methods used; as regard ELISA determination the antigen frequently found in our cases was lactoferrin (60%. Conclusions: Is still unclear the role of these “minor antigens” in the diagnosis and pathogenesis of IBD, but is clear that only morphologic evaluation is no more sufficient.

  15. Mycoplasma antigens as a possible trigger for the induction of antimitochondrial antibodies in primary biliary cirrhosis.

    Science.gov (United States)

    Berg, Christoph P; Kannan, Thirumalai R; Klein, Reinhild; Gregor, Michael; Baseman, Joel B; Wesselborg, Sebastian; Lauber, Kirsten; Stein, Gerburg M

    2009-07-01

    In primary biliary cirrhosis (PBC), autoreactivity mainly targets members of the pyruvate dehydrogenase complex (PDC). Because PDC subunits are expressed on the surface of mycoplasma and molecular mimicry may be one aetiological factor, we analysed the presence of mammalian and mycoplasma PDC-specific antibodies in PBC patients. Antibodies to porcine PDC and Mycoplasma pneumoniae (mp) antigens mpPDH-C (to be designated mpPDC-E2 chain), mpPDH-B (to be designated mpPDC-E1beta chain), mpCARDS TX and mpP1 were investigated in sera from 43 PBC patients, 19 patients with autoimmune hepatitis and 11 healthy controls by an enzyme-linked immunosorbent assay and Western blotting. To study the rate of acute mycoplasma infection, an adhesin P1-specific polymerase chain reaction (PCR) was performed. Immune reactivity to the mpPDC-E2 antigen was significantly enhanced in PBC patients (83.7%) as compared with controls (overall frequency of 36.7%), while antibodies to the porcine PDC-E2 chain were found only in PBC patients (88%) excluding a simple cross-reactivity of PDC-related antibodies. This observation was confirmed by inhibition studies demonstrating that porcine PDC did not inhibit mycoplasma PDC-specific antibodies and vice versa. The occurrence of antibodies to mpPDC seems to precede the occurrence of antibodies to porcine PDC. Infection with mycoplasma was equally distributed in the groups as evidenced by an antibody frequency comparable to CARDS TX and P1 and PCR reactivity. Because PBC patients show a significantly enhanced frequency of mpPDC-E2-related antibodies, besides other factors, molecular mimicry between surface molecules of mycoplasma and epitopes of the autoantigen may play a central role in the aetiopathology of PBC.

  16. New principle for the simultaneous detection of total and immunoglobulin M antibodies applied to the measurement of antibody to hepatitis B core antigen.

    OpenAIRE

    Angarano, G.; Monno, L; Santantonio, T A; Pastore, G.

    1984-01-01

    A new test principle for the simultaneous detection of total approximate titers and immunoglobulin M antibodies has been developed and applied to the detection of antibody to hepatitis B core antigen. The method is based on the combination of a competition radioimmunoassay, for the determination of total antibody titer, with an indirect enzyme-linked immunosorbent assay for the determination of single class antibodies. The interference of the rheumatoid factor was avoided by including heat-ag...

  17. Immunotherapy of hepatocellular carcinoma using chimeric antigen receptors and bispecific antibodies.

    Science.gov (United States)

    Hoseini, Sayed Shahabuddin; Cheung, Nai-Kong V

    2017-07-28

    Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide with an overall survival rate of less than 15% in developed countries. Despite attempts at new therapeutic strategies, the majority of patients succumb to this cancer. Buttressed by the highly successful clinical impact in melanoma, immunotherapy is gaining momentum as the next treatment modality for many human cancers. Chimeric antigen receptors (CAR) contain the antigen binding moieties of a monoclonal antibody and the co-stimulatory and signaling domains associated with effector receptor signaling. Bispecific antibodies (BsAb) combine the binding specificities of two different monoclonal antibodies, one activating a receptor on a killer effector cell, while the other engaging a tumor-associated antigen to initiate tumor cytotoxicity. In this review, we survey the HCC targets for which CARs and bispecific antibodies have been generated. The pros and cons of these targets for T-cell and Natural Killer cell based immunotherapy will be discussed. Published by Elsevier B.V.

  18. Microarray-based identification of human antibodies against Staphylococcus aureus antigens.

    Science.gov (United States)

    Kloppot, Peggy; Selle, Martina; Kohler, Christian; Stentzel, Sebastian; Fuchs, Stephan; Liebscher, Volkmar; Müller, Elke; Kale, Devika; Ohlsen, Knut; Bröker, Barbara M; Zipfel, Peter F; Kahl, Barbara C; Ehricht, Ralf; Hecker, Michael; Engelmann, Susanne

    2015-12-01

    The mortality rate of patients with Staphylococcus aureus infections is alarming and urgently demands new strategies to attenuate the course of these infections or to detect them at earlier stages. To study the adaptive immune response to S. aureus antigens in healthy human volunteers, a protein microarray containing 44 S. aureus proteins was developed using the ArrayStrip platform technology. Testing plasma samples from 15 S. aureus carriers and 15 noncarriers 21 immunogenic S. aureus antigens have been identified. Seven antigens were recognized by antibodies present in at least 60% of the samples, representing the core S. aureus immunome of healthy individuals. S. aureus-specific serum immunoglobulin G (IgG) levels were significantly lower in noncarriers than in carriers specifically anti-IsaA, anti-SACOL0479, and anti-SACOL0480 IgGs were found at lower frequencies and quantities. Twenty-two antigens present on the microarray were encoded by all S. aureus carrier isolates. Nevertheless, the immune system of the carriers was responsive to only eight of them and with different intensities. The established protein microarray allows a broad profiling of the S. aureus-specific antibody response and can be used to identify S. aureus antigens that might serve as vaccines or diagnostic markers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies.

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    Ben-Jiang Ma

    2011-09-01

    Full Text Available The HIV-1 gp41 envelope (Env membrane proximal external region (MPER is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL, or weakly bound (CON-S, 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.

  20. Porcine Cysticercosis and Risk Factors in The Gambia and Senegal

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    Arss Secka

    2010-01-01

    Full Text Available During a stratified cross-sectional survey, 1705 pigs were sampled from 279 randomly selected households, 63 randomly selected communities and villages, from four study areas in The Gambia and Senegal during the period October 2007 to January 2008. Porcine cysticercosis prevalence detected by tongue inspection at animal level per study area ranged from 0.1% to 1.0%. Using an antigen-detection ELISA the seroprevalence of cysticercosis at both community/village and animal levels for the four selected study areas is: Western region 80.0% (95%CI: 52.4%–93.6% and 4.8% (95%CI: 3.4%–6.5%, Bignona 86.7% (95%CI: 59.8%–96.6% and 8.9% (95%CI: 5.0%–15.5%, Kolda 82.4% (95%CI: 46.8%–96.1% and 13.2% (95%CI: 10.8%–16.0%, and Ziguinchor 81.3% (95%CI: 43.5%–96.1% and 6.4% (95%CI: 4.0%–10.1%, respectively. No risk factors for cysticercosis were found significant in this study. This study proved that porcine cysticercosis is endemic and distributed widely in the study areas though its incidence might be suppressed by the generalised use of toilets and latrines in the study areas.

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

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    Toshio Ariga

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

  2. Mobile myelographic filling defects: Spinal cysticercosis

    Energy Technology Data Exchange (ETDEWEB)

    Savoiardo, M.; Cimino, C.; Passerini, A.; La Mantia, L.

    1986-03-01

    Cysticercosis usually affects the brain and is easily demonstrated by CT. Spinal cysticercosis is much rarer and is usually diagnosed only at surgery. Myelographic demonstration of multiple rounded filling defects, some of which were mobile, allowed diagnosis of spinal extramedullary cysticercosis in an unsuspected case. The literature on spinal cysticercosis is briefly reviewed. Diagnosis is important in view of the recent development of medical treatment.

  3. Persisting antibody reaction in paragonimiasis after praziquantel treatment is elicited mainly by egg antigens

    Science.gov (United States)

    Kong, Yoon; Yun, Doo-Hee; Kang, Shin-Yong; Kim, Lee-Soo; Chung, Young-Bae; Yang, Hyun-Jong

    2000-01-01

    Antibody responses in serum and cerebrospinal fluid (CSF) samples from patients with active and chronic paragonimiasis and in sera from patients on whom follow-up studies were done after praziquantel treatment were analyzed using antigens of Paragonimus westermani prepared from eggs, metacercariae, juveniles of 4- and 7-week old, adult worms and recombinant protein of 28 kDa cruzipain-like cysteine protease (rPw28CCP). The patient sera/CSFs of active and chronic paragonimiasis revealed strong antibody reactions against the crude extracts of 4- and 7-week old juveniles as well as against those from egg and adult. rPw28CCP also showed specific reaction to the sera with active paragonimiasis. After the treatment, levels of specific antibodies in the sera gradually decreased to negative range in most patients. In some cases with persisting high antibody levels, however, the reactions at 27 kDa egg protein were sustained throughout the observation period of 34 months. The reactions at 35 and 32 kDa in adult extract and rPw28CCP disappeared rapidly after the treatment. Persistent antibody reactions even after successful treatment are provoked by continuous antigenic challenge from eggs which were not resolved by treatment. PMID:10905068

  4. Correlation of serum HIV antigen and antibody with clinical status in HIV-infected patients.

    Science.gov (United States)

    Paul, D A; Falk, L A; Kessler, H A; Chase, R M; Blaauw, B; Chudwin, D S; Landay, A L

    1987-08-01

    An enzyme immunoassay (EIA) has been developed which detects antigen(s) (Ag) of the human immunodeficiency virus (HIV) in the serum of patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC), and patients at high risk for HIV infection. The test has a sensitivity of approximately 50 pg/ml of HIV protein. The specificity of the assay was determined with various virus infected cell lines, normal human sera/plasma, and serum from patients not known to be at risk for HIV infection. No false-positive HIV-Ag results were seen. Sera from 69% of patients with AIDS were positive for HIV-Ag as were 46% of patients with ARC and 19% of asymptomatic, HIV-antibody-positive individuals. There were significant associations between the stage of HIV infection--ie, AIDS vs ARC vs asymptomatic--and the detection of HIV-Ag in serum (p less than 0.0001) and the lack of detection of antibody to HIV core Ag (p less than 0.0001). HIV-Ag was also found in the serum of two asymptomatic antibody-negative individuals who were at high risk for AIDS and who later developed HIV antibody. The presence of HIV-Ag in sera was confirmed by an inhibition procedure. Thus, HIV-Ag can be detected in the serum of infected individuals prior to antibody production and correlates with the clinical stage of HIV infection.

  5. Computational Docking of Antibody-Antigen Complexes, Opportunities and Pitfalls Illustrated by Influenza Hemagglutinin

    Directory of Open Access Journals (Sweden)

    Mattia Pedotti

    2011-01-01

    Full Text Available Antibodies play an increasingly important role in both basic research and the pharmaceutical industry. Since their efficiency depends, in ultimate analysis, on their atomic interactions with an antigen, studying such interactions is important to understand how they function and, in the long run, to design new molecules with desired properties. Computational docking, the process of predicting the conformation of a complex from its separated components, is emerging as a fast and affordable technique for the structural characterization of antibody-antigen complexes. In this manuscript, we first describe the different computational strategies for the modeling of antibodies and docking of their complexes, and then predict the binding of two antibodies to the stalk region of influenza hemagglutinin, an important pharmaceutical target. The purpose is two-fold: on a general note, we want to illustrate the advantages and pitfalls of computational docking with a practical example, using different approaches and comparing the results to known experimental structures. On a more specific note, we want to assess if docking can be successful in characterizing the binding to the same influenza epitope of other antibodies with unknown structure, which has practical relevance for pharmaceutical and biological research. The paper clearly shows that some of the computational docking predictions can be very accurate, but the algorithm often fails to discriminate them from inaccurate solutions. It is of paramount importance, therefore, to use rapidly obtained experimental data to validate the computational results.

  6. Antibody response against saliva antigens of Anopheles gambiae and Aedes aegypti in travellers in tropical Africa.

    Science.gov (United States)

    Orlandi-Pradines, Eve; Almeras, Lionel; Denis de Senneville, Laure; Barbe, Solenne; Remoué, Franck; Villard, Claude; Cornelie, Sylvie; Penhoat, Kristell; Pascual, Aurélie; Bourgouin, Catherine; Fontenille, Didier; Bonnet, Julien; Corre-Catelin, Nicole; Reiter, Paul; Pagés, Frederic; Laffite, Daniel; Boulanger, Denis; Simondon, François; Pradines, Bruno; Fusaï, Thierry; Rogier, Christophe

    2007-10-01

    Exposure to vectors of infectious diseases has been associated with antibody responses against salivary antigens of arthropods among people living in endemic areas. This immune response has been proposed as a surrogate marker of exposure to vectors appropriate for evaluating the protective efficacy of antivectorial devices. The existence and potential use of such antibody responses in travellers transiently exposed to Plasmodium or arbovirus vectors in tropical areas has never been investigated. The IgM and IgG antibody responses of 88 French soldiers against the saliva of Anopheles gambiae and Aedes aegypti were evaluated before and after a 5-month journey in tropical Africa. Antibody responses against Anopheles and Aedes saliva increased significantly in 41% and 15% of the individuals, respectively, and appeared to be specific to the mosquito genus. A proteomic and immunoproteomic analysis of anopheles and Aedes saliva allowed for the identification of some antigens that were recognized by most of the exposed individuals. These results suggest that antibody responses to the saliva of mosquitoes could be considered as specific surrogate markers of exposure of travellers to mosquito vectors that transmit arthropod borne infections.

  7. Use of a recombinant antigen in an indirect ELISA for detecting bovine antibody to capripoxvirus.

    Science.gov (United States)

    Carn, V M; Kitching, R P; Hammond, J M; Chand, P

    1994-10-01

    The gene coding for the capripoxvirus structural protein P32 was cloned, expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and purified on glutathione Sepharose. An indirect enzyme linked immunosorbent assay (ELISA) using this antigen was developed to screen bovine sera for antibodies to capripoxvirus. Sequential serum samples from experimentally infected animals tested by ELISA and by virus neutralisation test (VNT) showed that the ELISA was more sensitive and detected antibodies to capripoxvirus earlier post-infection than the VNT.

  8. Effect of antigen load on development of milk antibodies in infants allergic to milk.

    OpenAIRE

    Firer, M A; Hosking, C S; Hill, D J

    1981-01-01

    The phenomenon that large amounts of antigen, such as are absorbed during the neonatal period, suppress the IgE response while low-dose exposure enhances it was investigated by analysing the antibody responses of infants allergic to milk according to their degree of exposure to cows'-milk protein. IgG, IgA, and IgM milk-specific antibodies in these infants and in age-matched controls were measured by enzyme-linked immunosorbent assay. Milk-specific IgE and total IgE were also measured. Childr...

  9. REQUIREMENT FOR CONTINUOUS ANTIGENIC STIMULATION IN THE DEVELOPMENT AND DIFFERENTIATION OF ANTIBODY-FORMING CELLS

    Science.gov (United States)

    Hanna, M. G.; Nettesheim, P.; Francis, Mary W.

    1969-01-01

    The essential role of continuous antigenic stimulation in the development and differentiation of antibody-forming cells as defined in the X-Y-Z immune cell maturation scheme was examined in these studies. Mice were primed with sheep erythrocytes (SRBC) in an attempt to induce maximum immune progenitor cell conversion (X → Y). Subsequently antigen was depleted at 1 or 4 days after priming with isologous specific antibody in order to interrupt further immune cell differentiation (Y → Z). It was reasoned that this condition would result in depression of the functional antibody-producing cell compartment as measured in the intact mice and subsequently in enhancement of the sensitized (Y cell) compartment as measured in the spleen cell transfer system. These data were also correlated with systematic studies of the hyperplasia of the spleen germinal centers. The effect of passive antibody on the primary response to SRBC was a marked decrease indirect and indirect hemolysin-producing cells (DPFC and IPFC). However, there was a lack of correlation in the degree of antibody-mediated 19S and 7S immune cell suppression during the primary response, the DPFC being much less depressed than the IPFC. As measured in the transfer system there was an enhanced 19S sensitized cell compartment and a depressed 7S sensitized cell compartment in 1 day passively immunized mice. This was true whether or not transfers were performed 1, 2, or 4 wk after priming. Similarly, there was an enhanced 19S-sensitized cell compartment with little or no effect on the 7S-sensitized. cell compartment in 4 day passively immunized mice. These data suggest that progeny of the antigen-stimulated progenitor cells (X cell), as a consequence of lack of further antigenic stimulation, were forced into maturation arrest. These studies further demonstrate that isologous passive antibody suppresses germinal center growth regardless of whether the antibody is infused 1, 2, or 4 days after priming. In terms of

  10. 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

    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...... oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest...

  11. Human cysticercosis and Indian scenario: a review

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    (cysticercosis) including brain (NCC). Human and pig both acquire cysticercosis through ingestion of eggs excreted in faeces by human T. solium carrier. T. solium infection is also increasingly diagnosed in affluent countries owing to human migration from endemic areas (Garcia et al 1996). Cysticercosis is common in ...

  12. Use of a monoclonal antibody specific for a protein epitope of Echinococcus granulosus antigen 5 in a competitive antibody radioimmunoassay for diagnosis of hydatid disease.

    Science.gov (United States)

    Chamekh, M; Facon, B; Dissous, C; Haque, A; Capron, A

    1990-11-06

    A monoclonal antibody (mAb) designated as EG 02 154/12, specific for the major antigen (antigen 5) of Echinococcus granulosus was produced, and used to study the binding sites recognized by anti-antigen 5 antibodies from patients with hydatid disease. The nature of the target epitope was partially characterized. The antibody reactivity was analyzed towards sheep hydatid fluid antigens (SHF Ag) using ELISA, immunoelectrophoresis (IEP), Western blotting (WB), and immunoprecipitation (IP). In IEP, EG 02 154/12 mAb gave a single precipitin of Ag 5. The mAb and human hydatid patient sera recognized a major antigen of 64 kDa, in SHF Ag analyzed in non-reducing conditions. Both types of antibodies revealed two components of 37 and 22 kDa in reducing conditions. Deglycosylation and delipidation of SHF Ag did not affect the mAb binding. These results, together with the observation of mAb binding to in vitro translation products from protoscoleces messenger RNA, suggest the protein nature of the epitope recognized on the antigen 5. Using competitive antibody radioimmunoassay (CRIA), a competition between this mAb and hydatid patient sera, for the same epitope or closely related sites on antigen 5, was observed. No such competition was detected with the sera from other helminthiasis. The sensitivity and specificity of CRIA was compared to that of ELISA and CRIA found to be an improved diagnostic test for hydatid disease.

  13. Microfluidic cell surface antigen expression analysis using a single antibody type.

    Science.gov (United States)

    Zhang, Ye; Pappas, Dimitri

    2016-02-21

    Antigen expression plays a significant role in clinical studies, pathology, biology and chemistry. The type and degree of antigen expression can provide information for disease diagnosis/monitoring and is used for phenotype analysis of cells. In this work, an affinity capture method was developed to capture cells based on antigen expression differences in a single microfluidic chip. Microfluidic chips with two affinity regions-at different antibody concentrations-captured two cell types based on differences in the expression of a single antigen. Using herringbone-modified capture channels, a separation purity of 95% and a capture efficiency of 15% were achieved under continuous-flow conditions. We observed that the capture ratio of Ramos B lymphocytes and HuT 78 T lymphocytes matched the expression ratio of CD71 for the two cell lines (R(2) = 0.94). To further validate our analytical method, Ramos B lymphocytes were spiked into blood samples to demonstrate performance with a complex sample. Expression ratios matched conventional flow cytometry measurements over a 40-fold difference, and the sample enrichment was 9.5×. This method has proven to be a robust system to measure the differences in antigen expression, and can be used to distinguish cells without having a unique surface antigen if the expression level is sufficiently high in one cell type.

  14. Force-induced selective dissociation of noncovalent antibody-antigen bonds.

    Science.gov (United States)

    Yao, Li; Xu, Shoujun

    2012-08-23

    Specific noncovalent binding between antibody and antigen molecules is the basis for molecular recognition in biochemical processes. Quantitative investigation of the binding forces could lead to molecular specific analysis and potentially mechanical manipulation of these processes. Using our force-induced remnant magnetization spectroscopy, we revealed a well-defined binding force for the bonds between mouse immunoglobulin G and magnetically labeled α-mouse immunoglobulin G. The force was calibrated to be 120 ± 15 pN. In comparison, the binding force was only 17 ± 3 pN for physisorption and much higher than 120 pN for biotin-streptavidin bonds. A unique rebinding method was used to confirm the dissociation of the antibody-antigen bonds. A well-defined and molecule-specific binding force opens a new avenue for distinguishing different noncovalent bonds in biochemical processes.

  15. 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...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-05-01

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

  17. Correlation between antibodies to bisphenol A, its target enzyme protein disulfide isomerase and antibodies to neuron‐specific antigens

    Science.gov (United States)

    Vojdani, Aristo

    2016-01-01

    Abstract Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co‐occurrence of anti‐MBP and anti‐MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA‐human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd. PMID:27610592

  18. Humoral antibody response to glutaraldehyde-treated antigens of Dermatophilus congolensis.

    Science.gov (United States)

    Makinde, A A; Molokwu, J U; Ezeh, A O

    1986-04-01

    Glutaraldehyde-treated whole cell antigens (GA.WcA) of Dermatophilus congolensis induced in guinea pigs immunological memory in contrast to cell wall antigens treated similarly (GA.CwA). However, GA.WcA could not induce a secondary response in animals primed with untreated WcA while GA.CwA on the other hand did stimulate a secondary response in animals primed with untreated CwA. Primary antibody production was induced by both GA.CwA and untreated CwA to a similar level in their respective hosts but it was the secondary response that was found similar in response to GA.WcA and untreated WcA. However, both untreated WcA and CwA induced primary and secondary antibody production in their respective hosts though these responses were considerably higher in guinea pigs given untreated CwA. This study showed that both untreated and GA-treated antigens of D. congolensis are capable of stimulating antibody production in guinea pigs but they differ in their levels of stimulation.

  19. Validation of ELISA for the detection of African horse sickness virus antigens and antibodies.

    Science.gov (United States)

    Rubio, C; Cubillo, M A; Hooghuis, H; Sanchez-Vizcaino, J M; Diaz-Laviada, M; Plateau, E; Zientara, S; Crucière, C; Hamblin, C

    1998-01-01

    The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y Produccion Animal (LSPA) in Algete, Spain was charged with the responsibility of co-ordinating and supplying samples of viruses and antisera to the participating laboratories in Spain, France and the United Kingdom. The panels comprised 76 antigen samples for assay by indirect sandwich ELISAs and 53 serum samples for antibody detection by either indirect or competitive ELISAs. Results generated by ELISA for each laboratory were analysed in LSPA in terms of their relative sensitivities and specificities. There was a good agreement between the ELISAs used for either antigen or antibody detection. The participating groups agreed that any field sample giving a doubtful result would always be retested by ELISA and an alternative assay.

  20. A monoclonal antibody reactive with Marek's disease tumor-associated surface antigen.

    Science.gov (United States)

    Lee, L F; Liu, X; Sharma, J M; Nazerian, K; Bacon, L D

    1983-02-01

    An antibody-secreting hybridoma, RPH-6, to Marek's disease tumor-associated surface antigen (MATSA) was produced by somatic-cell hybridization between the mouse myeloma SP2/O-Ag/14 and spleen cells from MSB1 immunized mice. The antibody reacted with 95 to 100% of the cells from eight of 10 chicken MD cell lines and one of two turkey MD cell lines. It did not react with chicken MD cell lines RP1 and SK3, turkey MD cell line RP19, lymphoid leukosis (LL) cell lines, RP9 and RP12, and reticuloendotheliosis virus (REV) cell line RP14. A weak reaction was observed with REV cell line RP13 (10 to 20%), and a very slight reaction with normal chicken spleen cells (1 to 5%). RPH-6 produces immunoglobulin of the IgM class. Cell culture and mouse ascitic fluids have titers of 10(2) and 10(6), respectively, by fluorescent antibody (FA) test against MD tumor cell lines. The antibody did not react with Marek's disease virus (MDV) internal antigen or membrane antigen as detected by indirect FA test on chicken embryo fibroblast cultures grown on coverslips. The 51Cr release assay showed RPH-6 is highly cytotoxic (60% release) only against MD lymphoblastoid cell lines, with antibody prepared from mouse ascites having a cytotoxic titer approaching 10(6). Results from using RPH-6 for differential diagnosis of chicken lymphoid tumors of unknown origin were in complete agreement with those obtained with rabbit anti-MATSA reference serum and pathologic diagnosis.

  1. Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle

    Directory of Open Access Journals (Sweden)

    Stabel Judith R

    2008-01-01

    Full Text Available Abstract Background Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection. Results Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease. Conclusion Collectively these results demonstrate that M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens.

  2. 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

  3. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials

    National Research Council Canada - National Science Library

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2013-01-01

    .... 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...

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

    Science.gov (United States)

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

    2017-03-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

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

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

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

  7. Deciphering complement interference in anti-human leukocyte antigen antibody detection with flow beads assays.

    Science.gov (United States)

    Visentin, Jonathan; Vigata, Margaux; Daburon, Sophie; Contin-Bordes, Cécile; Fremeaux-Bacchi, Véronique; Dromer, Claire; Billes, Marc-Alain; Neau-Cransac, Martine; Guidicelli, Gwendaline; Taupin, Jean-Luc

    2014-09-27

    Anti-human leukocyte antigen (HLA) antibody detection in solid-phase flow beads assays can be quenched by complement activation, but the precise mechanism of this interference is not fully elucidated yet. Using the Luminex flow beads screening assay for detection of anti-HLA antibodies, we analyzed the binding of high concentrations of the pan class I anti-HLA monoclonal antibody W6/32 in neat normal, ethylenediaminetetraacetic acid-treated normal and complement factors C1q, C4/C3, C2, C3, factor B or C5-depleted human sera, using anti-mouse immunoglobulin G as the detection antibody. Complement activation and binding to beads were revealed using anti-human C1q, C4d, and C3d antibodies. To translate our findings to the human setting, we used the class I and class II HLA single-antigen flow beads assays and sera from four patients with high titers of antibodies. Detection of W6/32 did not suffer any interference with C1q and C4/C3-depleted sera. A partial quenching was observed with C2, C3, and factor B-depleted sera, but was more pronounced with the factor B-depleted serum. W6/32 was undetectable in presence of C5-depleted serum. The binding of activation products derived from C3 principally, and also from C4, impaired immunoglobulin G and C1q detection. Accordingly, C4d detection was hindered by deposition of activated C3. Similar findings were obtained with patients' sera. Binding of C4 and C3 activation products is the main responsible for complement interference in flow beads assays. A complete quenching requires complement activation through C3 cleavage and its amplification by the alternative pathway.

  8. Bovine cysticercosis situation in Brazil

    Directory of Open Access Journals (Sweden)

    Gabriel Augusto Marques Rossi

    2014-02-01

    Full Text Available The taeniasis-cysticercosis complex is a long known zoonotic parasitosis characteristic of underdeveloped countries. In addition to its public health significance, this parasitosis is cause of economic losses to the beef production chain, and synonymous of technical inadequacy in relation to the adoption of Good Agricultural Practices. The occurrences of both human teniasis and bovine cysticercosis could and should be controlled with basic sanitary measures. However, there is much variation in the occurrence of the disease in cattle, characterizing a low rate of technical development as well as problems related to the adoption of basic sanitation measures. This review describes, in details, the causative agent and its epidemiological chain, besides raising current information about the occurrence of bovine cysticercosis in different regions of Brazil, aiming at the adoption of prophylactic measures by different segments responsible.

  9. Development and characterization of monoclonal antibodies against excretory-secretory antigens of Fasciola gigantica.

    Science.gov (United States)

    Viyanant, V; Upatham, E S; Sobhon, P; Krailas, D; Ardseungnoen, P; Anatawara, S

    1997-01-01

    Monoclonal antibodies (MAbs) directed against Fasciola gigantica excretory-secretory (ES) antigens were developed from BALB/c mice. Four were selected for further study, from the panel of hybridomas. The antigen specificities of these MAbs were characterized and localized by enzyme-linked immunoeletrotransfer blot (EITB) and immunoperoxidase technique. The target epitopes of these MAbs are 66 kDa protein (MAb 2D10), 66 and 27-26 kDa proteins (MAbs 5D10 and 4F5) and 27-26 kDa protein (MAb 2D9). MAb 2D9 reacted to the antigenic components of the luminal content and epithelial cell lining the cecum, whereas MAb 2D10 reacted specifically to the antigens of the tegument and surface membrane. It was found that all MAbs cross-reacted to various degrees with the antigens extracted from Schistosoma mansoni, S. mekongi, S. spindale and Paramphistomum spp. However, when MAbs were diluted to 1:100 or 1:400 significant reduction of their cross-reactivities was observed.

  10. Simulation and Theory of Antibody Binding to Crowded Antigen-Covered Surfaces.

    Directory of Open Access Journals (Sweden)

    Cristiano De Michele

    2016-03-01

    Full Text Available In this paper we introduce a fully flexible coarse-grained model of immunoglobulin G (IgG antibodies parametrized directly on cryo-EM data and simulate the binding dynamics of many IgGs to antigens adsorbed on a surface at increasing densities. Moreover, we work out a theoretical model that allows to explain all the features observed in the simulations. Our combined computational and theoretical framework is in excellent agreement with surface-plasmon resonance data and allows us to establish a number of important results. (i Internal flexibility is key to maximize bivalent binding, flexible IgGs being able to explore the surface with their second arm in search for an available hapten. This is made clear by the strongly reduced ability to bind with both arms displayed by artificial IgGs designed to rigidly keep a prescribed shape. (ii The large size of IgGs is instrumental to keep neighboring molecules at a certain distance (surface repulsion, which essentially makes antigens within reach of the second Fab always unoccupied on average. (iii One needs to account independently for the thermodynamic and geometric factors that regulate the binding equilibrium. The key geometrical parameters, besides excluded-volume repulsion, describe the screening of free haptens by neighboring bound antibodies. We prove that the thermodynamic parameters govern the low-antigen-concentration regime, while the surface screening and repulsion only affect the binding at high hapten densities. Importantly, we prove that screening effects are concealed in relative measures, such as the fraction of bivalently bound antibodies. Overall, our model provides a valuable, accurate theoretical paradigm beyond existing frameworks to interpret experimental profiles of antibodies binding to multi-valent surfaces of different sorts in many contexts.

  11. Comparison of Platforms for Testing Antibody Responses against the Chlamydia trachomatis Antigen Pgp3.

    Science.gov (United States)

    Gwyn, Sarah; Cooley, Gretchen; Goodhew, Brook; Kohlhoff, Stephan; Banniettis, Natalie; Wiegand, Ryan; Martin, Diana L

    2017-12-01

    Antibody responses to Chlamydia trachomatis (CT) antigens may be useful tools for surveillance of trachoma by estimating cumulative prevalence of infection within a population. Data were compared from three different platforms-multiplex bead array (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA)-measuring antibody responses against the CT antigen protein plasmid gene product 3 (Pgp3). Sensitivity was defined as the proportion of specimens testing antibody positive from a set of dried blood spots from Tanzanian 1-9-year olds who were positive for CT nucleic acid of all nucleic acid amplification test (NAAT)-positive individuals (N = 103). The sensitivity of the LFA could not be determined because of the use of dried blood spots for this test; this specimen type has yet to be adapted to LFA. Specificity was defined as the proportion of sera from U.S. and Bolivian 1-9-year olds that had previously tested negative by the Chlamydia microimmunofluorescence (MIF) assay testing negative to Pgp3-specific antibodies (N = 154). The sensitivity for MBA and ELISA was the same-93.2 (95% confidence interval [CI]: 88.3-98.1). Specificity ranged across platforms from 96.1 (95% CI: 91.8-98.2) to 99.4% (95% CI: 98.2-100). ELISA performance was similar regardless of whether the plates were precoated or freshly coated with antigen. Sensitivity and specificity of control panels were similar if the cutoff was determined using receiver operator curves or a finite mixture model, but the cutoffs themselves differed by approximately 0.5 OD using the different methodologies. These platforms show good sensitivity and specificity and show good agreement between tests at a population level, but indicate variability for ELISA outcomes depending on the cutoff determination methodology.

  12. Molecular interference in antibody-antigen interaction studied with magnetic force immunoassay.

    Science.gov (United States)

    Dorokhin, D; van IJzendoorn, L J; de Jong, A M; Nieto, L; Brunsveld, L; Orsel, J G; Prins, M W J

    2015-09-25

    Molecular interferences are an important challenge in biotechnologies based on antibody-antigen interactions, such as sandwich immunoassays. We report how a sandwich immunoassay with magnetic particles as label can be used to probe interference by surfactants. Surfactants are often used to improve the performance of immunoassays, however the surfactants can affect the involved proteins and the mechanism of action of surfactant molecules on the antibody-antigen system is mostly unknown. As an example, we investigated molecular interference by a nonionic surfactant (Pluronic F-127) in a cardiac troponin (cTn) sandwich immunoassay with two monoclonal antibodies. The influence of the surfactant below the critical micelle concentration (0.00-0.04%) on dissociation properties was quantified in a magnetic tweezers setup, where a force is applied to the molecules via magnetic particle labels. The force-dependent dissociation curves revealed the existence of two distinct cTn-dependent bond types, namely a weak bond attributable to non-specific binding of cTn, and a strong bond attributable to the specific binding of cTn. The dissociation rate constant of the strong bonds increased with the surfactant concentration by about a factor of two. Circular dichroism spectroscopy data showed that the nonionic surfactant influences the conformation of cTn while not noticeably affecting the two monoclonal antibodies. This suggests that the surfactant-induced increase of the dissociation rate of the specific sandwich-type cTn binding may be related to a conformational change of the antigen molecule. The described methodology is an effective tool to study the influence of surfactants and other interferences on assays based on protein interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. [Epidemiology of cysticercosis and neurocysticercosis].

    Science.gov (United States)

    Bouteille, B

    2014-01-01

    Within the genus Taenia, three species are human parasites: T. solium, T. saginata and a new uncommon species, T. asiatica, described recently in Asia. T. saginata and T. solium live as adult tapeworms in human intestines, where they cause taeniasis. T. saginata is widely present worldwide, in all regions where cattle are bred. T. solium is endemic in many countries where livestock and consumption of pigs are common. Cattle and pigs become infected by ingesting eggs emitted by humans into the environment and serve as the respective intermediate hosts of these helminths and host larval forms, or metacestodes or cysticerci. Cysticerci develop into adult worms in the human intestines after a person has eaten contaminated raw or undercooked meat. In the T. solium, eggs are also human contaminants. Humans, like swine, can develop cysticercosis after ingesting eggs with water or contaminated food, or via dirty hands. The clinical manifestations of cysticercosis are highly variable both in kind and in severity. The period between initial infection and the onset of symptoms can also vary. The clinical expression of cysticercosis is generally dependent on the number, size and location of the cysts, as well as the host immune response to the parasite. The preferred locations are the muscles, subcutaneous tissues, central nervous system (CNS), and eyes. Subcutaneous and muscular forms are often asymptomatic. Severe cysticercosis is due to larvae located in human CNS - neurocysticercosis. The World Health Organization (WHO) lists neurocysticercosis as a neglected tropical disease. It estimates that about 50 million people worldwide have neurocysticercosis in the world and that it causes about 50,000 deaths each year. Its most frequent clinical manifestations are seizures, intracranial hypertension, neurological deficits, and sometimes psychiatric manifestations. It is also responsible for more than 50% of the cases of late-onset epilepsy in developing countries. The T

  14. Longitudinal fluctuation of antibodies to extractable nuclear antigens in systemic lupus erythematosus.

    Science.gov (United States)

    Faria, Alex Carvalho; Barcellos, Karin Spat Albino; Andrade, Luis Eduardo Coelho

    2005-07-01

    To examine the appearance, persistence, and disappearance of anti-extractable nuclear antigen (ENA, Sm, U1-RNP, Ro/SSA, and La/SSB) and anti-native DNA (dsDNA) antibodies during systemic lupus erythematosus (SLE) followup. One hundred and thirty patients who fulfilled American College of Rheumatology classification criteria for SLE with at least 5 yearly anti-ENA and dsDNA tests between 1987-2002 were retrospectively selected. Four longitudinal antibody data patterns were considered for each antibody: always absent, always present, absent at diagnosis with positive seroconversion, and present at diagnosis with negative seroconversion. Antibodies to Ro/SSA were present in 47%, U1-RNP in 36%, DNA in 32%, Sm in 23%, and La/SSB in 7% of patients. Among patients ever positive for a given autoantibody, the frequency of the "always present" pattern was 52% for anti-Ro/SSA, 38% for U1-RNP, 17% for Sm, 11% for La/SSB, and 9% for DNA antibodies; the frequency of positive seroconversion was 56% for anti-La/SSB, 33% for DNA, 26% for Sm, 21% for U1-RNP, and 15% for Ro/SSA. Time to positive seroconversion varied from 1 to 8 years after diagnosis. Among patients with a positive test at diagnosis the frequency of those remaining positive between the 2nd and 4th year of followup decreased to 39-78%, depending upon autoantibody specificity; between the 5th and 10th years this rate was 20-75%. Antibody data pattern frequency differed significantly among autoantibody specificities except between anti-U1-RNP and Ro/SSA (p = 0.15) and between anti-DNA and Sm antibodies (p = 0.29). The high frequency of longitudinal fluctuation in anti-ENA antibodies suggests that a periodic reappraisal may be appropriate in seronegative patients with a suspect diagnosis of SLE. The clinical significance of such fluctuation deserves future study.

  15. [Evaluation of four antigens for the detection of anti-Mycobacterium bovis antibodies by enzyme immunoassay].

    Science.gov (United States)

    Ritacco, V; Lopez, B; Barrera, L; Torrea, G; Nader, A; de Kantor, I N; Fliess, E

    1988-01-01

    An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine tuberculosis through the detection of specific seric antibodies has recently been developed in our laboratory. In order to assess its reproducibility and select the most adequate antigen, four bovine PPDs from different sources were evaluated in parallel: PPD M. bovis strain AN5, CEPANZO standard (CPZ), PPD M. bovis strain AN5, European Economic Community standard (EEC), PPD M. bovis strain AN5, prepared from non heated bacilli, killed by phenol (P) and PPD. M. bovis BCG strain prepared at the Pasteur Institute, Paris (BCG). Sera from 22 healthy cattle from tuberculosis free area and 20 bacteriologically confirmed tuberculous animals were employed in simultaneous assays. Antibody mean and standard deviations from healthy cattle expressed as optical density (OD) values were 45 +/- 22 when CPZ was used as antigen, 24 +/- 10 with EEC, 103 +/- 56 with P and 56 +/- 20 with BCG. Mean O.D. from tuberculous cattle were 588 +/- 158, 510 +/- 234, 782 +/- 138 and 441 +/- 189 with antigens CPZ, EEC, P and BCG respectively. A close correlation was observed when results obtained with EEC and P were compared with that of CPZ (r: 0.97 and 0.94 respectively). A lower specificity was achieved when BCG was used as antigen being also lower its correlation with the results obtained with CPZ (r: 0.87). It is concluded that our ELISA would achieve similar sensitivity and specificity if CPZ, EEC and P were used as antigens. On the other hand, BCG would not be suitable for this assay.

  16. Comparative evaluation of different immunoassays for the detection of Taenia solium cysticercosis in swine with low parasite burden

    Directory of Open Access Journals (Sweden)

    Andréia Bartachini Gomes

    2007-09-01

    Full Text Available Seven swine were experimentally infected with Taenia solium eggs and blood samples from each animal were periodically collected. At the end of the experiment (t140 the animals did not show clinical aspects of cysticercosis or parasites in tongue inspection. All animals were slaughtered and cut into thin slices in searching for cysts. The number of cysts found in each animal varied from 1 to 85. Enzyme-linked immunosorbent assay (ELISA tests for antibody (Ab detection and for antigen (Ag detection were performed, which presented respectively 71 and 57% of positivity. By immunoblot (IB, using 18/14(T. crassiceps Ag or lentil-lectin-purified glycoproteins from T. solium Ag (LLGP as Ag, five (71% and six (86% animals were positive, respectively. The association between Ag-ELISA with any IB (18/14 or LLGP allowed the detection of all animals at 140 days post-experimental infection (days p.e.i.. The use of IB 18/14 combined to the Ag-ELISA allowed the detection of all animals since 70 days p.e.i., and the association between IB LLGP and Ag-ELISA allowed the detection of all animals since 112 days p.e.i. While all animals could be considered healthy by conventional screening tests, the use of immunoassays for detecting Ab and Ag showed better accuracy; therefore it would be more useful than usual clinical examination for screening cysticercosis in slightly infected pigs.

  17. Relationship between antibody susceptibility and lipopolysaccharide O-antigen characteristics of invasive and gastrointestinal nontyphoidal Salmonellae isolates from Kenya.

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    Robert S Onsare

    2015-03-01

    Full Text Available Nontyphoidal Salmonellae (NTS cause a large burden of invasive and gastrointestinal disease among young children in sub-Saharan Africa. No vaccine is currently available. Previous reports indicate the importance of the O-antigen of Salmonella lipopolysaccharide for virulence and resistance to antibody-mediated killing. We hypothesised that isolates with more O-antigen have increased resistance to antibody-mediated killing and are more likely to be invasive than gastrointestinal.We studied 192 NTS isolates (114 Typhimurium, 78 Enteritidis from blood and stools, mostly from paediatric admissions in Kenya 2000-2011. Isolates were tested for susceptibility to antibody-mediated killing, using whole adult serum. O-antigen structural characteristics, including O-acetylation and glucosylation, were investigated. Overall, isolates were susceptible to antibody-mediated killing, but S. Enteritidis were less susceptible and expressed more O-antigen than Typhimurium (p<0.0001 for both comparisons. For S. Typhimurium, but not Enteritidis, O-antigen expression correlated with reduced sensitivity to killing (r = 0.29, 95% CI = 0.10-0.45, p = 0.002. Both serovars expressed O-antigen populations ranging 21-33 kDa average molecular weight. O-antigen from most Typhimurium were O-acetylated on rhamnose and abequose residues, while Enteritidis O-antigen had low or no O-acetylation. Both Typhimurium and Enteritidis O-antigen were approximately 20%-50% glucosylated. Amount of S. Typhimurium O-antigen and O-antigen glucosylation level were inversely related. There was no clear association between clinical presentation and antibody susceptibility, O-antigen level or other O-antigen features.Kenyan S. Typhimurium and Enteritidis clinical isolates are susceptible to antibody-mediated killing, with degree of susceptibility varying with level of O-antigen for S. Typhimurium. This supports the development of an antibody-inducing vaccine against NTS for Africa. No clear

  18. Exploring the energy landscape of antibody-antigen complexes: protein dynamics, flexibility, and molecular recognition.

    Science.gov (United States)

    Thielges, Megan C; Zimmermann, Jörg; Yu, Wayne; Oda, Masayuki; Romesberg, Floyd E

    2008-07-08

    The production of antibodies that selectively bind virtually any foreign compound is the hallmark of the immune system. While much is understood about how sequence diversity contributes to this remarkable feat of molecular recognition, little is known about how sequence diversity impacts antibody dynamics, which is also expected to contribute to molecular recognition. Toward this goal, we examined a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we selected six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. Sequencing revealed that two pairs of antibodies employ homologous heavy chains that were derived from common germline genes, while the other two heavy chains and all six of the light chains were derived from different germline genes and are not homologous. Interestingly, more than half of all the somatic mutations acquired during affinity maturation among the six antibodies are located in positions unlikely to contact fluorescein directly. To quantify and compare the dynamics of the antibody-fluorescein complexes, three-pulse photon echo peak shift and transient grating spectroscopy were employed. All of the antibodies exhibited motions on three distinct time scales, ultrafast motions on the <100 fs time scale, diffusive motions on the picosecond time scale, and motions that occur on time scales longer than nanoseconds and thus appear static. However, the exact frequency of the picosecond time scale motion and the relative contribution of the different motions vary significantly among the antibody-chromophore complexes, revealing a high level of dynamic diversity. Using a hierarchical model, we relate the data to features of the antibodies' energy landscapes as well as their flexibility in terms of elasticity and plasticity. In all, the data provide a consistent picture of antibody flexibility

  19. Prevalence of Toxoplasma gondii antibodies, circulating antigens and DNA in stray cats in Shanghai, China

    Directory of Open Access Journals (Sweden)

    Wang Quan

    2012-09-01

    Full Text Available Abstract Background Toxoplasma gondii is prevalent in most areas of the world and may cause abortions or neonatal complications in humans. As the only definitive host, cats play an important role in the epidemiology of the disease. Infection rates in cats, especially stray or free-living cats, are considered to be the best sentinels of the level of T. gondii in the environment. The T. gondii infection can be diagnosed in different ways with different methods depending on the target. However, little information on T. gondii infection in cats was available in Shanghai, China. Moreover reports on prevalence of circulating antigens, antibodies and DNA of T. gondii in the same study are rare. Methods In the present study, the presence of antibodies (Ab, circulating antigens (CA, and/or DNA of Toxoplasma gondii in samples from 145 stray or unwanted cats from 6 animal shelters in Shanghai (China was determined in order to estimate the prevalence of T. gondii infection, by Ab-ELISA, CA-ELISA, and nested-PCR, respectively. Results The positive rates for the antibodies, circulating antigen and DNA of T. gondii were 11.7% (17 of 145, 5.5% (8 of 145 and 5.71% (2 of 35, respectively. No cat tested was positive by both the Ab-ELISA and the CA-ELISA, but the results of the PCR were consistent with the CA-ELISA assay. Therefore, the overall estimated prevalence of toxoplasmosis was 17.2% (25 of 145. According to our results, the positive rates of specific antibodies and circulating antigen of T. gondii were significantly different between adult cats (>1 year old and juvenile cats (≤1 year old; the former was 13.5% versus 3.9% by Ab-ELISA, while the latter was 1.7% versus 23.1% by CA-ELISA. From the results obtained with all three detection methods used in this study, the rate of infection was not significantly different between male and female cats (P ≥0.05; and the overall rate was 17.9% for males versus 16.4% for females. Conclusions The results

  20. Evaluation of envelope domain III-based single chimeric tetravalent antigen and monovalent antigen mixtures for the detection of anti-dengue antibodies in human sera.

    Science.gov (United States)

    Batra, Gaurav; Nemani, Satish K; Tyagi, Poornima; Swaminathan, Sathyamangalam; Khanna, Navin

    2011-03-15

    Flavivirus cross-reactive antibodies in human sera interfere with the definitive identification of dengue virus (DENV) infections especially in areas with multiple co-circulating flaviviruses. Use of DENV envelope domain-III (EDIII) can partially resolve the problem. This study has examined the effect of (i) incorporating the EDIIIs of four DENV serotypes into a single chimeric antigen, and (ii) immobilizing the antigen through specific interaction on the sensitivity and specificity of anti-DENV antibody detection. A sera panel (n = 164) was assembled and characterized using commercial kits for infection by DENV and a host of other pathogens. Anti-DENV antibodies of both IgM and IgG classes in this panel were detected in indirect ELISAs using a mixture of monovalent EDIIIs, a chimeric EDIII-based tetravalent antigen, EDIII-T, and a biotinylated version of the latter as coating antigens. The sensitivity and specificity of these assays were compared to those obtained using the PanBio Dengue IgG/IgM ELISAs. The performance of dengue IgG and IgM indirect ELISAs, using either a physical mixture of four EDIIIs or the single chimeric EDIII-T antigen, were comparable. Coating of a biotinylated version of the tetravalent antigen on streptavidin plates enhanced sensitivity without compromising specificity. The incorporation of the EDIIIs of the four DENV serotypes into a single chimeric antigen did not adversely affect assay outcome in indirect ELISAs. Oriented, rather than random, immobilization of the tetravalent antigen enhanced sensitivity of detection of anti-DENV antibodies with retention of 100% specificity.

  1. Specificity and affinity of 26 monoclonal antibodies against the CA 125 antigen : First report from the ISOBM TD-1 workshop

    NARCIS (Netherlands)

    Nustad, K; Bast, RC; OBrien, TJ; Nilsson, O; Seguin, P; Suresh, MR; Saga, T; Nozawa, S; Bormer, OP; deBruijn, HWA; Vitali, A; Gadnell, M; Clark, J; Shigemasa, K; Karlsson, B; Kreutz, FT; Jette, D; Sakahara, H; Endo, K; Paus, E; Warren, D; Hammarstrom, S; Kenemans, P; Hilgers, J

    1996-01-01

    The specificity of 26 monoclonal antibodies against the CA 125 antigen was investigated in two phases of the ISOBM TD-1 workshop. The binding specificity was studied using CA 125 immunoextracted by specific antibodies immobilized on various solid phases, or on the surface of human cell lines.

  2. Antibodies from Patients with Melioidosis Recognize Burkholderia mallei but Not Burkholderia thailandensis Antigens in the Indirect Hemagglutination Assay

    OpenAIRE

    Tiyawisutsri, Rachaneeporn; Peacock, Sharon J.; Langa, Sayan; Limmathurotsakul, Direk; Allen C Cheng; Chierakul, Wirongrong; Chaowagul, Wipada; Day, Nicholas P. J.; Wuthiekanun, Vanaporn

    2005-01-01

    The indirect hemagglutination assay routinely used to detect antibodies to Burkholderia pseudomallei was modified to detect cross-reactivity of antibodies to B. pseudomallei, B. mallei, and B. thailandensis antigens. We demonstrate a lack of cross-reactivity between B. pseudomallei and B. thailandensis but marked cross-reactivity between B. pseudomallei and B. mallei.

  3. Plant virus particles carrying tumour antigen activate TLR7 and Induce high levels of protective antibody.

    Directory of Open Access Journals (Sweden)

    Jantipa Jobsri

    Full Text Available Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP, which are structurally analogous to VLP, coupled to a weak idiotypic (Id tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the "gold standard" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-α by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe "in-built" ssRNA adjuvant.

  4. Phage-displayed peptides as capture antigens in an innovative assay for Taenia saginata-infected cattle.

    Science.gov (United States)

    Fogaça, Rafaela L; Capelli-Peixoto, Janaína; Yamanaka, Isabel B; de Almeida, Rodrigo P M; Muzzi, João Carlos D; Borges, Mariangela; Costa, Alvimar J; Chávez-Olortegui, Carlos; Thomaz-Soccol, Vanete; Alvarenga, Larissa M; de Moura, Juliana

    2014-11-01

    Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex.

  5. Use of SpyTag/SpyCatcher to construct bispecific antibodies that target two epitopes of a single antigen.

    Science.gov (United States)

    Yumura, Kyohei; Akiba, Hiroki; Nagatoishi, Satoru; Kusano-Arai, Osamu; Iwanari, Hiroko; Hamakubo, Takao; Tsumoto, Kouhei

    2017-09-01

    Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies.

    Science.gov (United States)

    Li, Jinfeng; Hu, Feihuan; Chen, Shouyi; Luo, Peifang; He, Zuoping; Wang, Wenjing; Allain, Jean-Pierre; Li, Chengyao

    2017-05-15

    Brucellosis is a severe zoonotic disease worldwide. Detection and identification of Brucella species are essential to prevent or treat brucellosis in humans and animals. The outer membrane protein-31 (Omp31) is a major protein of Brucellae except for B. abortus, while the Omp31 antigenic epitopes have not been extensively characterized yet. A total of 22 monoclonal antibodies (mAbs) were produced against Omp31 of Brucella (B.) melitensis, of which 13 recognized five linear epitopes, 7 reacted with semi-conformational epitopes and 2 reacted with conformational epitopes, respectively. The mAb isotypes were 11 (50%) IgG2a, 5 (23%) IgG1 and 6 (27%) IgM. On the basis of epitope recognition and reactivity levels, 8 mAbs including 3 IgM and 5 IgG clones were considered as highly reactive and potentially diagnostic antibodies. Among these mAbs, 7A3 (IgG1), 5B1 (IgG2a), 2C1 (IgG2a) and 5B3 (IgG2a) reacted with differently conserved linear epitopes of B. melitensis, B. ovis, B. suis and B. canis strains, while 5H3 (IgG2a) highly reacted with a conformational epitope of Omp31 when tested with several immunoassays. These potent monoclonal antibodies can be used for identifying Omp31 antigens or detecting B. melitensis and other Brucella species beyond B. abortus in vitro or in vivo.

  7. Inexpensive designer antigen for anti-HIV antibody detection with high sensitivity and specificity.

    Science.gov (United States)

    Talha, Sheikh M; Salminen, Teppo; Chugh, Deepti A; Swaminathan, Sathyamangalam; Soukka, Tero; Pettersson, Kim; Khanna, Navin

    2010-03-01

    A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.

  8. Ontogeny of adaptive antibody response to a model antigen in captive altricial zebra finches.

    Directory of Open Access Journals (Sweden)

    Tess L Killpack

    Full Text Available Based on studies from the poultry literature, all birds are hypothesized to require at least 4 weeks to develop circulating mature B-cell lineages that express functionally different immunoglobulin specificities. However, many altricial passerines fledge at adult size less than four weeks after the start of embryonic development, and therefore may experience a period of susceptibility during the nestling and post-fledging periods. We present the first study, to our knowledge, to detail the age-related changes in adaptive antibody response in an altricial passerine. Using repeated vaccinations with non-infectious keyhole limpet hemocyanin (KLH antigen, we studied the ontogeny of specific adaptive immune response in altricial zebra finches Taeniopygia guttata. Nestling zebra finches were first injected at 7 days (7d, 14 days (14d, or 21 days post-hatch (21d with KLH-adjuvant emulsions, and boosted 7 days later. Adults were vaccinated in the same manner. Induced KLH-specific IgY antibodies were measured using ELISA. Comparisons within age groups revealed no significant increase in KLH-specific antibody levels between vaccination and boost in 7d birds, yet significant increases between vaccination and boost were observed in 14d, 21d, and adult groups. There was no significant difference among age groups in KLH antibody response to priming vaccination, yet KLH antibody response post-boost significantly increased with age among groups. Post-boost antibody response in all nestling age groups was significantly lower than in adults, indicating that mature adult secondary antibody response level was not achieved in zebra finches prior to fledging (21 days post-hatch in zebra finches. Findings from this study contribute fundamental knowledge to the fields of developmental immunology and ecological immunology and strengthen the utility of zebra finches as a model organism for future studies of immune ontogeny.

  9. Ontogeny of adaptive antibody response to a model antigen in captive altricial zebra finches.

    Science.gov (United States)

    Killpack, Tess L; Karasov, William H

    2012-01-01

    Based on studies from the poultry literature, all birds are hypothesized to require at least 4 weeks to develop circulating mature B-cell lineages that express functionally different immunoglobulin specificities. However, many altricial passerines fledge at adult size less than four weeks after the start of embryonic development, and therefore may experience a period of susceptibility during the nestling and post-fledging periods. We present the first study, to our knowledge, to detail the age-related changes in adaptive antibody response in an altricial passerine. Using repeated vaccinations with non-infectious keyhole limpet hemocyanin (KLH) antigen, we studied the ontogeny of specific adaptive immune response in altricial zebra finches Taeniopygia guttata. Nestling zebra finches were first injected at 7 days (7d), 14 days (14d), or 21 days post-hatch (21d) with KLH-adjuvant emulsions, and boosted 7 days later. Adults were vaccinated in the same manner. Induced KLH-specific IgY antibodies were measured using ELISA. Comparisons within age groups revealed no significant increase in KLH-specific antibody levels between vaccination and boost in 7d birds, yet significant increases between vaccination and boost were observed in 14d, 21d, and adult groups. There was no significant difference among age groups in KLH antibody response to priming vaccination, yet KLH antibody response post-boost significantly increased with age among groups. Post-boost antibody response in all nestling age groups was significantly lower than in adults, indicating that mature adult secondary antibody response level was not achieved in zebra finches prior to fledging (21 days post-hatch in zebra finches). Findings from this study contribute fundamental knowledge to the fields of developmental immunology and ecological immunology and strengthen the utility of zebra finches as a model organism for future studies of immune ontogeny.

  10. Study on the antigenicity of metallofullerenol: antibody production, characterization, and its enzyme immunoassay application.

    Science.gov (United States)

    Guo, Xihong; Yang, Shangyuan; Huang, Huan; Cui, Rongli; Li, Cheng; Yao, Huanli; Liu, Bing; Zhang, Lele; Xu, Binggang; Dong, Jinquan; Sun, Baoyun

    2017-11-01

    With their intriguing structures and properties, metallofullerenols have attracted considerable attention in biological and medical applications. Due to the increasing biomedical interest, effective detection methods are important to monitor and control metallofullerenols. However, the detection of metallofullerenols becomes very difficult after polyhydroxylated modification due to the lack of detectable features. Antibody-based immunoassay methods have been important tools for detection and will better meet the needs of analysis of metallofullerenols. Thus, the antigenicity of metallofullerenol has been studied for the first time. In this study, no immune response was detected when metallofullerenol Gd@C82(OH)x was used as immunogen. However, the polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen, indicating that metallofullerenol can act as hapten. The specificity of the obtained antibody was investigated. It has been found that the hydroxyl groups on the surface of the carbon cage, the encapsulated metal, and the size and shape of the carbon cage did not affect the recognition specificity of the antibody. Based on the obtained antibody, an indirect competitive enzyme immunoassay was developed for the determination of metallofullerenol with detection limits of 18 ng/mL in PBS. This enzyme immunoassay method was successfully used to detect metallofullerenol in serum. This work can provide an innovative way to determine metallofullerenols. Graphical abstract The polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen. Based on the obtained antibody, a competitive enzyme immunoassay was developed for the determination of metallofullerenol.

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

    DEFF Research Database (Denmark)

    Petersen, E; Høgh, B; Dziegiel, Morten Hanefeld

    1992-01-01

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

  12. Antigenic determinants of influenza virus hemagglutinin. XI. Conformational changes detected by monoclonal antibodies.

    Science.gov (United States)

    Jackson, D C; Nestorowicz, A

    1985-08-01

    At pH 5 influenza virus hemagglutinin undergoes an irreversible conformational change (J.J. Skehel, P. M. Bayley, E. B. Brown, S. R. Martin, M. D. Waterfield, J. M. White, I. A. Wilson, and D. C. Wiley (1982). Proc. Natl. Acad. Sci. USA 79, 968-972) which parallels the appearance of fusion activity of this molecule. This paper describes experiments which explore the conformational change using a panel of monoclonal antibodies which define four of the major antigenic sites of this protein. The results indicate that three of the major antigenic sites of hemagglutinin undergo changes when exposed to acid pH. These changes have little effect on the binding avidity of influenza virus to glycophorin, the major receptor present on the red blood cell surface. These findings have been used to postulate a mechanism where the molecule flexes around a central region resulting in rearrangement in space of its component domains on exposure to low pH.

  13. Seizures, cysticercosis and rural-to-urban migration: the PERU MIGRANT study.

    Science.gov (United States)

    Gonzales, Isidro; Miranda, J Jaime; Rodriguez, Silvia; Vargas, Victor; Cjuno, Alfredo; Smeeth, Liam; Gonzalez, Armando E; Tsang, Victor C W; Gilman, Robert H; Garcia, Hector H

    2015-04-01

    To examine the prevalence of seizures, epilepsy and seropositivity to cysticercosis in rural villagers (cysticercosis-endemic setting), rural-to-urban migrants into a non-endemic urban shanty town and urban inhabitants of the same non-endemic shanty town. Three Peruvian populations (n = 985) originally recruited into a study about chronic diseases and migration were studied. These groups included rural inhabitants from an endemic region (n = 200), long-term rural-to-urban migrants (n = 589) and individuals living in the same urban setting (n = 196). Seizure disorders were detected by a survey, and a neurologist examined positive respondents. Serum samples from 981/985 individuals were processed for cysticercosis antibodies on immunoblot. Epilepsy prevalence (per 1000 people) was 15.3 in the urban group, 35.6 in migrants and 25 in rural inhabitants. A gradient in cysticercosis antibody seroprevalence was observed: urban 2%, migrant 13.5% and rural group 18% (P < 0.05). A similarly increasing pattern of higher seroprevalence was observed among migrants by age at migration. In rural villagers, there was strong evidence of an association between positive serology and having seizures (P = 0.011) but such an association was not observed in long-term migrants or in urban residents. In the entire study population, compared with seronegative participants, those with strong antibody reactions (≥ 4 antibody bands) were more likely to have epilepsy (P < 0.001). It is not only international migration that affects cysticercosis endemicity; internal migration can also affect patterns of endemicity within an endemic country. The neurological consequences of cysticercosis infection likely outlast the antibody response for years after rural-to-urban migration. © 2015 The Authors. Tropical Medicine & International Health Published by John Wiley & Sons Ltd.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    Background Praziquantel treatment of schistosomiasis during pregnancy was only recommended in 2002; hence the effects of treatment during pregnancy are not fully known. We have therefore evaluated the effects on infection intensity and the immunological effects of praziquantel treatment against...... after delivery. Infection intensity after treatment was assessed by a single Kato-Katz examination of stool samples with duplicate slides and categorised as undetected, light (1-99 eggs per gram (epg)), moderate (100-399 epg) or heavy (=400 epg). Antibodies against S. mansoni worm and egg antigens were......E, but these boosts were less pronounced than in women whose treatment was delayed until after delivery. Praziquantel had limited effects on antibodies against egg antigens. Conclusion S mansoni antigen-specific antibody levels and praziquantel-induced boosts in antibody levels were broadly suppressed during...

  15. Nine-year longitudinal study of antibodies to variant antigens on the surface of Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

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

    1999-01-01

    PfEMP1 is an antigenically variable molecule which mediates the adhesion of parasitized erythrocytes to a variety of cell types and which is believed to constitute an important target for naturally acquired protective immune responses in malaria. For 9 years we have monitored individuals living...... in an area of low-intensity, seasonal, and unstable malaria transmission in eastern Sudan, and we have used this database to study the acquisition, specificity, and duration of the antibody response to variant parasitized erythrocyte surface antigens. Both the levels and the spectrum of reactivity...... of these antibodies varied considerably among individuals, ranging from low levels of antibodies recognizing only few parasitized erythrocyte surface antigens to high levels of broad-specificity antibodies. In general, episodes of clinical malaria were associated with increases in the levels of parasitized...

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

    DEFF Research Database (Denmark)

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

    1987-01-01

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

  17. Antigen-Binding Properties of Monoclonal Antibodies Reactive with EBNA1 and Use in Immunoaffinity Chromatography

    OpenAIRE

    Duellman, Sarah J.; Burgess, Richard R.

    2009-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1)) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, ...

  18. Seroepidemiological study of human cysticercosis with blood samples collected on filter paper, in Lages, State of Santa Catarina, Brazil, 2004-2005

    Directory of Open Access Journals (Sweden)

    Maria Márcia Imenes Ishida

    2011-06-01

    Full Text Available INTRODUCTION: Human serofrequency of antibodies against Taenia solium antigens was determined and risk factors for cysticercosis transmission were identified. METHODS: Individuals (n=878 from periurban and rural locations of Lages, SC, were interviewed to gather demographic, sanitary and health information. Interviews and blood sample collections by finger prick on Whatman filter paper were performed from August 2004 to May 2005. Observation determined that 850 samples were suitable for analysis and were tested by ELISA using vesicular fluid of Taenia crassiceps heterologous antigen. To ensure the reliability of the results, 77 samples of the dried blood were matched with sera. The reactive samples were submitted to a serum confirmatory immunoblot (IB test using purified Taenia crassiceps glycoproteins. RESULTS: The ELISA results for the dried blood and serum samples were statistically consistent. ELISA was positive in 186 (21.9% out of 850 individuals. A group of 213 individuals were asked to collect vein blood for IB (186 with positive result in ELISA and 27 with inappropriate whole blood samples and 130 attended the request. The IB was positive in 29 (3.4% out of 850 individuals. A significant correlation (p = 0.0364 was determined among individuals who tested positive in the IB assay who practiced both pig rearing and kitchen gardening. CONCLUSIONS: ELISA with dried blood eluted from filter paper was suitable for cysticercosis population surveys. In Lages, human infection was associated with pig rearing and kitchen gardening. The prevalence index was compatible with other Latin American endemic areas.

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

    Directory of Open Access Journals (Sweden)

    Savannah E. Howe

    2016-10-01

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

  20. Reduced antibody responses against Plasmodium falciparum vaccine candidate antigens in the presence of Trichuris trichiura

    DEFF Research Database (Denmark)

    Esen, Meral; Mordmüller, Benjamin; de Salazar, Pablo Martinez

    2012-01-01

    BACKGROUND: Helminth infections are highly prevalent in the tropics and may have an effect on immune responses to vaccines due to their immunomodulatory effect. The prevalence of helminth infections in young children, the target group for malaria and most other vaccines, is high. Therefore we...... assessed the influence of helminth infection on vaccine-induced immune responses in a phase I clinical trial of the malaria vaccine candidate GMZ2. METHODS: Twenty Gabonese preschool-age children were vaccinated with GMZ2, a blood stage malaria vaccine candidate. Humoral immune response against the vaccine...... antigens and parasitological status were assessed. Vaccine-specific antibody concentrations and memory B-cell numbers were compared in worm infected and non-infected participants. RESULTS: Antibody response to GMZ2 was 3.4-fold (95% confidence interval: 1.6, 7.4) higher in Trichuris trichiura negative...

  1. Antibodies elicited by influenza virus hemagglutinin fail to bind to synthetic peptides representing putative antigenic sites.

    Science.gov (United States)

    Nestorowicz, A; Tregear, G W; Southwell, C N; Martyn, J; Murray, J M; White, D O; Jackson, D C

    1985-02-01

    A number of peptides of the hemagglutinin (HA) of X-31 influenza virus have been synthesised. The amino acid sequences of some of these peptides represent regions of HA which have been postulated [Wiley et al., Nature, Lond. 289, 373-378 (1981)] to form the antigenic sites of this molecule. Animals were immunized with free peptide or peptide conjugated to a carrier and the resulting antisera examined for their capacities to bind to homologous peptide, whole HA, reduced and alkylated HA, and intact virus. Not all peptides examined in this way were immunogenic. Only antibodies raised against the C-terminus of HA1 peptide displayed binding to virus. This antiserum bound to the intact HA but not to the reduced and alkylated form of the molecule. These results raise questions as to the feasibility of using synthetic peptides of the influenza HA in short linear sequences to elicit neutralising antibody.

  2. ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci Teste ELISA para diagnóstico da cisticercose suína usando antígenos de larvas de Taenia solium e Taenia crassiceps

    Directory of Open Access Journals (Sweden)

    Paulo Sérgio de Arruda PINTO

    2000-04-01

    Full Text Available In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra and crude larvae extract (T-Tcra, and from Taenia solium extracts of scolex (S-Ts and of larvae (T-Ts. A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.Foi padronizado o teste ELISA para o diagnóstico da cisticercose suína. Após confirmação por exame post-mortem, os soros dos respectivos animais foram empregados como controles positivos e negativos. Soros de suínos portadores de infecções heterólogas foram ensaiados para determinação de reações cruzadas. Os quatro antígenos testados na fase de padronização foram líquido vesicular (VF e extrato total (T de larvas de Taenia crassiceps (Tcra e de extrato de escólex (S e de cisticercos (T de Taenia solium (Tso. A titulação em bloco das ótimas concentrações de antígenos e diluições de soros e de conjugado, bem como o emprego de Tween-20 e de leite desnatado nas

  3. Prevalence and risk factors for porcine cysticercosis in rural communities of eastern Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    Emilio C. Acevedo-Nieto

    Full Text Available ABSTRACT: Cysticercosis is caused by Taenia solium, a parasitic zoonosis that affects human and pigs raised free-range in developing countries. The epidemiology of the taeniosis cysticercosis complex in Brazil is poorly understood especially when it comes to field research. The aim of this study was to estimate the prevalence and identify the risk factors associated with porcine cysticercosis in rural communities located in the east of Minas Gerais (MG, Brazil. From 371 farms in the county of Tumiritinga/MG, 101 farms from 14 communities were randomly sampled. Blood samples from pigs were collected, and epidemiological questionnaires were carried out. The serum samples obtained were analyzed through immunodiagnosis techniques, including ELISA and Western Blot, both for the detection of antibodies. The data obtained by different surveys were analyzed using EpiInfo 3.5.1 software to determine seroprevalence and risk factors associated with cysticercosis. The prevalence of farms with porcine cysticercosis was 9.9% (10/101 and antibody-based seropositive was 5.3% (13/247. The results indicate that cysticercosis occurs in high level in the rural area never studied before. These results suggest the presence of tapeworm carriers contributing to the occurrence and maintenance of this zoonotic life cycle in the county. Regarding risk factors, the most significant determinants for porcine cysticercosis in the field were free-range pig management (OR: 17.4, p: 0.0001, the method of disposal of human faeces in the environmental (OR: 7.6; p 0.012, and the size of the farm. Porcine cysticercosis was diagnosed only in areas represented by Agrarian Reform Settlements. From the results, it is possible to recommend as a means of control and prevention the destination of human faeces in appropriate sanitary landfills and the production of pigs in an enclosed area. Additionally, improving education in the communities sampled will indirectly affect the spreading of

  4. Antigenic relationships within the genus Salmonella as revealed by anti-Salmonella enteritidis monoclonal antibodies.

    Science.gov (United States)

    Malik, M; Butchaiah, G; Bansal, M P; Siddiqui, M Z; Bakshi, C S; Singh, R K

    2002-04-01

    A panel of 38 monoclonal antibodies (MAbs) that react with outer membrane proteins (OMPs) of Salmonella enteritidis was produced. On the basis of their binding pattern in ELISA, the MAbs were divided into three groups. The first group, consisting of 15 MAbs, was found to be Salmonella-specific as they did not cross-react with Escherichia coli or Pasteurella multocida. The second group of 15 MAbs cross-reacted with E. coli but not with P. multocida, reflecting the closer antigenic relationship of E. coli with Salmonella. The third group of 8 MAbs cross-reacted with both E. coli and P. multocida, indicating that the antigenic determinants identified by these MAbs are conserved in all the three genera. The antigenic relationship of the Salmonella serovars (S. enteritidis, S. gallinarum, S. typhimurium, S. dublin, S. agona, S. indiana and S. choleraesuis) was studied using OMPs prepared from them and the anti-S. enteritidis MAbs. Three MAbs appeared to be specific for S. enteritidis as they did not cross-react with any of the other Salmonella serovars. Twelve of the 38 MAbs cross-reacted with all the serovars tested. Six of these were specific to the Salmonella genus as they did not cross-react with any of the other Gram-negative bacteria tested. The reactivity pattern of the other MAbs indicated that S. gallinarum was antigenically close to S. enteritidis, followed in order by S. dublin, S. agona, S. typhimurium and S. indiana, whereas S. choleraesuis seemed to be antigenically quite distant from S. enteritidis.

  5. Experimental Schistosoma bovis infection in goats. Circulating antigen and antibody responses to egg and adult worm antigens during infection and following treatment with praziquantel.

    Science.gov (United States)

    Johansen, M V; Fillié, Y; Monrad, J; Christensen, N O; Deelder, A

    1996-10-01

    Circulating antigen levels and antibody responses in Schistosoma bovis-infected West African Dwarf goats were evaluated during infection and following treatment with praziquantel (60 mg/kg) 13 weeks post-infection. One day, 1 week and 4 weeks post-treatment, subgroups of goats were sacrificed and perfused for worm recovery. For comparison, parasite-free control animals were included. Blood and faecal samples were collected biweekly. Two gut-associated schistosome antigens, circulating cathodic and circulating anodic antigen (CCA and CAA) and 3 specific antibody responses (total Ig, IgG and IgM) were measured. For specific antibody detection, crude S. bovis adult worm and egg homogenates were used. The level of CCA in the infected groups was significantly elevated from the time of onset of egg excretion onwards. However, following treatment, the CCA titres dropped to control levels within 1 week post-treatment. Strong positive correlations were found between CCA levels and worm counts and faecal egg counts during peak egg excretion. The correlations of CAA and specific antibody titres to egg and worm counts were poor. The antibody responses were all significantly elevated in the infected goats during patency, but only marginally affected by the treatment. Hence, CCA proved to be superior by correlating strongly to the level of infection and by being a sensitive indicator of the effect of treatment.

  6. HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies.

    Science.gov (United States)

    Ali, Ayub; Kitchen, Scott G; Chen, Irvin S Y; Ng, Hwee L; Zack, Jerome A; Yang, Otto O

    2016-08-01

    Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8(+) T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1

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

    Science.gov (United States)

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

    2017-08-01

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

  8. Tracking serum antibody response to viral antigens with arrayed imaging reflectometry

    Science.gov (United States)

    Mace, Charles R.; Rose, Robert C.; Miller, Benjamin L.

    2009-02-01

    Arrayed Imaging Reflectometry, or "AIR", is a new label-free technique for detecting proteins that relies on bindinginduced changes in the response of an antireflective coating on the surface of a silicon ship. Because the technique provides high sensitivity, excellent dynamic range, and readily integrates with standard silicon wafer processing technology, it is an exceptionally attractive platform on which to build systems for detecting proteins in complex solutions. In our early research, we used AIR chips bearing secreted receptor proteins from enteropathogenic E. coli to develop sensors for this pathogen. Recently, we have been exploring an alternative strategy: Rather than detecting the pathogen directly, can one immobilize antigens from a pathogen, and employ AIR to detect antibody responses to those antigens? Such a strategy would provide enhanced sensitivity for pathogen detection (as the immune system essentially amplifies the "signal" caused by the presence of an organism to which it responds), and would also potentially prove useful in the process of vaccine development. We describe herein preliminary results in the application of such a strategy to the detection of antibodies to human papillomavirus (HPV).

  9. Antibody response to Plasmodium vivax antigens in Fy-negative individuals from the Colombian Pacific coast.

    Science.gov (United States)

    Herrera, Sócrates; Gómez, Andrés; Vera, Omaira; Vergara, Juana; Valderrama-Aguirre, Augusto; Maestre, Amanda; Méndez, Fabián; Wang, Ruobing; Chitnis, Chetan E; Yazdani, Syed S; Arévalo-Herrera, Myriam

    2005-11-01

    The Duffy antigen (Fy) is necessary for Plasmodium vivax invasion of human erythrocytes. Some populations have a highly prevalent Fy-negative phenotype; such persons are naturally protected from P. vivax blood infection but are expected to completely support the P. vivax pre-erythrocytic cycle, representing a valuable model for studying the immune response during these parasitic stages. We typed 214 individuals, mostly Afro-Colombians, from a P. vivax-endemic area for Fy expression and determined the antibody response to P. vivax pre-erythrocytic (sporozoites and CS) and blood-stage antigens (blood forms, P. vivax merozoite surface protein 1, and P. vivax Duffy binding protein [PvDBP]). Antibody titers to P. vivax circumsporozoite protein, P11, and N-terminal peptides and the number of responders were similar in Fy-negative and Fy-positive individuals. The number of responders to sporozoites, blood forms, and PvDBP were different between these groups. Thus, Fy-negative individuals from malaria-endemic areas can be used to study the immune response to the P. vivax liver phase without interference of the erythrocytic cycle.

  10. Prevalence of G class antibodies to antigens of lyme disease causes in dogs in Vojvodina, Serbia

    Directory of Open Access Journals (Sweden)

    Potkonjak Aleksandar

    2013-01-01

    Full Text Available Lyme disease is a multisystemic disease, zoonotic in nature, caused by the Borrelia burgdorferi sensu lato complex. In the continent of Europe, these spirochetes are predominantly transmitted by ticks of the genus Ixodes. Small mammals and birds have particular significance as reservoirs of the cause of lyme disease. The objective of these epidemiological investigations was to determine the value of IgG seroprevalence to Borrelia burgdorferi and to secure the geographic distribution of seropositive dogs in Vojvodina. The investigations covered 135 dogs that were not vaccinated against lyme disease. The indirect ELISA test was used to determine IgG prevalence to Borrelia burgdorferi antigens. Reactive blood serums of dogs were tested again using the rapid immunochromatographic and immunoblot test. A seroprevalence of G class antibodies to antigens of lyme disease causes of 8.1% (11/135 was established in the examined dog population of Vojvodina. The biggest number of positive results was recorded for the South Bačka District. The presented value for the seroprevalence of anti-Borrelia burgdorferi antibodies in the dog population indicates the exhistence of a significant risk of humans becoming infected with the cause of lyme disease in Vojvodina.

  11. Cowpea mosaic virus-based systems for the expression of antigens and antibodies in plants.

    Science.gov (United States)

    Sainsbury, Frank; Liu, Li; Lomonossoff, George P

    2009-01-01

    This chapter describes the use of Cowpea mosaic virus-based vectors for the production of foreign proteins such as antigens and antibodies in plants. The systems include vectors based on both full-length and deleted versions of RNA-2. In both cases, the modified RNA-2 is replicated by coinoculation with RNA-1. The constructs based on full-length RNA-2 retain the ability to spread systemically throughout an inoculated plant and the infection can be passaged. The vector based on a deleted version of RNA-2 can stably incorporate larger inserts but lacks the ability to move systemically. However, it has the added advantage of biocontainment. In both cases, vector constructs modified to contain a foreign gene of interest can be delivered by agroinfiltration to obtain transient expression of the foreign protein. If required, the same constructs can also be used for stable nuclear transformation. Both types of vector have proved effective for the production in plants of a diverse range of proteins including antigens and antibodies.

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

    Science.gov (United States)

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

    2018-01-05

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

  13. Detection of specific IgE antibodies to major and minor antigenic determinants in sera of penicillin allergic patients.

    Science.gov (United States)

    Zhao, Yongxing; Qiao, Hailing

    2003-12-01

    To investigate the mechanism(s) of penicillins allergic reaction. The radioallergosorbent test (RAST) was used to detect 9 specific IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO), phenoxomethylpenicilloyl (PVO) and flucloxacilloyl (FLUO), and minor antigenic determinants: benzylpenicillanyl (BPA), amoxicillanyl (AXA), 6-aminopenicillanic (APA) and phenoxomethylpenicillany (PVA), in the sera of 32 penicillin allergic patients. The relationship between specific IgE antibodies and penicillins chemical structures was studied by radioallergosorbent inhibition test. Nineteen of 32 patients (59.4%) were RAST positive, among whom, five cases were positive only to one or two antigenic minor determinants, and three cases were positive only to one or three major antigenic determinants. The remaining 11 patients were positive not only to major antigenic determinants but also minor antigenic determinants. In 9 specific IgE antibodies, the positive rate of PVA-IgE was the highest (34.38%), followed by BPO-IgE (31.25%). The positive rate of FLUO-IgE was the lowest (15.63%). Of the total patient group, 53.13% were positive to one or more minor antigenic determinants, while 37.5% (12/32) were positive to one or more major antigenic determinants. The percentage of patients with urticarial reactions who were positive to minor antigenic determinants (63.16%) was significantly higher than observed in the anaphylactic shock group (38.5%, P allergic reaction. The combining sites of the specific IgE antibodies were likely to be the side-chain of drug or the overwhelming drug molecule.

  14. IgG subclass and vaccination stimulus determine changes in antigen specific antibody glycosylation in mice.

    Science.gov (United States)

    Kao, Daniela; Lux, Anja; Schaffert, Anja; Lang, Roland; Altmann, Friedrich; Nimmerjahn, Falk

    2017-08-02

    Immunoglobulin G (IgG) glycosylation can modulate antibody effector functions. Depending on the precise composition of the sugar moiety attached to individual IgG glycovariants either pro- or anti-inflammatory effector pathways can be initiated via differential binding to type I or type II Fc-receptors. However, an in depth understanding of how individual IgG subclasses are glycosylated during the steady state and how their glycosylation pattern changes during vaccination is missing. To monitor IgG subclass glycosylation during the steady state and upon vaccination of mice with different T-cell dependent and independent antigens, tryptic digests of serum, and antigen-specific IgG preparations were analyzed by reversed phase-liquid chromatography-mass spectrometry. We show that there is a remarkable difference with respect to how individual IgG subclasses are glycosylated during the steady state. More importantly, upon T-cell dependent and independent vaccinations, individual antigen-specific IgG subclasses reacted differently with respect to changes in individual glycoforms, suggesting that the IgG subclass itself is a major determinant of restricting or allowing alterations in specific IgG glycovariants. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

    Science.gov (United States)

    Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

    2010-10-01

    Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

  16. Defining influenza A virus hemagglutinin antigenic drift by sequential monoclonal antibody selection.

    Science.gov (United States)

    Das, Suman R; Hensley, Scott E; Ince, William L; Brooke, Christopher B; Subba, Anju; Delboy, Mark G; Russ, Gustav; Gibbs, James S; Bennink, Jack R; Yewdell, Jonathan W

    2013-03-13

    Human influenza A virus (IAV) vaccination is limited by "antigenic drift," rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Flow cytometry and monoclonal antibodies identify normal liver cell populations antigenically related to oval cells.

    Science.gov (United States)

    Agelli, M; Halay, E D

    1995-01-01

    Oval cells, a non-parenchymal cell population induced to rapidly proliferate in animals treated with carcinogens, are thought to be related to the hypothesized liver stem cells. In normal liver there are poorly defined cells antigenically related to oval cells. These oval cell antigen positive (OCAP) cells present in normal animals are thought to include hepatocyte and bile duct cell precursors. To isolate them, we modified the existing protocols designed for oval cells and used it on normal neonatal rat livers. Using flow cytometry, the percentage of normal liver OCAP-cells varied with the monoclonal antibody (MoAb) to the different oval cell membrane markers used: 12% (MoAb 18.2), 23% (MoAb 270.38), 27% (MoAb 18.11), 31% (MoAb 18.13), and 37% (MoAb 374.3). Macrophages consisted 10% of the cells (MoAb MCA 275); hepatocytes were essentially absent ( < 1%, MoAb 236.4). Our results demonstrate that is possible to obtain significant numbers of normal cells antigenically related to oval cells and that using different MoAbs, different cell populations can be sorted for use in experimental studies testing liver progenitor cell hypothesis.

  18. Performance evaluation of a new fourth-generation HIV combination antigen-antibody assay.

    Science.gov (United States)

    Mühlbacher, A; Schennach, H; van Helden, J; Hebell, T; Pantaleo, G; Bürgisser, P; Cellerai, C; Permpikul, P; Rodriguez, M I; Eiras, A; Alborino, F; Cunningham, P; Axelsson, M; Andersson, S; Wetlitzky, O; Kaiser, C; Möller, P; de Sousa, G

    2013-02-01

    Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys(®) HIV combi PT assay is a fourth-generation antigen-antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay's specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys(®) assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3-7.1 days observed with comparators. The analytical sensitivity of the Elecsys(®) HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys(®) assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys(®) assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys(®) HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.

  19. Plant Virus Particles Carrying Tumour Antigen Activate TLR7 and Induce High Levels of Protective Antibody

    Science.gov (United States)

    Jobsri, Jantipa; Allen, Alex; Rajagopal, Deepa; Shipton, Michael; Kanyuka, Kostya; Lomonossoff, George P.; Ottensmeier, Christian; Diebold, Sandra S.; Stevenson, Freda K.; Savelyeva, Natalia

    2015-01-01

    Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the “gold standard” Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-α by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe “in-built” ssRNA adjuvant. PMID:25692288

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

    Directory of Open Access Journals (Sweden)

    Ruud M Buijs

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

  1. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens.

    Directory of Open Access Journals (Sweden)

    Christoph B Geier

    Full Text Available Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

  2. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens

    Science.gov (United States)

    Geier, Christoph B.; Piller, Alexander; Linder, Angela; Sauerwein, Kai M. T.; Eibl, Martha M.; Wolf, Hermann M.

    2015-01-01

    Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency. PMID:26186701

  3. King cobra (Ophiophagus hannah) bites in Myanmar: venom antigen levels and development of venom antibodies.

    Science.gov (United States)

    Tun-Pe; Aye-Aye-Myint; Warrell, D A; Tin-Myint

    1995-03-01

    Venom, venom IgG and IgM antibody and total serum IgG levels following king cobra bites in two reptile handlers were measured by enzyme immunoassay. The patient in case 1 received antivenom while the patient in case 2 did not. Case 1 made a complete recovery following the bite and produced a high titre short-lived antibody. Venom antigen was not detected in the sample taken 11 hr after antivenom. Case 2 had experienced two recent minor king cobra bites and had received traditional immunization 4 weeks before the accident reported here. He had developed only local swelling and suffered no neurological symptoms. Venom antigen measured at 1.45 hr after the bite was 132 ng/ml; this rapidly fell to 45 ng/ml over the next 30 min, and was no longer detectable 14 hr after the bite. The pattern of venom IgG and IgM antibody responses in both cases was comparable, except that in case 2 the venom IgG peak was maintained for 13 days, compared with 1 day in case 1; in case 2 it subsequently fell to low levels 8 weeks after the bite. Venom IgM appeared 1 day after the bite, peaked at day 7-9, rapidly tailed off on day 12-16 and was then undetectable from day 20 onwards in both. Total IgG level remained within normal limits in both. It is possible that previous bites and recent immunization contributed to the boosting of the venom IgG response in case 2.

  4. Structural consequences of mutations in interfacial Tyr residues of a protein antigen-antibody complex. The case of HyHEL-10-HEL

    National Research Council Canada - National Science Library

    Shiroishi, Mitsunori; Tsumoto, Kouhei; Tanaka, Yoshikazu; Yokota, Akiko; Nakanishi, Takeshi; Kondo, Hidemasa; Kumagai, Izumi

    Tyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies...

  5. Kai 1 and Kai 2: Characterization of these dog erythrocyte antigens by monoclonal antibodies.

    Science.gov (United States)

    Lee, Jae Ho; Giger, Urs; Kim, Hee Young

    2017-01-01

    Dog Erythrocyte Antigens (DEA) have thus far been found by sensitizing dogs with canine allogeneic blood and are clinically important regarding blood transfusion incompatibilities, but remain poorly defined. The goals of this study were to discover and characterize two DEAs, named as Kai 1 and Kai 2. The monoclonal antibodies were produced by mouse hybridoma techniques and examined by ELISA isotyping, immunoblotting, and affinity chromatography. Canine blood samples were typed and the development of alloantibodies was examined in transfused dogs. The monoclonal Kai 1 and Kai 2 antibodies were isotyped as IgM kappa and IgG3 lamda, respectively, and identified two different erythrocyte membrane proteins of 200 kDa and 80 kDa in molecular weights, respectively. Either Kai 1 or Kai 2 can be expressed but not both in an individual dog. There were no naturally occurring anti-Kai 1 or Kai 2 alloantibodies. In addition, Kai 1- and/or Kai 2- dogs developed Kai 1 and Kai 2 alloantibodies, respectively, when transfused with mismatched blood. This is the first discovery of canine blood types by screening monoclonal antibodies. Kai 1 and Kai 2 are novel blood types which can induce anti-Kai 1 or anti-Kai 2 alloantibodies when Kai 1- and/or Kai 2- dogs are transfused with Kai 1+ or Kai 2+ blood. These canine blood types may explain some of the blood incompatibilities and transfusion reactions observed in dogs in clinical practice.

  6. Detection of Cysticercus antigens and antibodies in cerbrospinal fluid of patients with chronic meningitis Detecção de antígenos e anticorpos de Cysticercus em fluido cerebrospinal de pacientes com meningite crônica

    Directory of Open Access Journals (Sweden)

    Subhash Chandra Parija

    2007-10-01

    Full Text Available Chronic meningitism is a less frequent manifestation of neurocysticercosis caused by Taenia solium cysticerci. In the present study we used Co-agglutination (Co-A, a simple and rapid slide agglutination test to detect specific Cysticercus antigen in the 67 cerebrospinal fluid (CSF samples from patients with chronic meningitis of unknown etiology. The results were compared with that of ELISA for detection of antibodies. Among these samples four (5.97% were positive for Cysticercus antigen by Co-A test and six (8.95% were positive for antibodies by ELISA. Two samples were positive by both Co-A and ELISA, two were positive only by Co-A and four were positive only by ELISA. In the present study, although Cysticercus antigen and antibodies were present in CSF samples from eight (11.94% patients, we cannot affirm that all the cases of chronic meningitis are due to cysticercosis, but for any case of chronic meningitis of unknown origin, it would be useful to consider the possibility of cysticercal meningitis.Meningite crônica é manifestação pouco freqüente de neurocisticercose causada por cisticerco de Taenia solium. No presente estudo utilizamos co-aglutinação (Co-A um teste simples e rápido de aglutinação para detectar antígeno específico de Cysticercus nas 67 amostras de fluido cerebrospinal (CSF de pacientes com meningite crônica de etiologia desconhecida. Os resultados foram comparados com os de ELISA para detecção de anticorpos. Dentre estas amostras quatro (5,97% foram positivas para antígenos de Cysticercus pelo teste Co-A e seis (8,95% foram positivas para anticorpos por ELISA. Duas amostras foram positivas por ambos Co-A e ELISA, duas foram positivas somente por Co-A e quatro foram positivas somente por ELISA. No presente estudo embora antígenos e anticorpos de Cysticercus estivessem presentes nas amostras de CSF de oito pacientes (11,94%, não podemos afirmar que todos os casos de meningite crônica sejam devidos

  7. [Detection of antibodies to Campylobacter jejuni in pediatrics patients with Gullain-Barré syndrome using different antigen preparations].

    Science.gov (United States)

    Rokosz, Natalia; Rastawicki, Waldemar; Jagielski, Marek; Hetkowska-Abramczyk, Zofia

    2011-01-01

    Diagnosis of previous C. jejuni infections in GBS patients are mostly based on serological findings. However, there are not consensus what kind of antigen should be used in the serological assays. In our study we used ELISA with four different antigen preparations for investigation of specific antibodies to C. jejuni in serum samples obtained from 6 children with GBS. In all patients the high level of IgA and IgG antibodies to LPS were diagnosed. The antibodies in particular classes of immunoglobulins to recombinant proteins (Mikrogen), termostabile antigen and whole-cell antigen (Virion/Serion) of C. jejuni were diagnosed only in some of the children with GBS. However, in comparison to the control groups, the ELISA with recombinant proteins was most specific. Moreover, in two of the patients a characteristic decline of the level of antibodies to recombinant proteins in sera obtained in acute and chronic phase of disease have been observed. The results of this study showed that serodiagnosis, specially based on recombinant antigens, may be a reliable marker of recent or previous infection caused by C. jejuni in patients with GBS.

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

    DEFF Research Database (Denmark)

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

    1987-01-01

    count. Both antigenaemia and the disappearance of antibodies to the core protein were associated with development of the acquired immune deficiency syndrome (AIDS) or AIDS related complex and depletion of CD4 cells. Thus AIDS or AIDS related complex developed in eight out of 16 patients...... and 16 months after the estimated time of seroconversion. These results show that the late stages of HIV infection are characterised by increased production of antigen and a decrease in antibodies directed against the core protein. Antigenaemia indicates a poor prognosis; and as the antigen test...... is simple to do and interpret, it may therefore be useful for selecting patients for antiviral treatment....

  9. Antibodies to Antigenic Site A of Influenza H7 Hemagglutinin Provide Protection against H7N9 Challenge

    OpenAIRE

    Falko Schmeisser; Anupama Vasudevan; Swati Verma; Wei Wang; Esmeralda Alvarado; Carol Weiss; Vajini Atukorale; Clement Meseda; Weir, Jerry P.

    2015-01-01

    Identifying major antigenic and protective epitopes of the H7 hemagglutinin (HA) will be important for understanding the antibody response to vaccines developed against the novel influenza H7N9 viruses that emerged in China in 2013. To facilitate antigenic characterization of the H7N9 HA and to develop reagents for evaluation of H7N9 candidate vaccines, we generated a panel of murine monoclonal antibodies (mAbs) to the HA of A/Shanghai/2/2013 using mammalian cell-derived virus-like particles ...

  10. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

    Directory of Open Access Journals (Sweden)

    Luciana Pereira Silva

    2003-07-01

    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

  11. The indirect fluorescent antibody test based on schizont antigen for study of the sheep parasite Theileria lestoquardi.

    Science.gov (United States)

    Leemans, I; Hooshmand-Rad, P; Uggla, A

    1997-04-01

    An indirect fluorescent antibody test (IFAT), based on schizont-infected lymphoblastoid cells, was applied to study the course of antibody production in adult sheep inoculated with attenuated, in vitro grown, Theileria lestoquardi (Theileria hirci) infected cells. Bright fluorescence of the intracellular schizonts could first be demonstrated 15 days after inoculation. A 32-64-fold rise in antibody titres was recorded 1 month after infection, and substantial titres were still observed 90 days after inoculation. Fluorescence was absent with negative control sera and background staining was minimal. No serological cross-reactions were detected with sheep sera positive for Babesia motasi, Babesia ovis or Toxoplasma gondii. Results obtained did not differ when antigens prepared from three different strains of T. lestoquardi infected lymphoid cells were compared. Testing for reactivity to non-pathogenic Theileria species of sheep revealed a low degree of cross-reaction of a Theileria ovis and a Theileria separata antiserum to T. lestoquardi antigen. Cross-reactions were also observed with bovine sera positive for Theileria annulata and Theileria parva. Moreover, T. lestoquardi positive sera reacted almost equally strongly with bovine T. annulata antigen as with their homologous antigen, whereas cross-reaction with bovine T. parva antigen was less pronounced. These results indicate a close antigenic relationship between ovine T. lestoquardi and T. annulata of cattle.

  12. [Evaluation of new fourth-generation human immunodeficiency virus antigen and antibody detection assay with enzyme-linked fluorescent immunoassay].

    Science.gov (United States)

    Shima, Takako; Sudo, Koji; Kondo, Makiko; Kurai, Hanako; Sagara, Hiroko; Imai, Mitsunobu

    2007-09-01

    We evaluated the fourth-generation HIV screening assay VIDAS HIV DUOII (DUOII) based on ELFA for simultaneous detection of anti-HIV-1 and anti-HIV-2 antibodies and HIV-1 p24 antigen through comparison with other HIV antigen-antibody detection assays. Materials were 1228 HIV-negative specimens, 95 HIV-antibody-positive specimens, and HIV commercial panels. The specificity of DUOII was 99.8% and sensitivity 100%, detecting all of HIV-1 group M subtype A, B, B', C, D, A/E, F, G, B/D, HIV-1 group O, and HIV-2. The sensitivity test to HIV-1 p24 antigen was 5pg/ mL, higher than other assays. DUOII was equivalent to or superior in detecting results earlier than other assays in an evaluation using 10 commercial HIV-1 seroconversion panels of primary infection. DUOII detects anti-HIV IgM antibody, so no negative sample was found in the second window between p24 antigen disappearance and raised anti-HIV IgG antibody. DUOII has sufficient specificity and sensitivity for HIV screening, and detects primary infection sooner than other assays. These results indicate that DUOII is useful and reliable in HIV screening.

  13. Measurement of antibodies to varicella-zoster virus using a virus-free fluorescent-antibody-to-membrane-antigen (FAMA) test.

    Science.gov (United States)

    Park, Rackhyun; Hwang, Ji Young; Lee, Kang Il; Namkoong, Sim; Choi, Seuk-Keun; Park, Songyong; Park, Hosun; Park, Junsoo

    2015-02-01

    The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

  14. A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus.

    Science.gov (United States)

    Reddington, J J; Reddington, G M; MacLachlan, N J

    1991-04-01

    A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave greater than 20% inhibition) that ranged from 32 to greater than 1.024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by cELISA. The sensitivity and specificity of the cELISA test was further confirmed by analysis of a panel of bovine test sera supplied by the National Veterinary Services Laboratories, indicating that the cELISA is a superior test for detection of BTV group-specific antibodies in sera from ruminants in the United States.

  15. High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on Staphylococcus aureus Antigen MntC.

    Directory of Open Access Journals (Sweden)

    Alexey V Gribenko

    2016-09-01

    Full Text Available The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 - group 1, mAB 305-78-7 - group 2, and mAB 305-101-8 - group 3 were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS. All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7 with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity.

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

    Directory of Open Access Journals (Sweden)

    Masato Kiyoshi

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

  17. Development of a highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses enzyme-labeled antigen.

    OpenAIRE

    Seppänen, H

    1990-01-01

    An enzyme immunoassay (EIA) for serum immunoglobulin M (IgM) antibodies to rubella virus based on enzyme labeling of viral antigen was developed. The sensitivity of the EIA for the detection of recent rubella virus infection was evaluated by using 115 rubella-IgM-antibody-positive serum specimens, which were confirmed as positive by Rubazyme M (Abbott Diagnostics). In addition, 12 individuals, 2 of whom were exposed to rubella through vaccination and 10 of whom were exposed through natural in...

  18. Pig Production System, Marketing Chain and Cysticercosis ...

    African Journals Online (AJOL)

    Publications on pig production and cysticercosis in The Gambia and Senegal are very scant. Hence, this survey was implemented to characterise the pig production systems and marketing chain, and to assess people's awareness of cysticercosis. The survey sites were Western region and Kanifing Municipality of The ...

  19. Antibody production by injection of living cells expressing non self antigens as cell surface type II transmembrane fusion protein.

    Science.gov (United States)

    Nizet, Yannick; Gillet, Laurent; Schroeder, Hélène; Lecuivre, Corinne; Louahed, J; Renauld, J-C; Gianello, Pierre; Vanderplasschen, Alain

    2011-03-31

    Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related protein 1 (secreted protein) and CD94 (type II transmembrane protein). Using this vector, we developed a method to produce antibodies without antigen production. The flowchart of this method is as follows: (i) cloning of the antigen in the pCD1-CD134L vector; (ii) production of a histocompatible cell line stably expressing the CD134L-antigen fusion protein; (iii) testing for cell surface expression of the fusion protein by targeting the CD134L carrier; and (iv) prime-boost immunisation with living cells expressing the fusion protein. This method was successfully used for production of polyclonal antibodies raised against Ixodes ricinus calreticulin (secreted protein) in mice and for production of monoclonal antibodies raised against an epitope of Vaccinia virus A56 (type I transmembrane protein) protein in rat. The present study is the first to demonstrate the use of a type II transmembrane protein as a carrier for cell surface display of antigens. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue functional antibody assays.

    Science.gov (United States)

    Yamanaka, Atsushi; Suzuki, Ryosuke; Konishi, Eiji

    2014-07-23

    Dengue fever and dengue hemorrhagic fever are endemic throughout tropical and subtropical countries. Four serotypes of dengue viruses (DENV-1 to DENV-4), each with several genotypes including various subclades, are co-distributed in most endemic areas. Infection-neutralizing and -enhancing antibodies are believed to play protective and pathogenic roles, respectively. Measurement of these functional antibodies against a variety of viral strains is thus important for evaluating coverage and safety of dengue vaccine candidates. Although transportation of live virus materials beyond national borders is increasingly limited, this difficulty may be overcome using biotechnology that enables generation of an antibody-assay antigen equivalent to authentic virus based on viral sequence information. A rapid system to produce flavivirus single-round infectious particles (SRIPs) was recently developed using a Japanese encephalitis virus (JEV) subgenomic replicon plasmid. This system allows production of chimeric SRIPs that have surface proteins of other flaviviruses. In the present study, SRIPs of DENV-1 (D1-SRIPs) were evaluated as an antigen for functional antibody assays. Inclusion of the whole mature capsid gene of JEV into the replicon plasmid provided higher D1-SRIP yields than did its exclusion in cases where a DENV-1 surface-protein-expressing plasmid was used for co-transfection of 293T cells with the replicon plasmid. In an assay to measure the balance between neutralizing and enhancing activities, dose (antibody dilution)-dependent activity curves in dengue-immune human sera or mouse monoclonal antibodies obtained using D1-SRIP antigen were equivalent to those obtained using DENV-1 antigen. Similar results were obtained using additional DENV-2 and DENV-3 systems. In a conventional Vero-cell neutralization test, a significant correlation was shown between antibody titers obtained using D1-SRIP and DENV-1 antigens. These results demonstrate the utility of D1-SRIPs as

  1. MDR-TB Antibody Response (Western Blot) to Fractions of Isoniazid and Rifampicin Resistant Antigens of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hadizadeh Tasbiti, Alireza; Yari, Shamsi; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Bahrmand, Ahmadreza

    2015-12-01

    Drug-resistant TB poses a major threat to control of TB worldwide. Despite progress in the detection of Multidrug-resistant TB (MDR-TB) cases, a major diagnostic gap remains: 55% of reported TB patients estimated to have MDR-TB were not detected in 2013. MDR-TB antigens were conjugated to CNBr-activated Sepharose 4B. Specific polyclonal antibodies against MDR-TB Ags were prepared in rabbits using two boosted injections of the MDR-TB antigen. The antibodies were purified and treated with susceptible TB to remove any non-specific and cross-reactive antibodies. In the present study, comparative analysis of electrophoretic pattern of different antigens of INH/RIF-resistant TB were studied for identifying protein profiles. A RIF-resistant TB antigen was shown here to have different protein profiles from INH-resistant TB isolate. The results of Western blotting analysis showed that in the RIF- and INH-resistant antigenic fractions some bands of 14.4 and 45 kDa as immunogenic were common. Moreover, four bands of RIF-resistant TB antigen fractions (16, 19, 21, and 45 KDa) and one band of INH-resistant TB (about 26 KDa) were detected as diagnostic antigens. This study suggests that the Western blot is an accurate test to survey INH- and RIF-resistant TB antigens of M. tuberculosis infection. These findings indicate that MDR-TB diagnosis (based on Ag detection) could be useful in the identification of disease stages that precede symptomatic and microbiologically positive TB, such as subclinical and incipient TB.

  2. Two new monoclonal antibodies to human monocytes and granulocytes: isolation of membrane antigens and lack of effects of antibodies on leukocyte functions in vitro.

    Science.gov (United States)

    Stevenson, H C; Kimball, E; Schroff, R W; Buescher, S; Clarke, G; Gregorio, T; Wilburn, S; Foon, K A

    1984-01-01

    Mice were immunized with purified human monocytes or granulocytes obtained by leukapheresis and isolated on dextran gradients or by countercurrent centrifugation-elutriation. A monoclonal antibody, Mo95, was generated in response to monocytes and was found to react strongly with monocytes, large granular lymphocytes (LGL), granulocytes, eosinophils, and some myelomonocytic leukemia cells, but not with normal T or B lymphocytes, platelets, red cells, or leukemic cell lines. Mo95 is an IgG1 antibody, which precipitated a 95 kD molecular weight antigen. Addition of the Mo95 antibody to monocytes in the absence of complement did not inhibit lysozyme secretion nor did it affect superoxide production, C3b-rosetting, nitrotetrazolium blue reduction, phagocytosis, or chemotactic responses. A second antibody, PMN70, was found to react exclusively with granulocytes and not with monocytes, lymphocytes, LGL, platelets, red cells, or any of the myelomonocytic, T-cell-derived or B-cell-derived leukemic cell lines tested. The PMN70 antibody immunoprecipitated a 70 kD molecular weight antigen found only on mature granulocytes. Mo95 and PMN70 appear to be distinct from five other tested monoclonal antibodies reactive to monocytes and/or granulocytes on the basis of the fluorescent cell sorter and immunoprecipitation studies performed.

  3. Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses.

    Science.gov (United States)

    Burkala, E J; Narayani, I; Hartaningsih, N; Kertayadnya, G; Berryman, D I; Wilcox, G E

    1998-09-01

    Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

  4. Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

    Science.gov (United States)

    de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

    2017-01-18

    We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, K D eff . Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

  5. Probing the effects of surface hydrophobicity and tether orientation on antibody-antigen binding

    Science.gov (United States)

    Bush, Derek B.; Knotts, Thomas A.

    2017-04-01

    Antibody microarrays have the potential to revolutionize molecular detection for many applications, but their current use is limited by poor reliability, and efforts to change this have not yielded fruitful results. One difficulty which limits the rational engineering of next-generation devices is that little is known, at the molecular level, about the antibody-antigen binding process near solid surfaces. Atomic-level structural information is scant because typical experimental techniques (X-ray crystallography and NMR) cannot be used to image proteins bound to surfaces. To overcome this limitation, this study uses molecular simulation and an advanced, experimentally validated, coarse-grain, protein-surface model to compare fab-lysozyme binding in bulk solution and when the fab is tethered to hydrophobic and hydrophilic surfaces. The results show that the tether site in the fab, as well as the surface hydrophobicity, significantly impacts the binding process and suggests that the optimal design involves tethering fabs upright on a hydrophilic surface. The results offer an unprecedented, molecular-level picture of the binding process and give hope that the rational design of protein-microarrays is possible.

  6. Prevalence of Dirofilaria immitis antigen and antibodies to Leishmania infantum in cats from southern Portugal.

    Science.gov (United States)

    Maia, Carla; Ramos, Cláudia; Coimbra, Mónica; Cardoso, Luís; Campino, Lenea

    2015-04-01

    Vector-borne diseases (VBD) are caused by a range of pathogens transmitted by arthropods and have emerged in recent years, showing a wider geographic distribution and increased global prevalence. In addition to their veterinary medical importance, cats play a central role in the transmission cycles of some VBD agents by acting as reservoirs, amplifying hosts or sentinels. The aim of this study was to determine the prevalence of Dirofilaria immitis antigen and of antibodies to Leishmania infantum in a sample of 271 cats from southern Portugal. Thirteen (4.8%) cats were positive to D. immitis, while antibodies to L. infantum were detected in 10 (3.7%) animals. The prevalence of D. immitis and L. infantum in the feline population from southern Portugal should alert for the need to implement control measures to protect animals and people from these zoonotic parasites. Furthermore, both parasitoses must be included in the differential diagnosis in feline clinical practice. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ramesh Venkatachalam

    2015-06-01

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

  8. Long-term treatment of pigs with low doses of monoclonal antibodies against porcine CD4 and CD8 antigens

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Eriksen, Lis

    2006-01-01

    In vivo depletion of lymphocyte subsets allows investigation of the role of specific subsets in protective immunity. In the present study we evaluated the effects of long-term, low-dose treatment with murine monoclonal antibodies (mAbs) against porcine CD4 and CD8 surface antigens on lymphocyte...

  9. Detection of genome, antigen, and antibodies in oral fluids from pigs infected with foot-and-mouth disease virus.

    Science.gov (United States)

    Senthilkumaran, Chandrika; Yang, Ming; Bittner, Hilary; Ambagala, Aruna; Lung, Oliver; Zimmerman, Jeffrey; Giménez-Lirola, Luis G; Nfon, Charles

    2017-04-01

    Virus nucleic acids and antibody response to pathogens can be measured using swine oral fluids (OFs). Detection of foot-and-mouth disease virus (FMDV) genome in swine OFs has previously been demonstrated. Virus isolation and viral antigen detection are additional confirmatory assays for diagnosing FMDV, but these methods have not been evaluated using swine OF. The objectives of this study were to further validate the molecular detection of FMDV in oral fluids, evaluate antigen detection and FMDV isolation from swine OFs, and develop an assay for isotypic anti-FMDV antibody detection in OFs. Ribonucleic acid (RNA) from FMDV was detected in OFs from experimentally infected pigs by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) from 1 day post-infection (dpi) to 21 dpi. Foot-and-mouth disease virus (FMDV) was isolated from OFs at 1 to 5 dpi. Additionally, FMDV antigens were detected in OFs from 1 to 6 dpi using a lateral flow immunochromatographic strip test (LFIST), which is a rapid pen-side test, and from 2 to 3 dpi using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). Furthermore, FMDV-specific immunoglobulin A (IgA) was detected in OFs using an isotype-specific indirect ELISA starting at dpi 14. These results further demonstrated the potential use of oral fluids for detecting FMDV genome, live virus, and viral antigens, as well as for quantifying mucosal IgA antibody response.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Duque-Beltrán Sofía

    2002-01-01

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

  12. The sickle cell trait is associated with enhanced immunoglobulin G antibody responses to Plasmodium falciparum variant surface antigens.

    NARCIS (Netherlands)

    Cabrera, G.; Cot, M.; Migot-Nabias, F.; Kremsner, P.G.; Deloron, P.; Luty, A.J.F.

    2005-01-01

    The sickle cell trait (HbAS) protects against severe Plasmodium falciparum malaria in young African children. We investigated the extent of the association between HbAS and antibodies directed to parasite-derived variant surface antigens (VSAs) on the membrane of infected erythrocytes. We measured

  13. Inhibition, by vinca alkaloids and colchicine, of antigenic modulation induced by anti-CD19 monoclonal antibodies

    NARCIS (Netherlands)

    de Rie, M. A.; Zeijlemaker, W. P.; von dem Borne, A. E.

    1988-01-01

    Several clinical trials have been reported in which monoclonal antibodies (McAb) were used for therapy of lymphoid malignancies. Such trials have shown that infusion of McAb recognizing lymphoid antigens, is well-tolerated, and leads to the coating of tumor cells and tumor regression in some

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    is maintained at low densities. Here, we test the hypothesis that the presence or absence of antibodies against variant antigens on the surface of P. falciparum-infected erythrocytes protect individuals against some infectious challenges and render them susceptible to others. Plasma collected in Daraweesh...

  15. Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

    Directory of Open Access Journals (Sweden)

    ESPÍNDOLA Noeli M.

    2000-01-01

    Full Text Available We describe the production of the potential monoclonal antibodies (MoAbs using BALB/c mice immunized with vesicular fluid (VF-Tcra (T. crassiceps antigen. Immune sera presented anti-VF-Tcra (<20kD IgG and IgM antibodies with cross-reactivity with T. solium (Tso antigen (8-12, 14, and 18 kD. After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

  16. Experimental infection in cattle: kinetics of production of IgM and IgG against bovine cysticercosis and inflammatory response

    Directory of Open Access Journals (Sweden)

    Rafaella Paola Meneguete dos Guimarães-Peixoto

    2015-04-01

    Full Text Available Bovine cysticercosis is a zoonosis that affects humans in their adult form (taeniasis and its larval is found inserted in the musculature of infected cattle (cysticerci. It is still not entirely clear how animal immune response against infection occurs, being the comprehension of this process necessary for the enhancement of diagnostic capacity and disease prevention. This work aimed to evaluate the evolution of the immune response in experimentally infected cattle, compared with findings in cell response by optical microscopy. Nine animals were infected at a rate of 120,000 eggs of Taenia saginata. Five of the animals were similar in the kinetics of antibody production against cysticerci, with maximal levels of IgG and IgM. The other four animals showed an immune response different from the majority, with two of them showing delayed response to infection by cysticerci while the others apparently did not have initial contact with antigens secreted by cysticerci. Regarding the cellular response, it was found that, in lesions of viable cysticerci, inflammatory cells predominated, whereas in nonviable cysticerci there were tissue repair cells in the most part, being possible to notice that the amount of migratory calcareous corpuscles are related to the death stage of the parasite. These findings are important for the understanding immune response of cattle infected with cysticercosis.

  17. A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells.

    Science.gov (United States)

    Piccione, Emily C; Juarez, Silvia; Liu, Jie; Tseng, Serena; Ryan, Christine E; Narayanan, Cyndhavi; Wang, Lijuan; Weiskopf, Kipp; Majeti, Ravindra

    2015-01-01

    Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an 'antigen sink' that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.

  18. Characterization of antigens expressed in normal baboon trophoblast and cross-reactive with HIV/SIV antibodies.

    Science.gov (United States)

    Langat, D K; Johnson, P M; Rote, N S; Wango, E O; Owiti, G O; Isahakia, M A; Mwenda, J M

    1999-01-01

    Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles in normal primate placental tissues. These particles have ultrastructural similarities to type C retroviral particles and are mainly associated with the trophoblast. In normal human placental tissues, they have antigenic similarity with exogenous retroviruses, such as the human immunodeficiency virus (HIV), and may have a role to play in the regulation of cellular gene expression, syncytiotrophoblast formation or pregnancy-related immunosuppression. In this study, a panel of antibodies (polyclonal and monoclonal antibodies) against viral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) proteins were assessed by immunohistochemistry and immunoblotting, for their cross-reactivity with ERV particles isolated from normal baboon placental tissues. The antibodies (anti-HERV-K RT, anti-ERV3 env, anti-HIV-1 p17, anti-HIV-2 gp120) reacted positively with the syncytiotrophoblast and each antibody recognized one or two proteins of molecular weights (MW) 38, 58 or 64 kDa present in the baboon placental villous tissues and SIV-infected molt-4 Cl8 cells, but not in uninfected cells. The results of this study confirm the specific expression of retroviral cross-reactive antigens in normal baboon placental tissues and suggest placental cellular proteins may have antigenic similarity with those recognized by anti-HIV/SIV antibodies. The role of these retroviral-related proteins expressed at the maternal-fetal interface remain unclear.

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  1. Suppression and contrasuppression in the induction of contact sensitivity by the administration of cellbound antigen-antibody complexes.

    Science.gov (United States)

    Ptak, W; Bereta, M; Ptak, M; Iverson, G M; Green, D R

    1985-10-01

    The tolerogenic signal produced by the i.v. injection of haptenated peritoneal exudate cells can be converted to an immunogenic signal by treating the cells with antibody to the hapten before administration. We examined this phenomenon and found that immunity induced by antigen-antibody complexes, as opposed to skin sensitization, is resistant to suppressor T cell influences. This resistance to suppression is due to the activation of an I-J+, Ly-1 T cell population which adheres to the Vicia villosa lectin, all characteristics of contrasuppressor T cells. Because haptenated cells can induce immunity if injected subcutaneously or into cyclophosphamide-pretreated recipients (thereby avoiding the induction of suppressor cells), we suggest that the activation of contrasuppressor cells by antigen-antibody complexes overrides suppressive influences in the host, allowing immunity to become dominant. The possible roles of suppression and contrasuppression in channeling the effector arm of the immune response (e.g., contact sensitivity vs humoral immunity) are discussed.

  2. Relevant peptides of Taenia crassiceps for the diagnosis of bovine cysticercosis by immunoblot

    Directory of Open Access Journals (Sweden)

    L.F. Silva

    2015-06-01

    Full Text Available Given the limited knowledge about the diagnosis of bovine cysticercosis by immunoblot, the aim of this study was to assess the applicability of this test, identifying key peptides with diagnostic value. Immunoblot assays were performed using total larval antigen of Taenia crassiceps and 60 sera of positive bovines for cysticercosis (30 naturally and 30 experimentally infected with T. saginata eggs, 30 sera of negative bovines for cysticercosis and 30 sera of bovines with other diseases (fascioliasis, hydatidosis and tuberculosis. The peptides of greater diagnostic importance, in descending order of accuracy (%, were as follows: 6-8kDa (90.8%, 129-143kDa (74.2%, 99-105kDa (71.7% and 14-19kDa (71.1%. Cross-reactions, due to fascioliasis and hydatidosis, were observed in the four intervals of peptides highlighted. The results demonstrate that the total antigen of T. crassiceps has peptides with a high diagnostic potential; therefore, the immunoblot is useful in the diagnosis of bovine cysticercosis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-06-15

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

  4. Localization of distinct surface antigens on Chlamydia trachomatis HAR-13 by immune electron microscopy with monoclonal antibodies.

    Science.gov (United States)

    Clark, R B; Nachamkin, I; Schatzki, P F; Dalton, H P

    1982-01-01

    Monoclonal antibodies were produced against the HAR-13 strain (A) of Chlamydia trachomatis. By an indirect immunofluorescence technique, three clones (1-7, 2-8, 9F) were found to produce antibody that reacted only with serotype A strains. Clone 3-5 produced antibody that cross-reacted with serotypes A, C, H, I, and J. Using an indirect immunoferritin technique, we examined the binding of these antibodies to strain HAR-13 by electron microscopy. Antibodies from all four clones were shown to bind to the outer membrane surface of both reticulate and elementary bodies. Two distinct binding patterns were demonstrated. Antibodies from clones 1-7 and 9F bound to the outer membrane surface in a homogeneous pattern, whereas antibodies 2-8 and 3-5 bound to the outer membrane surface in an irregular, patchy distribution. There was a direct correlation between the distribution of antigen on the outer membrane surface and neutralization of C. trachomatis HAR-13 infectivity in vitro. Neutralization of HAR-13 infectivity by antibodies 1-7 and 9F was complement dependent, whereas antibodies 2-8 and 3-5 did not neutralize under any conditions. Images PMID:7152670

  5. New principle for the simultaneous detection of total and immunoglobulin M antibodies applied to the measurement of antibody to hepatitis B core antigen.

    Science.gov (United States)

    Angarano, G; Monno, L; Santantonio, T A; Pastore, G

    1984-06-01

    A new test principle for the simultaneous detection of total approximate titers and immunoglobulin M antibodies has been developed and applied to the detection of antibody to hepatitis B core antigen. The method is based on the combination of a competition radioimmunoassay, for the determination of total antibody titer, with an indirect enzyme-linked immunosorbent assay for the determination of single class antibodies. The interference of the rheumatoid factor was avoided by including heat-aggregated immunoglobulin G in the dilution buffer. The specificity, sensitivity, and clinical application of the test are discussed. The results presented suggest that the simultaneous detection of total and immunoglobulin M antibody to hepatitis B core antigen might be helpful in the differentiation between previous and recent or ongoing hepatitis B infection, as well as in the differential diagnosis of acute hepatitis, in monitoring viral activity in chronic infections, and in helping to differentiate acute from chronic infections. The test principle appears applicable in the accurate diagnosis of other infectious diseases by a single test on only one serum sample.

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

    DEFF Research Database (Denmark)

    Askjaer, N; Maxwell, C; Chambo, W

    2001-01-01

    variant surface antigens (VSA) are important in the development of naturally acquired immunity to Plasmodium falciparum malaria and may thus be good indicators of immune status. We have compared the levels of VSA antibodies in plasma from children who have used ITN for 4 years to levels in plasma from...... children from a nearby village not using ITN. A total of 97 plasma samples were analyzed using 13 different P. falciparum isolates. We found that the children using ITN had significantly lower VSA antibody levels and recognized a smaller proportion of the VSA expressed by the tested parasite isolates than...... children not using ITN....

  7. Requirement for continuous antigenic stimulation in the development and differentiation of antibody-forming cells. The effect of passive antibody on the primary and secondary response.

    Science.gov (United States)

    Hanna, M G; Nettesheim, P; Francis, M W

    1969-05-01

    The essential role of continuous antigenic stimulation in the development and differentiation of antibody-forming cells as defined in the X-Y-Z immune cell maturation scheme was examined in these studies. Mice were primed with sheep erythrocytes (SRBC) in an attempt to induce maximum immune progenitor cell conversion (X --> Y). Subsequently antigen was depleted at 1 or 4 days after priming with isologous specific antibody in order to interrupt further immune cell differentiation (Y --> Z). It was reasoned that this condition would result in depression of the functional antibody-producing cell compartment as measured in the intact mice and subsequently in enhancement of the sensitized (Y cell) compartment as measured in the spleen cell transfer system. These data were also correlated with systematic studies of the hyperplasia of the spleen germinal centers. The effect of passive antibody on the primary response to SRBC was a marked decrease indirect and indirect hemolysin-producing cells (DPFC and IPFC). However, there was a lack of correlation in the degree of antibody-mediated 19S and 7S immune cell suppression during the primary response, the DPFC being much less depressed than the IPFC. As measured in the transfer system there was an enhanced 19S sensitized cell compartment and a depressed 7S sensitized cell compartment in 1 day passively immunized mice. This was true whether or not transfers were performed 1, 2, or 4 wk after priming. Similarly, there was an enhanced 19S-sensitized cell compartment with little or no effect on the 7S-sensitized. cell compartment in 4 day passively immunized mice. These data suggest that progeny of the antigen-stimulated progenitor cells (X cell), as a consequence of lack of further antigenic stimulation, were forced into maturation arrest. These studies further demonstrate that isologous passive antibody suppresses germinal center growth regardless of whether the antibody is infused 1, 2, or 4 days after priming. In terms of

  8. Detection of antibodies to hepatitis B surface antigen (HBsAg) using monoclonal antibody and the avidin-biotin system.

    Science.gov (United States)

    Korec, E; Hlozánek, I; Mach, O; Stará, J; Nĕmecek, V; König, J

    1986-01-01

    A direct ELISA using biotinylated HBsAg and a competitive ELISA using biotinylated monoclonal antibody were developed for the detection of antibodies to HBsAg. Both tests are capable of detecting 0.1 I.U. of anti-HBsAg antibody/ml. The direct ELISA was compared with a SPRIA test for anti-HBsAg antibody in human sera.

  9. Disseminated cysticercosis with huge muscle hypertrophy

    Directory of Open Access Journals (Sweden)

    Bandyopadhyay Debabrata

    2009-01-01

    Full Text Available Cysticercosis is caused by cysticercus cellulose, which is the larva of Taenia solium , the pork tapeworm. The larvae are carried in the blood stream after penetrating the walls of the alimentary tract and they lodge in different tissues like the skin, skeletal muscles, brain, fundus and heart, to cause disseminated cysticercosis. Cases of disseminated cysticercosis have rarely been reported in the literature. They may inhabit the muscles and cause muscular hypertrophy, which, at times, may assume gross proportions. Morbidity is usually caused by the involvement of the central nervous system or the eyes.

  10. 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.

  11. Histological features of rheumatoid arthritis patients having antibodies to enterobacterial common antigens: correlation of antibody levels with semiquantitative histologic scores and laboratory markers.

    Science.gov (United States)

    Aoki, S; Mitsui, T

    2005-01-01

    To ascertain whether clinically diagnosed rheumatoid arthritis (RA) patients having antibodies to enterobacterial common antigens (ECA) in synovial fluid (SF) have a histological appearance characteristic of RA synovitis. Twenty-five RA patients for which synovial biopsy specimens were preserved, were selected from 58 patients with RA tested for antibodies to ECA in SF The synovial tissue specimens were examined histologically using a semiquantitative scoring system with quantitative counts. The correlation of anti-ECA antibody levels with total scores for synovitis and laboratory markers in three RA patient groups (markedly positive, moderately to slightly positive, and negative according to anti-ECA antibody levels) was analyzed statistically. Histologic examination in the markedly positive RA group (total score for synovitis, range 18-20 points) revealed typical histological features of rheumatoid synovitis. Total scores for synovitis were significantly higher in both the markedly positive and moderately to slightly positive RA groups than in the negative RA group. A comparison of anti-ECA antibody levels with total scores for synovitis revealed a strong and significant correlation. Furthermore, levels of anti-ECA antibodies were also correlated with rheumatoid factor and C-reactive protein. Clinically diagnosed RA patients having anti-ECA antibodies in SF showed typical or characteristic histological features of RA synovitis. Our data suggest that a group of RA patients with an entrobacterial etiology exists in larger groups of patients with RA which is thought to be a heterogeneous disease.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  13. Generation of human monoclonal antibodies against ganglioside antigens and their applications in the diagnosis and therapy of cancer

    Energy Technology Data Exchange (ETDEWEB)

    Alfonso, M. [Dept. of Tumor Cell Biology, Div. of Cancer Biology, Danish Cancer Society, Copenhagen (Denmark)]|[Dept. of Research and Development, Center of Molecular Immunology, Havana (Cuba); Zeuthen, J. [Dept. of Tumor Cell Biology, Div. of Cancer Biology, Danish Cancer Society, Copenhagen (Denmark)

    1996-10-01

    Different approaches to generating human monoclonal antibodies (MAbs) against tumor-associated ganglioside antigens have been carried out in several laboratories. A specific goal addressed by our laboratory is to produce human MAbs to several ganglioside antigens of relevance as therapeutic targets, such as the GM2, GD2, GD3 and GM3 gangliosides in melanoma. In vitro immunization of human B lymphocytes from normal donors was performed using liposomes containing gangliosides as the immunizing antigen combined with either complete tetanus toxoid or a synthetic peptide corresponding to a T helper epitope to stimulate in vitro immunization. Specific human anti-ganglioside antibodies were obtained, indicating that the antibdoy response found in vitro was antigen-driven. To overcome the widely reported problems concerning stability of immunoglobulin production by the antibody-secreting cell lines, a method of positive selection using GM3-coated magnetic beads has been developed in order to rescue unstable clones. Development of new methods to reproducibly generate ganglioside-specific human MAbs will amplify the possibilities for diagnostic and therapeutic applications. (orig.).

  14. A monoclonal-antibody-defined adhesion-related antigen on bovine neutrophils is required for neutrophil aggregation.

    Science.gov (United States)

    Bochsler, P N; Doré, M; Neilsen, N R; Slauson, D O

    1990-10-01

    Surface adhesion molecules present on human leukocytes are known to regulate certain adhesion-related events, such as adhesion to endothelium, extravasation, and aggregation. We have used a mouse anti-human monoclonal antibody designated 60.3 (MAb 60.3) and indirect immunofluorescence technique to identify an antigen on bovine neutrophils (PMNs). MAb 60.3 bound to resting and stimulated bovine PMN in a surface-oriented pattern. Immunofluorescence flow cytometric analysis indicated that warming the PMNs from 4 degrees C to 37 degrees C slightly increased (13.9%) expression of the antigen recognized by MAb 60.3. Zymosan-activated serum (ZAS, 10%) increased antigen expression by 12.4% over those PMNs in buffer alone, and phorbol 12-myristate 13-acetate (PMA; 100 ng/ml) by 65.6%. Bacterial lipopolysaccharide (LPS; 1 micrograms/ml) from E. coli 0111:B4 did not enhance antigen expression. The functional nature of this antigen was demonstrated by use of MAb 60.3 and PMN aggregation. Preincubation of bovine PMN with MAb 60.3 for 10 min resulted in nearly complete inhibition of PMN-PMN aggregation upon subsequent stimulation with PMA (100 ng/ml); preincubation with a control antibody did not inhibit aggregation. These results indicate that bovine PMNs possess surface molecule(s) that may function in adhesion-related events, and surface expression may be enhanced by PMN stimulation.

  15. Timing of the human prenatal antibody response to Plasmodium falciparum antigens.

    Directory of Open Access Journals (Sweden)

    Samuel Tassi Yunga

    Full Text Available Plasmodium falciparum (Pf-specific T- and B-cell responses may be present at birth; however, when during fetal development antibodies are produced is unknown. Accordingly, cord blood samples from 232 preterm (20-37 weeks of gestation and 450 term (≥37 weeks babies were screened for IgM to Pf blood-stage antigens MSP1, MSP2, AMA1, EBA175 and RESA. Overall, 25% [95% CI = 22-28%] of the 682 newborns were positive for IgM to ≥1 Pf antigens with the earliest response occurring at 22 weeks. Interestingly, the odds of being positive for cord blood Pf IgM decreased with gestational age (adjusted OR [95% CI] at 20-31 weeks = 2.55 [1.14-5.85] and at 32-36 weeks = 1.97 [0.92-4.29], with ≥37 weeks as reference; however, preterm and term newborns had similar levels of Pf IgM and recognized a comparable breadth of antigens. Having cord blood Pf IgM was associated with placental malaria (adjusted OR [95% CI] = 2.37 [1.25-4.54]. To determine if in utero exposure occurred via transplacental transfer of Pf-IgG immune complexes (IC, IC containing MSP1 and MSP2 were measured in plasma of 242 mother-newborn pairs. Among newborns of IC-positive mothers (77/242, the proportion of cord samples with Pf IC increased with gestational age but was not associated with Pf IgM, suggesting that fetal B cells early in gestation had not been primed by IC. Finally, when cord mononuclear cells from 64 term newborns were cultured in vitro, only 11% (7/64 of supernatants had Pf IgM; whereas, 95% (61/64 contained secreted Pf IgG. These data suggest fetal B cells are capable of making Pf-specific IgM from early in the second trimester and undergo isotype switching to IgG towards term.

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

    Directory of Open Access Journals (Sweden)

    Amanda Maestre

    2010-07-01

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

  17. Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children

    DEFF Research Database (Denmark)

    Dodoo, D; Staalsoe, T; Giha, H

    2001-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand...... to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of PfEMP...... during the season. The prevalences of antibodies to both the conserved PfEMP1 peptide and the variant epitopes were greater than 50%, and the levels of immunoglobulin G (IgG) correlated with age. The levels of antibodies to both the conserved peptide and the variant epitopes were higher in protected than...

  18. Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum.

    Directory of Open Access Journals (Sweden)

    Franziska Hempel

    Full Text Available Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO(2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies.

  19. Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum

    Science.gov (United States)

    Hempel, Franziska; Lau, Julia; Klingl, Andreas; Maier, Uwe G.

    2011-01-01

    Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies. PMID:22164289

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

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

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

    Directory of Open Access Journals (Sweden)

    Ravindra P Singh

    2013-01-01

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

  2. Antibodies produced by mice immunized with recombinant vaccinia viruses expressing two different types of a major Theileria sergenti surface antigen (p32) react with the native surface antigen.

    Science.gov (United States)

    Takasima, Y; Xuan, X; Matsumoto, Y; Onuma, M; Otsuka, H

    1999-07-01

    A 32 kDa major surface antigen, p32, of Theileria sergenti at the piroplasm stage is the main target of the host immune response. The immunogenic property of the p32 varies in some strains among the population of Theileria sergenti in Japan where the Chitose type and the Ikeda type are the most common varieties. We have constructed vaccinia virus recombinants vv/p32C and vv/p32I which harbor the Chitose and Ikeda types of p32 gene, respectively. It was found that vv/p32C and vv/p32I produced type-specific p32 which did not cross react with the monoclonal antibodies (mAbs) against the other type of p32. When mice were immunized with vv/p32C and vv/p32I, antibodies against p32 were detectable 2 weeks after the immunization, and these antibodies reacted with the native surface antigen in purified T. sergenti merozoite.

  3. Cerebral cysticercosis in a cat : clinical communication

    Directory of Open Access Journals (Sweden)

    E.V. Schwan

    2002-07-01

    Full Text Available The metacestode of Taenia solium, Cysticercus cellulosae, was recovered from the brain of a cat showing central nervous clinical signs ante mortem. This is the first record of cerebral cysticercosis in a cat in South Africa.

  4. Allergen-specific IgE antibodies against antigenic components in cow milk and milk substitutes.

    Science.gov (United States)

    Gjesing, B; Osterballe, O; Schwartz, B; Wahn, U; Løwenstein, H

    1986-01-01

    Crossed radioimmunoelectrophoresis (CRIE) was used to study the presence of serum IgE against antigenic components of cow milk in 21 selected milk-allergic patients. The amount of each IgE specificity was estimated by a scoring system. The milk-allergic children had mainly IgE against alpha-lactalbumin, beta-lactoglobulin, albumin and immunoglobulin, the four major proteins of bovine whey, as well as IgE against three casein components. A serum pool from 1000 normal adults had IgE against the same whey protein, but in smaller amounts, and no IgE against the casein components. Eight cow milk-based formulae, commonly used for infant feeding, and goat milk were studied by the same method. It was found that six of the milk substitutes did not differ significantly from cow milk in antibody binding, but the two hydrolysed casein products, Nutramigen and Pregestimil, consisted of such small molecules that the rabbit antisera could not precipitate the hydrolysed proteins in the gels on the CRIE plates. It was therefore not possible to study their IgE binding, if any, by this method.

  5. Recurrent acute hemolytic transfusion reactions by antibodies against Doa antigens, not detected by cross-matching.

    Science.gov (United States)

    Baumgarten, Ruben; van Gelder, Warry; van Wintershoven, Joyce; Maaskant-Van Wijk, Petra A; Beckers, Erik A M

    2006-02-01

    An 81-year-old male patient suffered from recurrent acute hemolytic transfusion reactions after transfusion with phenotyped cross-match-negative red blood cells (RBCs). Extensive posttransfusion workup eventually revealed Dombrock (a) (Do(a)) antibodies. Because commercially available cell panels do not allow for identification of anti-Do(a) and owing to the lack of Do(a) typing serum samples, selection of matched units of RBCs is dependent on negative cross-match results. In this case, selection of Do(a-) units by cross-matching failed, indicating that serologic methods were not reliable. A polymerase chain reaction with sequence-specific priming assay was used to detect DOA and DOB alleles, which encode Do(a) and Do(b) antigens, respectively. The patient was confirmed to be DOB/DOB by DNA sequencing. Furthermore, the involved mismatched units in each of the three hemolytic episodes were shown to be Do(a+). In the presenting case, DNA typing appeared to be superior to serologic methods in selecting matched RBC units in the presence of anti-Do(a).

  6. Structural and Antigenic Definition of Hepatitis C Virus E2 Glycoprotein Epitopes Targeted by Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Giuseppe Sautto

    2013-01-01

    Full Text Available Hepatitis C virus (HCV is the major cause of chronic liver disease as well as the major indication for liver transplantation worldwide. Current standard of care is not completely effective, not administrable in grafted patients, and burdened by several side effects. This incomplete effectiveness is mainly due to the high propensity of the virus to continually mutate under the selective pressure exerted by the host immune response as well as currently administered antiviral drugs. The E2 envelope surface glycoprotein of HCV (HCV/E2 is the main target of the host humoral immune response and for this reason one of the major variable viral proteins. However, broadly cross-neutralizing monoclonal antibodies (mAbs directed against HCV/E2 represent a promising tool for the study of virus-host interplay as well as for the development of effective prophylactic and therapeutic approaches. In the last few years many anti-HCV/E2 mAbs have been evaluated in preclinical and clinical trials as possible candidate antivirals, particularly for administration in pre- and post-transplant settings. In this review we summarize the antigenic and structural characteristics of HCV/E2 determined through the use of anti-HCV/E2 mAbs, which, given the absence of a crystal structure of this glycoprotein, represent currently the best tool available.

  7. Antibodies to Bordetella pertussis antigens in maternal and cord blood pairs: a Thai cohort study

    Directory of Open Access Journals (Sweden)

    Nasamon Wanlapakorn

    2017-11-01

    Full Text Available Background Pertussis is a vaccine-preventable disease, yet an increasing incidence of pertussis occurs in many countries. Thailand has a long-standing pertussis vaccination policy, therefore most expectant mothers today had received vaccines as children. The resurgence of pertussis among Thai infants in recent years led us to examine the pre-existing antibodies to Bordetella pertussis antigens in a cohort of 90 pregnant women. Methods We evaluated the IgG to the Pertussis toxin (PT, filamentous hemagglutinin (FHA and pertactin (PRN in maternal and cord blood sera using commercial enzyme-linked immunosorbent assays (ELISA. Results When values of >10 IU/ml were accepted as potential protective concentrations, we found that the percentages of unprotected infants were 73.3%, 43.3% and 75.5% for anti-PT, anti-FHA and anti-PRN IgG, respectively. Discussion These results may explain the susceptibility for pertussis among newborn infants in Thailand and support the requirement for a pertussis booster vaccine during pregnancy, which may contribute to the passive seroprotection among newborns during the first months of life.

  8. Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody.

    Science.gov (United States)

    Malito, Enrico; Biancucci, Marco; Faleri, Agnese; Ferlenghi, Ilaria; Scarselli, Maria; Maruggi, Giulietta; Lo Surdo, Paola; Veggi, Daniele; Liguori, Alessia; Santini, Laura; Bertoldi, Isabella; Petracca, Roberto; Marchi, Sara; Romagnoli, Giacomo; Cartocci, Elena; Vercellino, Irene; Savino, Silvana; Spraggon, Glen; Norais, Nathalie; Pizza, Mariagrazia; Rappuoli, Rino; Masignani, Vega; Bottomley, Matthew James

    2014-12-02

    Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.

  9. Muscular cysticercosis: Case report and imaging findings

    Energy Technology Data Exchange (ETDEWEB)

    Olmo, Neide Regina Simoes; Fiorio, Ulysses Ferreira; Clemente, Marcel Andreazza, E-mail: neideolmo@yahoo.com.br [Clinica Mult Imagem, Santos, SP (Brazil); Bastos, Eder Amaral [Universidade Metropolitana de Santos (UNIMES), Santos, SP (Brazil); Mendes, Gustavo Gomes [AC Camargo Cancer Center, Sao Paulo, SP (Brazil)

    2016-11-15

    Cysticercosis is a parasitic disease caused by a worm of the Cestoda class. The most prevalent form affects the nervous system. This case report is from a 78-year old female patient evaluated at Clinica Mult Imagem, in the city of Santos, Brazil, who presented a form of the disease that differed from the classic neurocysticercosis, in this case muscular cysticercosis. This and other forms of manifestation justify further studies to ensure adequate recognition, diagnosis and treatment of this parasitic disease. (author)

  10. The many faces of cysticercosis. Pictorial review

    Energy Technology Data Exchange (ETDEWEB)

    Rahalkar, M.D.; Shetty, D.D.; Kelkar, A.B.; Kelkar, A.A.; Kinare, A.S.; Ambardekar, S.T

    2000-09-01

    Cysticercosis in humans results from infestation with the larval stage of the parasite Cysticercus cellulosae of the tapeworm Taenia solium. Man normally acts as a definitive host. However, man can occasionally be the intermediate host, when cysticercosis becomes clinically manifest. Larvae lodge in the target organs, the brain, eyes, spine and skeletal muscles, where their appearances are highly suggestive or specific. We present a spectrum of such images, as encountered in Western India. Rahalkar, M.D. (2000)

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

    Science.gov (United States)

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

    2017-12-01

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

  12. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D. (NIH)

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  13. Development of an Antigen-DNAzyme Based Probe for a Direct Antibody-Antigen Assay Using the Intrinsic DNAzyme Activity of a Daunomycin Aptamer

    Directory of Open Access Journals (Sweden)

    Noorsharmimi Omar

    2013-12-01

    Full Text Available G-Quadruplex (G-4 structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays.

  14. Evaluation of a polyclonal antibody based sandwich ELISA for the detection of faecal antigens in Schistosoma spindale infection in bovines.

    Science.gov (United States)

    Sreenivasa Murthy, G S; D'Souza, Placid E; Shrikrishna Isloor, K

    2013-04-01

    Schistosomosis is a common parasitic infection in animals prevalent in cattle in Asia and Africa, where it is estimated that at least 165 million animals are infected. Out of the 10 species reported to naturally infect cattle only Schistosoma nasale and Schistosoma spindale have received particular attention, because of their recognized veterinary significance. Although animal schistosomes may, under rare conditions favouring intensive transmission, act as important pathogens in endemic areas occur at a subclinical level, causing significant losses due to long term effects on animal growth and productivity. The detection of Schistosoma antigens in serum or stool could be more valuable in diagnosis, hence early treatment before irreparable damage. In this study, fresh adult worms of S. spindale were collected from the mesenteric blood vessels, whole worm antigen was prepared. These were immunized to rabbit and guinea pig to raise antibodies against S. spindale. Polyclonal antibodies of rabbit are further used as primary capture antibodies to coat ELISA plates. The capture of antibodies of guinea pig was conjugation with horse reddish peroxidase was used as secondary antibodies. Sandwich ELISA was performed to detect Schistosoma antigens in faecal samples collected from a total of 86 infected cattle and buffaloes. The working dilutions of capture antibody, detecting antibody and conjugate were found to be 1:32, 1:20 and 1:5,000 respectively by checker board titration method. The dilution of faecal supernatant antigens of S. spindale antibodies was 1:80. Out of 86 faecal samples, 77 samples were positive by Sandwich ELISA indicating 89.54 % infection. Where as in control samples none of the samples was positive. In mixed infection out of 20 samples positive for fasciola, amphistome and hydatid, Out of 20 samples 2 samples were positive indicating 10 % infection rate. The overall sensitivity of this test is 88.65 % and specificity was 90.90 %. It could be concluded

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...

  16. 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

    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...

  17. Effect of pronase on high-incidence blood group antigens and the prevalence of antibodies to pronase-treated erythrocytes.

    Science.gov (United States)

    Reid, M E; Greeen, C A; Hoffer, J; Øyen, R

    1996-01-01

    Pronase is a useful and relatively nonspecific protease that cleaves many red blood cell (RBC) membrane proteins that carry blood group antigens. Unexpected findings in tests using pronase-treated RBCs during the investigation of a patient's blood sample led us to test which high-incidence blood group antigens were sensitive and which were resistant to pronase treatment, and to determine the prevalence of antipronase in the serum of blood donors. Our results show that antigens in the Cromer and Lutheran blood group systems and the JMH antigen were sensitive to pronase treatment of RBCs. Antigens in the Dombrock blood group system and Sc1 were either sensitive to or markedly weakened by pronase treatment of RBCs. The following high-incidence antigens were resistant to treatment of RBCs with pronase: AnWj, Ata, Coa, Co3, Dib, EnaFR, Era, Fy3, Jk3, Jra, k, Kpb, Jsb, K14, Lan, Oka, Rh17, U, Vel, and Wrb. Over half of the serum samples from normal blood donors contained antibodies to pronase-treated RBCs. When testing human serum against pronase-treated RBCs, it is essential either to use an autocontrol or to perform the testing with an eluate.

  18. Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision

    Directory of Open Access Journals (Sweden)

    Patrick R. Adair

    2017-09-01

    Full Text Available Human regulatory CD4+ T cells (Tregs are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs, and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

  19. Diverse monoclonal antibodies against the CA 19-9 antigen show variation in binding specificity with consequences for clinical interpretation.

    Science.gov (United States)

    Partyka, Katie; Maupin, Kevin A; Brand, Randall E; Haab, Brian B

    2012-07-01

    The CA 19-9 antigen is currently the best individual marker for the detection of pancreatic cancer. In order to optimize the CA 19-9 assay and to develop approaches to further improve cancer detection, it is important to understand the specificity differences between CA 19-9 antibodies and the consequential affect on biomarker performance. Antibody arrays enabled multiplexed comparisons between five different CA 19-9 antibodies used in the analysis of plasma samples from pancreatic cancer patients and controls. Major differences were observed between antibodies in their detection of particular patient samples. Glycan array analysis revealed that certain antibodies were highly specific for the canonical CA 19-9 epitope, sialyl-Lewis A, while others bound sialyl-Lewis A in addition to a related structure called sialyl-Lewis C and modification with Nue5Gc. In a much larger patient cohort, we confirmed the binding of sialyl-Lewis C glycan by one of the antibodies and showed that the broader specificity led to the detection of an increased number of cancer patients without increasing detection of pancreatitis patient samples. This work demonstrates that variation between antibody specificity for cancer-associated glycans can have significant implications for biomarker performance and highlights the value of characterizing and detecting the range of glycan structures that are elevated in cancer. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Antibody response patterns to Bordetella pertussis antigens in vaccinated (primed) and unvaccinated (unprimed) young children with pertussis.

    Science.gov (United States)

    Cherry, James D; Heininger, Ulrich; Richards, David M; Storsaeter, Jann; Gustafsson, Lennart; Ljungman, Margaretha; Hallander, Hans O

    2010-05-01

    In a previous study, it was found that the antibody response to a nonvaccine pertussis antigen in children who were vaccine failures was reduced compared with the response in nonvaccinated children who had pertussis. In two acellular pertussis vaccine efficacy trials in Sweden, we studied the convalescent-phase enzyme-linked immunosorbent assay (ELISA) geometric mean values (GMVs) in response to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM 2/3) in vaccine failures and controls with pertussis. In Germany, the antibody responses to Bordetella pertussis antigens PT, FHA, PRN, and FIM-2 were analyzed by ELISA according to time of serum collection after onset of illness in children with pertussis who were vaccine failures or who were previously unvaccinated. Antibody values were also compared by severity of clinical illness. In Sweden, infants who had received a PT toxoid vaccine and who were vaccine failures had a blunted response to the nonvaccine antigen FHA compared with the response in children who had received a PT/FHA vaccine. Similarly, infants who had pertussis and who had received a PT/FHA vaccine had a blunted response to the nonvaccine antigens PRN and FIM 2/3 compared with the response in children who were vaccine failures and who had received a PT, FHA, PRN, and FIM 2/3 vaccine. In Germany, in sera collected from 0 to 15 days after pertussis illness onset, the GMVs for all 4 antigens (PT, FHA, PRN, and FIM-2) were significantly lower in an unvaccinated group than in children who were diphtheria-tetanus-acellular pertussis (DTaP) vaccine failures. In the unvaccinated group, the GMV of the PT antibody rose rapidly over time so that it was similar to that of the DTaP vaccine recipients at the 16- to 30-day period. In contrast, the antibody responses to FHA, PRN, and FIM-2 at all time periods were lower in the diphtheria-tetanus vaccine (DT) recipients than in the DTaP vaccine failures. In both Sweden and Germany

  1. Seroprevalence of antibodies against fHbp and NadA, two potential vaccine antigens for Neisseria meningitidis.

    Science.gov (United States)

    Jacobsson, Susanne; Mölling, Paula; Olcén, Per

    2009-09-25

    The IgG antibody levels directed against fHbp and NadA, two potential vaccine antigens for Neisseria meningitidis, were examined in order to investigate the extent of natural immunisation against these antigens in different age groups. As a comparison, the IgG antibody levels against Haemophilus influenzae type b were examined. In the two youngest age groups, below 10 years of age, relatively low levels of both anti-fHbp and anti-NadA were measured. A 15-fold higher geometric mean concentration of anti-fHbp was noted in the age group 20-29 years as compared to the age group 15-19 years. The peak concentration was found at 30-39 years, followed by decreased levels with age. Anti-NadA showed a certain increase up to 9 years followed by an even increase up to 40-49 years.

  2. Comparison of two indirect ELISA coating antigens for the detection of dairy cow antibodies against Pasteurella multocida.

    Science.gov (United States)

    Tankaew, Pallop; Srisawat, Wanwisa; Singhla, Tawatchai; Tragoolpua, Khajornsak; Kataoka, Yasushi; Sawada, Takuo; Sthitmatee, Nattawooti

    2018-02-01

    The ELISA is recognized as an efficient diagnostic tool for antibody detection, but there is no standard ELISA assay for detection of antibodies against hemorrhagic septicemia (HS) in cattle. The present study reports on an indirect ELISA assay for antibody detection of HS in dairy cows, and evaluates the sensitivity (Se) and specificity (Sp) of the method using a Bayesian approach. An indirect ELISA was developed with two types of heat extract antigens, Pasteurella multocida strains P-1256 and M-1404, as coating antigens. A checkerboard titration was employed using dairy cow sera immunized with P. multocida bacterin and colostrum-deprived calf sera. The concentrations of heat extract antigen (160μg/mL), sample serum (1:100) and goat anti-bovine immunoglobulin G labeled with horseradish peroxidase (1:2000) were optimal for the assay. The cut-off values were 0.147 and 0.128 for P-1256 and M-1404 coating antigens, and there were no differences in the results of tests with positive and negative sera (p<0.05). The characteristics of three diagnostic tests were evaluated using a one-population Bayesian model, assuming conditional dependence between two types of coating antigen-based ELISAs and indirect hemagglutination assay (IHA). A total of 415 sera samples from dairy cows without HS vaccination and no history of disease were tested. The Se and Sp of the P-1256 and M-1404 ELISAs were higher than those of the IHA. The Se and Sp of the P-1256 ELISA were 90.3% and 90.1%, while the Se and Sp of the M-1404 ELISA were 92.1% and 71.9%. The median values of Se and Sp from the IHA were 36.0% and 58.2%. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Level and specificity of antibodies evoked by crude and purified antigens of poliovirus I and echovirus 7.

    Science.gov (United States)

    Frommhagen, L H

    1965-11-01

    Preparations of poliovirus I and echovirus 7, purified by density gradient centrifugation, liquid-phase partition, and anion exchange (diethylaminoethyl) chromatography, have been shown to evoke high antibody levels of substantial specificity in the complement-fixation assay. Certain practical aspects of the three purification methods are discussed. These results argue for the use of purified viral antigens, particularly in view of the simplicity of the purification methods now available.

  4. Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

    OpenAIRE

    Marusic, Carla; Vitale, Allessandro; Pedrazzini, Emanuela; Donini, Marcello; Frigerio, Lorenzo; Bock, Ralph; Dix, Philip J.; McCabe, Matthew S.; Bellucci, Michele; Benvenuto, Eugenio

    2009-01-01

    The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10?years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to ...

  5. Effect of oral antigen and antibody exposure at birth on subsequent immune status. A study in neonatal pigs.

    Science.gov (United States)

    Haverson, Karin; Corfield, Gaynor; Jones, Philip H; Kenny, Martin; Fowler, Jenny; Bailey, Mick; Stokes, Christopher R; Miller, Bevis G

    2009-01-01

    The purpose of this work was to investigate the effects of early low-level exposure to either antigen or antibody alone on subsequent immune responses in entirely immunologically naïve animals. This is impossible in species with a permeable placenta such as rodents or humans, where both antigen and antibody can be transferred in utero. It is, however, possible in pigs, due to the impermeable placenta of the sow. Thus, neonatal piglets were used for this study. Newborn piglets were exposed to ovalbumin (OVA) at dosages similar to those used in rodents to sensitise, as well as to serum containing anti-OVA antibodies. Both single low doses of OVA (10 and 1,000 mg per animal) induced classical oral tolerance following a systemic challenge: both doses reduced specific systemic IgG responses and tertiary in vitro recall proliferative responses by splenocytes and especially by mesenteric lymph node (MLN) cells. Additionally, dietary challenge had phenotypic effects on helper T cells in MLN, which could be reversed by OVA at birth. In contrast, giving antibody as serum collected from hyperimmune or orally tolerant pigs had no functional effects. Overall, our data support the hypothesis that contrary to previous work in rodents, very early exposure of neonatal pigs to a single small dose of antigen can reduce subsequent immune responses. This may have implications for human health. However, although these data point to a reducing/regulatory effect of low doses of antigen in very young animals, they cannot be extrapolated directly to allergy. (c) 2009 S. Karger AG, Basel.

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

    Science.gov (United States)

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

    2016-08-01

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

  7. Statistical prediction of immunity to placental malaria based on multi-assay antibody data for malarial antigens

    DEFF Research Database (Denmark)

    Siriwardhana, Chathura; Fang, Rui; Salanti, Ali

    2017-01-01

    Background Plasmodium falciparum infections are especially severe in pregnant women because infected erythrocytes (IE) express VAR2CSA, a ligand that binds to placental trophoblasts, causing IE to accumulate in the placenta. Resulting inflammation and pathology increases a woman’s risk of anemia......, miscarriage, premature deliveries, and having low birthweight (LBW) babies. Antibodies (Ab) to VAR2CSA reduce placental parasitaemia and improve pregnancy outcomes. Currently, no single assay is able to predict if a woman has adequate immunity to prevent placental malaria (PM). This study measured Ab levels...... levels to the majority of malaria antigens. Individually though, these malarial antigens did not achieve reasonably high performances in terms of predicting the PM status. Utilizing multiple antigens in predictive models considerably improved discrimination power compared to individual assays. Among...

  8. Reactivity of commercially available monoclonal antibodies to human CD antigens with peripheral blood leucocytes of dromedary camels (Camelus dromedarius

    Directory of Open Access Journals (Sweden)

    Jamal Hussen

    2017-05-01

    Full Text Available Monoclonal antibodies (mAbs to cell surface molecules have been proven as a key tool for phenotypic and functional characterization of the cellular immune response. One of the major difficulties in studying camel cellular immunity consists in the lack of mAbs that dtect their leukocyte differentiation antigens. In the present study two-parameter flow cytometry was used to screen existing commercially available mAbs to human leukocyte antigens and major histocompatibility molecules (MHC for their reactivity with camel leukocytes. The comparison of patterns of reactivity obtained after labelling human and camel leukocytes have shown that mAbs specific to human cluster of differentiation (CD 18, CD11a, CD11b and CD14 are predicted to be cross-reactive with homologous camel antigens.

  9. Intra-Blood-Brain Barrier Synthesis of Human Immunodeficiency Virus Antigen and Antibody in Humans and Chimpanzees

    Science.gov (United States)

    Goudsmit, Jaap; Epstein, Leon G.; Paul, Deborah A.; van der Helm, Hayo J.; Dawson, George J.; Asher, David M.; Yanagihara, Richard; Wolff, Axel V.; Gibbs, Clarence J.; Carleton Gajdusek, D.

    1987-06-01

    The presence of human immunodeficiency virus (HIV) antigens in cerebrospinal fluid (CSF) was associated with progressive encephalopathy in adult and pediatric patients with acquired immunodeficiency syndrome (AIDS). HIV antigen was detected in CSF from 6 of 7 AIDS patients with progressive encephalopathy. By contrast, HIV antigen, whether free or complexed, was detected in CSF from only 1 of 18 HIV antibody seropositive patients without progressive encephalopathy and from 0 of 8 experimentally infected chimpanzees without clinical signs. Intra-blood-brain barrier synthesis of HIV-specific antibody was demonstrated in the majority of patients with AIDS (9/12) or at risk for AIDS (8/13) as well as in the experimentally infected chimpanzees, indicating HIV-specific B-cell reactivity in the brain without apparent neurological signs. In 6 of 11 patients with HIV infection, antibodies synthesized in the central nervous system were directed against HIV envelope proteins. Active viral expression appears to be necessary for both the immunodeficiency and progressive encephalopathy associated with HIV infection.

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

    Directory of Open Access Journals (Sweden)

    Victor Greiff

    2017-05-01

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

  11. Cross-reactive Legionella antigens and the antibody response during infection

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Shand, G; Pearlman, E

    1991-01-01

    In order to define cross-reactive Legionella antigens suitable for diagnostic purposes, we investigated sonicate antigens from two Legionella species, including two serogroups of L. pneumophila. The antigens were reacted with heterologous and homologous rabbit antisera in Western blot. Sera from...

  12. Immunological Detection of Newcastle Disease Viral Antigen in the Naturally Infected Chickens by Monoclonal Antibodies against Fusion-2 Protein

    Directory of Open Access Journals (Sweden)

    Nyoman Mantik Astawa

    2014-08-01

    Full Text Available Monoclonal antibodies (mAbs against Fusion (F2 protein of Newcastle disease virus (NDV wereproduced for the detection of the viral antigen in infected chickens. Cells derived from spleen of Balb/c miceimmunized with the virus were fused with mouse myeloma cells to generate hybridomas capable ofproducing mAbs against the virus. The hybridomas were screened by enzyme-linked immunosorbent assay(ELISA for anti-NDV specific mAbs using crude viral antigen (allantoic fluid of NDV-infected fertile eggsand normal uninfected allantoic fluid of fertile eggs as negative control. The NDV proteins reactive withmAbs were then determined by Western Blotting using purified NDV as antigen. The mAbs reactive withF2 (12.5 KDa protein of NDV were then used for the detection of NDV antigen in both the allantoic fluidof NDV- infected chicken embryos and in organs of naturally infected chickens. The results showed that 2out of 5 mAbs produced were against F2 protein of NDV. By indirect ELISA, the mAbs were able to detectthe viral antigen in allantoic fluid of NDV infected fertile chicken eggs at the titre as low as 2-2 to 2-4 HAunits per 0.1 mL. NDV–antigen was also detected by immunoperoxidase staining in paraffin-embeddedtissues of NDV-infected chickens but not in normal uninfected chickens. The most prominent infection wasdetected in the gastrointestinal tract and the lung. The NDV antigen was also detected in other organssuch as the brain, spleen, and several other tissues. It is evident that mAbs produced against F2 proteinof NDV were applicable for use in the detection of NDV antigen in infected chickens.

  13. Enzyme linked immunosorbent assay for the diagnosis of cerebral cysticercosis in Reunion island: comparison with computerized tomography scan

    Energy Technology Data Exchange (ETDEWEB)

    Michault, A.; Coubes, P.; Laporte, J.P.; Bouillan-Linet, E.; Leroy, D.

    1988-03-01

    An immunoenzymologic (Elisa) serodiagnosis of cysticercosis is evaluated in 75 encephalic cysticercotic patients whose diagnosis of the disease and its progression is assessed by tomodensitometry. A Taenia solium antigen is used. Only Ig G are investigated. The sensibility of serodiagnosis is 85 % and specificity 87 % when there is a progression of the disease; no difference is noticed in the patients without any progression of the disease and in control normal subjects. This serodiagnosis of cysticercosis appears of value for the evaluation of the activity of the disease.

  14. Taenia solium cysticercosis in Bali, Indonesia: serology and mtDNA analysis.

    Science.gov (United States)

    Sudewi, A A R; Wandra, T; Artha, A; Nkouawa, A; Ito, A

    2008-01-01

    An active Taenia solium cysticercosis case in Bali, Indonesia, was followed-up by serology and computed tomography. Serology using semi-purified glycoprotein and recombinant antigens showed a drastic drop in titers after calcification of the cysts. Three paraffin-embedded cysts, prepared for histopathological examination, from three other patients were used for mtDNA analysis. The sequences of cox1 gene from T. solium cysticerci from Bali differed from those in Papua and other Asian countries.

  15. Recognition of antigens of three different stages of the Trichinella spiralis by antibodies from pigs infected with T. spiralis.

    Science.gov (United States)

    Bien, Justyna; Cabaj, Wladyslaw; Moskwa, Bozena

    2013-06-01

    Infective muscle larvae (ML), adults (Ad) and new born larvae (NBL) of Trichinella spiralis express many immunogenic proteins which can elicit a host protective response, and may be useful in the diagnosis of Trichinella infected humans and animals. The present study was carried out to identify T. spiralis antigens recognized by antibodies from pigs infected with T. spiralis. To that end, the crude extracts of ML, Ad, NBL and ML excretory-secretory (E-S) and Ad E-S proteins were analyzed by sodium dodecyl sulfate polycrystalline gel electrophoresis (SDS-PAGE). To identify antigens of T. spiralis that are recognized by host antibodies, crude extracts and E-S proteins were subjected to immunoblot with antisera derived from pigs experimentally infected with 200 or 20,000 T. spiralis ML. Searching for T. spiralis antigens with diagnostic potential, immunoblots showed that all T. spiralis antisera, regardless of the infective dose, recognized common proteins in each examined life stage with molecular weights around 20-27 kDa, 41 kDa and 197-105 kDa. Interestingly, all the common proteins were detected by T. spiralis sera throughout the infection, from 5 days post infection (dpi) to 60 dpi. These results extend our knowledge of specific antigenic components of T. spiralis. The finding of common components among all T. spiralis life stages may be useful in the preparation of parasite antigens for diagnostic use, as these antigens are relevant regardless of infection phase. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-11-01

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

  17. Murine CR1/2 targeted antigenized single-chain antibody fragments induce transient low affinity antibodies and negatively influence an ongoing immune response.

    Science.gov (United States)

    Prechl, József; Molnár, Eszter; Szekeres, Zsuzsanna; Isaák, Andrea; Papp, Krisztián; Balogh, Péter; Erdei, Anna

    2007-01-01

    We have generated a single-chain antibody which recognizes murine CR1/2 and carries a genetically fused influenza hemagglutinin derived peptide. Theoretically such a construct is able to crosslink the B cell antigen receptor and CR1/2 on peptide specific B cells. The construct was able to reach its CR1/2 positive target cells, yet intraperitoneal delivery of the construct elicited an IgM response only slightly exceeding that induced by the free peptide. Providing T cell help by the injection of peptide specific lymphocytes did not alter the response in essence, that is anti-peptide IgG was not detectable even after booster immunizations. When used as a booster vaccine following injection of the peptide in adjuvant, the construct even inhibited the development of IgG1 and IgG3 anti-peptide antibodies. These data indicate that although targeting of antigen to CR1/2 on B cells can enhance transient proliferation or differentiation of antigen specific B cells it cannot induce strong, longlasting humoral immune responses. Furthermore, CR1/2 targeting constructs may negatively influence an ongoing immune reaction.

  18. Refinement of immunizing antigens to produce functional blocking antibodies against the AniA nitrite reductase of Neisseria gonorrhoeae.

    Science.gov (United States)

    Shewell, Lucy K; Jen, Freda E-C; Jennings, Michael P

    2017-01-01

    The emergence of multi-drug resistant Neisseria gonorrhoeae has generated an urgent need for novel therapies or a vaccine to prevent gonococcal disease. In this study we investigate the potential of targeting the surface exposed nitrite reductase, AniA, to block activity by producing functional blocking antibodies. AniA activity is essential for anaerobic growth and biofilm formation of N. gonorrhoeae and functional blocking antibodies may prevent colonisation and disease. Seven peptides covering regions adjacent to the active site were designed based on the AniA structure. Six of the seven peptide conjugates generated immune responses. Peptide 7, GALGQLKVEGAEN, was able to elicit antibodies capable of blocking AniA activity. Antiserum raised against the peptide 7 conjugate detected AniA in 20 N. gonorrhoeae clinical isolates. Recombinant AniA protein antigens were also assessed in this study and generated high-titre, functional blocking antibody responses. Peptide 7 conjugates or truncated recombinant AniA antigens have potential for inclusion in a vaccine against N. gonorrhoeae.

  19. Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

    Directory of Open Access Journals (Sweden)

    Myriam Arévalo-Herrera

    Full Text Available Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

  20. Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

    Science.gov (United States)

    Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

    2017-05-01

    Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Antigen-binding properties of monoclonal antibodies reactive with EBNA1 and use in immunoaffinity chromatography.

    Directory of Open Access Journals (Sweden)

    Sarah J Duellman

    Full Text Available Epstein-Barr virus (EBV nuclear antigen 1 (EBNA1 was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1 and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6 fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.

  2. Antigen-binding properties of monoclonal antibodies reactive with EBNA1 and use in immunoaffinity chromatography.

    Science.gov (United States)

    Duellman, Sarah J; Burgess, Richard R

    2009-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1)) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.

  3. 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

    with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment......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...... purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody...

  4. Taenia solium Cysticercosis Hotspots Surrounding Tapeworm Carriers: Clustering on Human Seroprevalence but Not on Seizures

    Science.gov (United States)

    Lescano, Andres G.; Garcia, Hector H.; Gilman, Robert H.; Gavidia, Cesar M.; Tsang, Victor C. W.; Rodriguez, Silvia; Moulton, Lawrence H.; Villaran, Manuel V.; Montano, Silvia M.; Gonzalez, Armando E.

    2009-01-01

    Background Neurocysticercosis accounts for 30%–50% of all late-onset epilepsy in endemic countries. We assessed the clustering patterns of Taenia solium human cysticercosis seropositivity and seizures around tapeworm carriers in seven rural communities in Peru. Methodology The presence of T. solium–specific antibodies was defined as one or more positive bands in the enzyme-linked immunoelectrotransfer blot (EITB). Neurocysticercosis-related seizures cases were diagnosed clinically and had positive neuroimaging or EITB. Principal Findings Eleven tapeworm carriers were identified by stool microscopy. The seroprevalence of human cysticercosis was 24% (196/803). Seroprevalence was 21% >50 m from a carrier and increased to 32% at 1–50 m (p = 0.047), and from that distance seroprevalence had another significant increase to 64% at the homes of carriers (p = 0.004). Seizure prevalence was 3.0% (25/837) but there were no differences between any pair of distance ranges (p = 0.629, Wald test 2 degrees of freedom). Conclusion/Significance We observed a significant human cysticercosis seroprevalence gradient surrounding current tapeworm carriers, although cysticercosis-related seizures did not cluster around carriers. Due to differences in the timing of the two outcomes, seroprevalence may reflect recent T. solium exposure more accurately than seizure frequency. PMID:19172178

  5. Epidemiological investigation of Taenia solium taeniasis and cysticercosis in a rural village of Michoacan state, Mexico.

    Science.gov (United States)

    Sarti, E; Schantz, P M; Plancarte, A; Wilson, M; Gutierrez, O I; Aguilera, J; Roberts, J; Flisser, A

    1994-01-01

    We performed a survey for taeniasis and cysticercosis among persons living in a Mexican village where Taenia solium infection in pigs was known to be enzootic. A standardized questionnaire was administered in all 577 households to obtain medical histories and information on demographic and environmental factors and on risk factors associated with transmission of infection. Serum and/or stool specimens were obtained from 1005 volunteers and examined for cysticercosis antibodies and intestinal parasites. Faecal examination of 828 participants revealed infection by Taenia sp. in 2 (0.2%). Three additional cases of taeniasis were detected in individuals who evacuated proglottids after treatment with praziquantel. Of 1005 human serum specimens, 49 (4.9%) were positive in the cysticercosis immunoblot assay. Seropositivity increased with age and reached a peak in subjects aged 46-55 years (P < 0.05). A history of seizures was significantly associated with seropositivity (P < 0.05); approximately 25% of persons with such histories were seropositive. Histories of headache, dizziness, trembling, blurred vision, and vomiting were also significantly associated with positive immunoblot assays. This study has demonstrated previously undiagnosed morbidity associated with T. solium neurocysticercosis and identified community behavioural and environmental practices that must be modified to prevent continued transmission of cysticercosis and taeniasis.

  6. Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

    Directory of Open Access Journals (Sweden)

    Maria Domina

    Full Text Available We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb epitopes. For this purpose, we used a novel mAb (designated 31E10/E7 directed against Neisserial Heparin-Binding Antigen (NHBA, a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128 in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.

  7. A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle.

    Science.gov (United States)

    Magnarelli, Louis A; Bushmich, Sandra L; Sherman, Bruce A; Fikrig, Erol

    2004-08-01

    Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.

  8. De Novo Donor-Specific Anti-Human Leukocyte Antigen Antibody Detection in Long-Term Adult Liver Transplantation.

    Science.gov (United States)

    San Segundo, D; Alonso, C; Ruiz, P; Roman, I; Arias-Loste, M T; Cuadrado, A; Puente, A; Casafont, F; López-Hoyos, M; Crespo, J; Fábrega, E

    2016-11-01

    Information about the consequences of de novo donor-specific anti-human leukocyte antigen (DSA) antibody development in the long term after adult liver transplantation (LT) is scarce. We conducted a cross-sectional study in LT patients with a follow-up of at least 6 years. A total of 28 adult LT patients were included, with a median follow-up of 77 months (range, 63 to 96) and without preformed anti- human leukocyte antigen (HLA) antibodies prior to LT. The anti-HLA identification was performed with LABScreen Single Antigen, whereas the ability to fix the complement was demonstrated with C1q test (One Lambda). In both assays, a value >3.500 mean fluorescence intensity (MFI) was considered positive. The anti-HLA antibody specificities were compared with donor HLA antigens to confirm them as DSA. Hepatic fibrosis was assessed by transient elastography. In 5 patients (17.8%), de novo DSA were detected, all them against DQ locus. In all of these cases (100%) the complement fixation was confirmed by C1q binding. The grade of hepatic fibrosis in de novo DSA patients was significantly higher compared with No-DSA patients (13.2 ± 9.2 KPa vs 7.3 ± 3.7 KPa; P = .02). It is noteworthy that in both groups of patients the levels of liver function tests (LFT) at the time of the study were normal or near the normal range with no difference between patients with or without de novo DSA. Our preliminary results are consistent with those previously demonstrated in pediatric LT, where de novo DSA production and humoral response could contribute to the liver fibrosis observed in the long term after LT in pediatric patients with normal or near-normal LFT. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. THE DNA-BINDING PROTEIN PUL57 OF HUMAN CYTOMEGALOVIRUS IS A MAJOR TARGET ANTIGEN FOR THE IMMUNOGLOBULIN-M ANTIBODY-RESPONSE DURING ACUTE INFECTION

    NARCIS (Netherlands)

    VORNHAGEN, R; HINDERER, W; SONNEBORN, HH; BEIN, G; MATTER, L; THE, TH; JAHN, G; PLACHTER, B

    A small polypeptide from pUL57 of human cytomegalovirus was identified as a major target for the immunoglobulin M antibody response. This antigen seems to be superior to antigenic fragments from pp150 and p52 in the identification of sera from acutely infected patients. It may therefore represent an

  10. REFINEMENT OF AN INDIRECT IMMUNOTOXIN ASSAY OF MONOCLONAL-ANTIBODIES RECOGNIZING THE HUMAN SMALL-CELL LUNG-CANCER CLUSTER-2 ANTIGEN

    NARCIS (Netherlands)

    DERBYSHIRE, EJ; DELEIJ, L; WAWRZYNCZAK, EJ

    Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line

  11. Comparison of a rapid immunoassay for antibodies to the C6 antigen with conventional tests for antibodies to Borrelia burgdorferi in dogs in Europe.

    Science.gov (United States)

    Gerber, B; Haug, K; Eichenberger, S; Reusch, C E; Wittenbrink, M M

    2009-11-14

    A commercial immunoassay for antibodies to the C6 antigen of Borrelia burgdorferi was evaluated against an IgG in-house ELISA in combination with a Western blot assay to examine 104 samples of serum from 53 healthy Bernese mountain dogs, which were suspected to have a breed predisposition to Lyme borreliosis, and 55 samples from 30 healthy large-breed longhair dogs. The two test methods correlated in 125 (79 per cent) of the samples with an agreement of kappa=0.571 (Pdogs (k=0.681) than with the samples from the control dogs (k=0.347).

  12. Review of Cancer Immunotherapy: Application of Chimeric Antigen Receptor T Cells and Programmed Death 1/Programmed Death-ligand 1 Antibodies

    OpenAIRE

    Tengfei Zhang; Ling Cao; Zhen Zhang; Dongli Yue; Yu Ping; Hong Li; Lan Huang; Yi Zhang

    2015-01-01

    Cancer immunotherapy strategies based on chimeric antigen receptor (CAR) transduced T cells or antibodies against immune checkpoints, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), achieved significant successes from bench to clinic in the past 2 years. CARs are artificial engineered receptors that can specifically target tumor cell surface antigen, activate T cell and further enhance T cell function, independent of major histocompatibility complex. CAR T ...

  13. The kinetics of antibody binding to Plasmodium falciparum VAR2CSA PfEMP1 antigen and modelling of PfEMP1 antigen packing on the membrane knobs

    DEFF Research Database (Denmark)

    Joergensen, Lars M; Salanti, Ali; Dobrilovic, Tina

    2010-01-01

    on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target. METHODS: A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno...... molecules. CONCLUSIONS: High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18nm sweep radius of an antibody. The large size...

  14. Novel chemiluminescent Western blot blocking and antibody incubation solution for enhanced antibody-antigen interaction and increased specificity.

    Science.gov (United States)

    Schwartz, Kimberly; Bochkariov, Dmitry

    2017-10-01

    Western blotting is a ubiquitous tool used in protein and molecular biology research, providing information about the presence, size, relative abundance, and state of a protein in a mixture. First, the proteins in a sample are separated by size using SDS-PAGE then transferred onto a membrane for detection with a set of primary and secondary antibodies. High-quality Western data requires high signal-to-noise ratios, which depend upon reduction of nonspecific antibody interactions. Blocking is a critical step in the Western blot method as it prevents the antibodies from binding nonspecifically to the membrane and irrelevant proteins. A solution of nonfat dry milk (NFDM) in physiological buffer is commonly used for this purpose, but does not perform well with every type of antibody and is not optimal for low-abundance proteins. We present a novel blocking solution for chemiluminescent Western blots, AdvanBlock™-chemi, which outperforms NFDM in experiments with 20 unique antibodies by increasing signal-to-noise ratios and minimizing nonspecific binding. This solution enhances protein detection by Western blot and provides consistent results for detection of low abundant and modified proteins. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Geographical and temporal conservation of antibody recognition of Plasmodium falciparum variant surface antigens

    DEFF Research Database (Denmark)

    Nielsen, Morten A; Vestergaard, Lasse S; Lusingu, John

    2004-01-01

    The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunity...... (VSASM) that were better recognized by plasma IgG than VSA expressed by other parasites, but importantly, VSASM-type antigens also appeared to show substantial antigenic homogeneity. Our finding that the repertoire of immunologically distinct VSA in general, and in particular that of VSASM...

  16. Kai 1 and Kai 2: Characterization of these dog erythrocyte antigens by monoclonal antibodies

    National Research Council Canada - National Science Library

    Jae Ho Lee; Urs Giger; Hee Young Kim

    2017-01-01

    Dog Erythrocyte Antigens (DEA) have thus far been found by sensitizing dogs with canine allogeneic blood and are clinically important regarding blood transfusion incompatibilities, but remain poorly defined...

  17. Reduction of the diagnostic window with a new combined p24 antigen and human immunodeficiency virus antibody screening assay.

    Science.gov (United States)

    Gürtler, L; Mühlbacher, A; Michl, U; Hofmann, H; Paggi, G G; Bossi, V; Thorstensson, R; G-Villaescusa, R; Eiras, A; Hernandez, J M; Melchior, W; Donie, F; Weber, B

    1998-11-01

    In order to reduce the window phase between time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new fourth generation screening assays which permit a simultaneous detection of HIV antigen and antibody have been developed. In a multicenter study, a new automated fourth generation assay, Enzymun-Test HIV Combi (Boehringer Mannheim GmbH) was compared to third generation assay, p24 antigen tests and Western blot. A total of 37 seroconversion panels, samples of the early infection (n = 42), HIV-1 antibody positive sera, including subtypes A E, and O (n = 1118), HIV-2 positive samples (n = 252) and cell culture supernatants infected with different HIV-1 subtypes and HIV-2 (n = 50), blood donors (n = 6649), hospitalized patients (n = 475), HIV neg. sera with indeterminate Western blot (n = 32), potentially cross reactive serum samples (n = 435) and HIV negative specimens from Cameroon (n = 68) were tested. A total of 16 of 29 seroconversions were detected on average 8.5 days earlier with Enzymun-Test HIV Combi than HIV-1/HIV-2 3rd generation EIA (Abbott Laboratories). Overall, in the 29 panels investigated comparatively with the two assays, the mean time delay between Enzymun-Test HIV Combi and HIV-1/HIV-2 3rd generation EIA was 4.7 days. HIV antigen was detected in three out of 35 seroconversions one bleed earlier with HIV-1 Ag Monoclonal than with Enzymun-Test HIV Combi. Enzymun-Test HIV Combi showed a sensitivity of 100% for HIV antibody detection for HIV-1 group M and O and HIV-2 positive specimens. While p24 antigen of different HIV-1 subtypes was detected with Enzymun-Test HIV Combi in all the 49 cell culture supernatants, HIV Ag was not detected in an HIV-2 virus lysate. A total of 66 false positive results out of 7659 HIV negative samples were obtained with the Enzymun-Test HIV Combi. The specificity for unselected blood donors was 99.6%. The Enzymun-Test HIV Combi permits an earlier diagnosis of HIV infection than third generation

  18. Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Aβ antibodies

    Directory of Open Access Journals (Sweden)

    Ugen Kenneth E

    2007-03-01

    Full Text Available Abstract Background Prior work from our group found that acid dissociation (pH 2.5 incubation of serum from APP transgenic mice vaccinated against Aβ increased the apparent anti-Aβ titers, suggesting antibody masking by antigen in the ELISA assay. Subsequently, we found that pH 2.5 incubation of serum from unvaccinated non-transgenic mice showed antibody binding to Aβ1–42, but no increase when other proteins, including shorter Aβ peptides, coated the ELISA plate. To investigate further the effects of low pH incubation on apparent anti-Aβ1–42 signals, we examined normal sera from nonTg unvaccinated mice, nonTg mice vaccinated with Aβ peptide (to produce authentic anti-Aβ antibodies or a monoclonal antibody against Aβ (6E10 using competitive-inhibition ELISA and Aβ epitope mapping assays. In addition, we examined use of a less stringent low pH procedure at pH 3.5, to ascertain if it had the same effects as the pH 2.5 procedure. Results We believe there are three distinct effects of pH 2.5 incubation.; A an artifactual increase in binding to full length Aβ by mouse immunoglobulin which has low affinity for Aβ, B an inactivation of anti-Aβ antibodies that is time dependent and C unmasking of high affinity anti-Aβ antibodies when high levels of circulating Aβ is present in APP transgenic mice. All three reactions can interact to produce the final ELISA signal. Incubation of sera from unvaccinated nonTg mice at pH 2.5 enhanced ELISA signals by process A. Conversely, pH 2.5 incubation of sera from vaccinated nonTg mice with caused a time dependent reduction of antibody signal by process B (overcoming the increase caused by A. The artifactual anti-Aβ ELISA signal enhanced by pH 2.5 incubation of normal mouse sera could not be effectively competed by low to moderate concentrations of Aβ, nor bind to shorter Aβ peptides in a manner similar to authentic anti-Aβ antibodies. Incubation of mouse sera at pH 3.5 caused neither an apparent

  19. Enhanced antigen detection in immunohistochemical staining using a 'digitized' chimeric antibody.

    Science.gov (United States)

    Eng, Hui-Yan; Wang, Cheng-I; Xue, Yuezhen; Lee, Chia-Yin; Zulkifli, Sarah Binte; Chiam, Poh-Cheang; Ghadessy, Farid J; Lane, David P

    2016-01-01

    The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. INFLUENCE OF AGING ON ANTIBODY-FORMATION INVIVO AFTER IMMUNIZATION WITH THE PRIMARY T-CELL DEPENDENT ANTIGEN HELIX-POMATIA HEMOCYANIN

    NARCIS (Netherlands)

    DEGREEF, GE; KALLENBERG, CGM; VANSTAALDUINEN, GJ; REMARQUE, EJ; TJANDRA, YI; HIJMANS, W

    1992-01-01

    The in vivo antibody response to the primary T-cell dependent antigen Helix pomatia Haemocyanin (HPH) was studied, in order to detect the possible presence of a humoral immune deficiency in ageing. The IgG subclass distribution of the specific antibodies was also determined. In order to define a

  1. Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry

    NARCIS (Netherlands)

    Rispens, T.; te Velthuis, H.; Hemker, P.; Speijer, H.; Hermens, W.; Aarden, L.

    2011-01-01

    Assessment of high-affinity antibody-antigen binding parameters is important in such diverse areas as selection of therapeutic antibodies, detection of unwanted hormones in cattle and sensitive immunoassays in clinical chemistry. Label-free assessment of binding affinities is often carried out by

  2. Subclass restriction pattern of antigen-specific antibodies in donors with defective expression of IgG or IgA subclass heavy chain constant region genes

    NARCIS (Netherlands)

    Hammerström, L.; Carbonara, A.O.; DeMarchi, M.; LeFranc, G.; Möller, G.; Smith, C.I.E.; Zegers, B.J.M.

    1987-01-01

    We have developed a method for the measurement of the IgG and IgA subclass distribution of antigen-specific human antibodies. The controls for the specificity of the assay include the use of a number of monoclonal human antibodies and sera from individuals with deletions of particular immunoglobulin

  3. [The occurrence of antibodies to the glycoprotein antigen (gp70) of the enzootic leukemia virus in a leukemic herd].

    Science.gov (United States)

    Hofírek, B

    1980-08-01

    The serological examination of 175 head of cattle in a herd suffering from leucosis included the study of the occurrence of antibodies to the glycoprotein antigen (gp70) of the virus of enzootic leucosis (BLV). At the same time, these antibodies were studied, as occurring in the F1 generation of the progeneis of 51 positive and 38 negative cows. The results demonstrate two important facts: increasing age brings about a higher percentage of animals having precipitating antibodies in the leucosis-affected herd; the other important finding is that the positive reaction of a cow has no significant influence on the occurrence of the antibodies in the progeny. It was found that under the actual conditions of the mentioned herd, the horizontal transmission of enzootic leucosis was predominant and that precipitating antibodies were detected by the immunodiffusion method in animals at the age from 9 to 12 months. It is desirable from the viewpoint of diagnostics and eradication of enzootic bovine leucosis to apply with utmost consistency the serological methods of examination and individual diagnostics. There is no reason for destroying whole families of animals in which the disease occurred, because no genetically conditioned occurrence of enzootic leucosis was demonstrated in the progenies of cows suffering from the disease. These facts should be respected in amending the instructions for controlling enzootic bovine leucosis.

  4. Structure-activity relations of water-in-oil vaccine formulations and induced antigen-specific antibody responses.

    Science.gov (United States)

    Jansen, Theo; Hofmans, Marij P M; Theelen, Marc J G; Schijns, Virgil E J C

    2005-01-11

    Water-in-oil (W/O) emulsions are known as most effective adjuvants to generate high and durable antibody responses to vaccine antigens following a single immunization. However, their structural requirements remain poorly understood. Here we addressed the significance of certain pharmaceutical characteristics including water/oil ratios--ranging from 60/40 to 30/70 (w/w(%))--droplet size and type of oil, i.e. non-metabolizable (mineral oil) versus metabolizable (Miglyol 840). Stability of emulsions was accomplished by the use of a polymeric emulsifier. Distinct W/O emulsions were formulated with inactivated (i) infectious bronchitis virus (iIBV) and Newcastle disease virus (iNDV), and evaluated in immunized chickens for magnitude and duration of in vivo antiviral antibody formation and local reactions. A high mineral oil content proved most effective for antibody response formation. In general, a larger droplet size evoked higher antibody responses for both oil types. Inoculum residues proved lower using biodegradable Miglyol, when compared to mineral oil, for all emulsion variants. Especially water-to-oil ratio and droplet size may provide useful parameters for improving (antiviral) antibody production by W/O emulsions.

  5. Evaluation of a synthetic peptide from the Taenia saginata 18kDa surface/secreted oncospheral adhesion protein for serological diagnosis of bovine cysticercosis.

    Science.gov (United States)

    Guimarães-Peixoto, Rafaella Paola Meneguete; Pinto, Paulo Sérgio Arruda; Santos, Marcus Rebouças; Polêto, Marcelo Depólo; Silva, Letícia Ferreira; Silva-Júnior, Abelardo

    2016-12-01

    Bovine cysticercosis is a zoonotic infection widely spread throughout Brazil, creating a burden on hygiene maintenance and the economy. Diagnosis of cysticercosis usually relies on post mortem inspection of carcasses in slaughterhouses. This detection method provides only low sensitivity. Recent advancements have improved the performance of serologic tests, such as ELISA, providing greater sensitivity and specificity. The objective of the current study was to identify and evaluate a synthetic peptide derived from the Taenia saginata 18kDa oncospheric surface protein for the diagnosis of bovine cysticercosis in ELISA. Test performance of the identified peptide was compared to an ELISA based on a heterologous crude Taenia crassiceps antigen (Tcra), widely used for the sero-diagnosis of bovine cysticercosis. Based on the primary sequence of an in silico structural model of the 18kDa protein, an epitope region designated EP1 was selected (46-WDTKDMAGYGVKKIEV-61). The peptide derived from this region yielded 91.6% (CI=80-96%) sensitivity and 90% (CI=82-95%) specificity when used in an ELISA, whereas the crude antigen yielded 70% (CI=56-8%) sensitivity and 82% (CI=73-89%) specificity. Thus, we conclude that EP1 has higher diagnostic potential for detecting bovine cysticercosis than the crude antigen Tcra. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Specific Antibodies for the Detection of Alternaria Allergens and the Identification of Cross-Reactive Antigens in Other Fungi

    Science.gov (United States)

    Twaroch, Teresa E.; Curin, Mirela; Sterflinger, Katja; Focke-Tejkl, Margit; Swoboda, Ines; Valenta, Rudolf

    2017-01-01

    Background The mould Alternaria alternata is an important source of respiratory allergens. A. alternata extracts show great variations regarding allergenic potency. The aim of this study was to generate antibody probes specific for important Alternaria allergens and to use them to study allergen expression, depending on different culture conditions, as well as to search for cross-reactive allergens in other mould species. Methods Synthetic peptides from antigenic regions of A. alternata allergens (Alt a 1, Alt a 2, Alt a 3, Alt a 6 and Alt a 8) were used to raise highly specific rabbit antibodies. These antibodies and IgE from allergic patients were used to detect allergens by immunoblotting in extracts of 4 A. alternata strains grown under varying culturing conditions, in commercial skin-prick extracts and in closely (Cladosporium herbarum and Aureobasidium pullulans) or distantly related (Aspergillus niger and Penicillium chrysogenum) mould species. Results There was a wide variation of expression of the individual A. Alternata allergens, depending on the strain and culture conditions, but the antibody probes allowed us to distinguish strains and culture conditions with low and high allergen expression. In the commercial skin-prick solutions, varying levels of Alt a 1 were found, but no other allergens were detectable. Alt a 1 was identified as species-specific A. Alternata allergen, whereas Alt a 3, 6- and Alt a 8-cross-reactive antigens were found in C. herbarum and/or A. pullulans. Conclusions and Clinical Relevance Peptide-specific antibodies are useful to analyze diagnostic and therapeutic mould extracts, to study the presence of A. Alternata allergens in biological samples and to search for cross-reactive allergens in other mould species. PMID:27780168

  7. Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination

    NARCIS (Netherlands)

    Tam, Hok Hei; Melo, Mariane B.; Kang, Myungsun; Pelet, Jeisa M.; Ruda, Vera M.; Foley, Maria H.; Hu, Joyce K.; Kumari, Sudha; Crampton, Jordan; Baldeon, Alexis D.; Sanders, Rogier W.; Moore, John P.; Crotty, Shane; Langer, Robert; Anderson, Daniel G.; Chakraborty, Arup K.; Irvine, Darrell J.

    2016-01-01

    Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose.

  8. Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa

    Directory of Open Access Journals (Sweden)

    P. C.B. Turnbull

    2008-08-01

    Full Text Available TURNBULLP, P.C.B. DIEKMANNM,M., KILIAN, J.W., VERSFELDW, W.,DE VOS, V., ARNTZENL, L.,WOLTER, K., BARTELS, P. & KOTZE, A. 2008.N aturally acquired antibodies to Bacillusa nthracisp rotective antigeni n vultureso f southern Africa. Onderstepoort Journal of Veterinary Research, T5:95-102 Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories in North West Province, South Africa, were examined by an enzyme-linked immunosorbenats say( ELISAf or antibodiesto the Bacillus anthracis toxin protective antigen (PA. As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63% wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a hole and the other groups (P < 0.001 and no significant difference between the South African and control groups (P > 0.05. Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheress, six out of ten Whitebacked Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypiust racheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. lt is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and

  9. Isolated cysticercosis of the cauda equina

    Directory of Open Access Journals (Sweden)

    Maurizio Iacoangeli

    2013-01-01

    Full Text Available Cysticercosis is the most common parasitic infection of the central nervous system. It is an endemic condition in developing countries, but the incidence rate is increasing in developed countries as well because of rising immigration. Spinal involvement is quite rare and it is usually associated with concomitant intracranial infective lesions. We present an unusual case of a 44-year-old woman who experienced a cauda equina syndrome. Magnetic resonance imaging disclosed two intradural cystic lesions at L4-L5 level. Only after histological examination the diagnosis of cysticercosis was definitively determined. The entire neuraxis evaluation confirmed that it was a rare form of isolated intradural racemosus type cysticercosis of the cauda equina. Steroids and albendazole were administered and post-operative course was uneventful. In this paper we discuss clinical, pathogenic and therapeutic aspects of this infective pathology.

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

    Science.gov (United States)

    Saleem, Iram; Widger, William; Chu, Wei-Kan

    2017-07-01

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

  11. Myelin-reactive antibodies initiate T cell-mediated CNS autoimmune disease by opsonization of endogenous antigen.

    Science.gov (United States)

    Kinzel, Silke; Lehmann-Horn, Klaus; Torke, Sebastian; Häusler, Darius; Winkler, Anne; Stadelmann, Christine; Payne, Natalie; Feldmann, Linda; Saiz, Albert; Reindl, Markus; Lalive, Patrice H; Bernard, Claude C; Brück, Wolfgang; Weber, Martin S

    2016-07-01

    In the pathogenesis of central nervous system (CNS) demyelinating disorders, antigen-specific B cells are implicated to act as potent antigen-presenting cells (APC), eliciting waves of inflammatory CNS infiltration. Here, we provide the first evidence that CNS-reactive antibodies (Ab) are similarly capable of initiating an encephalitogenic immune response by targeting endogenous CNS antigen to otherwise inert myeloid APC. In a transgenic mouse model, constitutive production of Ab against myelin oligodendrocyte glycoprotein (MOG) was sufficient to promote spontaneous experimental autoimmune encephalomyelitis (EAE) in the absence of B cells, when mice endogenously contained MOG-recognizing T cells. Adoptive transfer studies corroborated that anti-MOG Ab triggered activation and expansion of peripheral MOG-specific T cells in an Fc-dependent manner, subsequently causing EAE. To evaluate the underlying mechanism, anti-MOG Ab were added to a co-culture of myeloid APC and MOG-specific T cells. At otherwise undetected concentrations, anti-MOG Ab enabled Fc-mediated APC recognition of intact MOG; internalized, processed and presented MOG activated naïve T cells to differentiate in an encephalitogenic manner. In a series of translational experiments, anti-MOG Ab from two patients with an acute flare of CNS inflammation likewise facilitated detection of human MOG. Jointly, these observations highlight Ab-mediated opsonization of endogenous CNS auto-antigen as a novel disease- and/or relapse-triggering mechanism in CNS demyelinating disorders.

  12. Immunoelectron microscopic identification and localization of Streptococcus sanguis with peroxidase-labeled antibody: localization of surface antigens in pure cultures.

    Science.gov (United States)

    Lai, C H; Listgarten, M A; Rosan, B

    1975-01-01

    An indirect method of localizing antigens with horseradish peroxidase-labeled antibody was used to identify and localize surface antigens of Streptococcus sanguis at the ultrastructural level. An electron dense layer surrounding the cell wall could be distinguished without any additional electron microscope staining. This labeled layer represents an immune complex consisting of bacterial surface antigens, specific rabbit antisera, and peroxidase-labeled goat anti-rabbit globulins. Although with undiluted antisera slight cross-reactions occurred with S. salivarius and S. miteor (mitis), these could be readily distinguished from the more intense homologous reaction by their patchiness and the difference in distribution of the label. These cross-reactions were eliminated by appropriate dilutions of the antiserum. No cross-reactions occurred with S. mutans, S. faecalis, Actinomyces species, or Bacterionema, microorganisms wents indicated that horseradish peroxidase can become non-specifically adsorbed to the membrane of certain bacterial cells. Appropriate controls must, therefore, be included for localization of membrane associated antigens with horseradish peroxidase. Images PMID:1090524

  13. Association of serum Epstein-Barr nuclear antigen-1 antibodies and intrathecal immunoglobulin synthesis in early multiple sclerosis.

    Science.gov (United States)

    Pfuhl, Catherina; Oechtering, Johanna; Rasche, Ludwig; Gieß, René M; Behrens, Janina R; Wakonig, Katharina; Freitag, Erik; Pache, Florence C; Otto, Carolin; Hofmann, Jörg; Eberspächer, Bettina; Bellmann-Strobl, Judith; Paul, Friedemann; Ruprecht, Klemens

    2015-08-15

    Multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection. A characteristic feature of MS is an intrathecal synthesis of immunoglobulin (Ig)G. In 90 patients with clinically isolated syndromes/early relapsing-remitting MS, serum antibodies to Epstein-Barr nuclear antigen-1, but not to EBV viral capsid antigen, rubella, or varicella zoster virus, were higher (p=0.03) in those with than those without a calculated intrathecal IgG synthesis >0% and correlated with the percentage (r=0.27, p=0.009) and concentration (r=0.27, p=0.012) of intrathecally produced IgG. These findings suggest a link between EBV infection and the events leading to intrathecal IgG synthesis in patients with MS. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. [Wegener's granulomatosis with anti-neutrophil cytoplasmic antibodies against anti-cathepsin G antigen].

    Science.gov (United States)

    Ocaña Pérez, E; Peña Casas, A M; del Campo Muñoz, T; Avila Casas, A; Luque Barona, R

    2013-12-01

    Wegener's granulomatosis belongs to the group of small vessel vasculitis associated with anti-neutrophil cytoplasmic antibodies characterized by granulomatous inflammation and necrotising vasculitis in various organs with particular involvement of the upper and lower respiratory tracts and kidneys. Wegener's granulomatosis is a rare disorder in childhood and early diagnosis of this disease is critical to the long-term prognosis of the disease. The presence of positive cytoplasmic antineutrophil cytoplasmic antibody staining or a high titre of proteinase 3 antibodies were added as new criteria of vasculitis in childhood. This article presents a case of Wegener's granulomatosis, with the presence of anti-neutrophil cytoplasm antibodies with cytoplasmic pattern with absence of anti-proteinase 3 antibodies and presence of high levels of anti-cathepsin G antibodies, rarely described in Wegener's granulomatosis. Copyright © 2012 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  15. Muscular cysticercosis: Case report and imaging findings

    Directory of Open Access Journals (Sweden)

    Neide Regina Simões Olmo

    Full Text Available Summary Cysticercosis is a parasitic disease caused by a worm of the Cestoda class. The most prevalent form affects the nervous system. This case report is from a 78-year-old female patient evaluated at Clínica Mult Imagem, in the city of Santos, Brazil, who presented a form of the disease that differed from the classic neurocysticercosis, in this case muscular cysticercosis. This and other forms of manifestation justify further studies to ensure adequate recognition, diagnosis and treatment of this parasitic disease.

  16. Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

    DEFF Research Database (Denmark)

    Shilova, N V; Navakouski, M J; Huflejt, M

    2011-01-01

    The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted...... pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization....

  17. Seroprevalence of antibodies and antigens against hepatitis A-E viruses in refugees and asylum seekers in Germany in 2015.

    Science.gov (United States)

    Jablonka, Alexandra; Solbach, Philipp; Wöbse, Michael; Manns, Michael P; Schmidt, Reinhold E; Wedemeyer, Heiner; Cornberg, Markus; Behrens, Georg M N; Hardtke, Svenja

    2017-08-01

    Migration because of miscellaneous political crises in countries in the Middle East and Africa is a global challenge for whole Europe from an economic, social, and public health view. There is an urgent need to generate comprehensive, evidence-based data to expedite further screening and vaccination strategies. A total of 604 individuals ranging in age from 2 to 68 years who enrolled at a single reception center were tested for the prevalence of serologic markers for hepatitis virus types A, B, C, D, and E (HAV, HBV, HCV, HDV, HEV), respectively. Anti-HAV antibody prevalence was 91.2 and 70.3% in children younger than 18 years of age. The prevalence of anti-HEV antibodies was 20.1% among the individuals. 3.0% were positive for hepatitis B surface antigen, whereas 15.2% tested positive for anti-hepatitis B core antigen. None of the refugees tested positive for anti-HDV. 14.1% of refugees were vaccinated against hepatitis B and had a protective anti-hepatitis B surface level of at least 10 mIU/ml. Significant differences in vaccination status were found between the regions (Eastern Mediterranean Region with 77/482 (16.0%; 95% confidence interval=12.7-19.3%) versus African Region with 1/55 (1.8%; 95% confidence interval=0-5.0%). The prevalence of anti-HCV antibodies was 1.2% (n=7), with 0.7% HCV RNA positivity; 16.7% of hepatitis B surface antigen-positive individuals were HCV coinfected (n=3). The prevalence of refugees with previous exposure to hepatitis viruses was higher than that in the general German population, but lower than in other migrant populations in Germany. The vaccination status against hepatitis B was poor.

  18. Novel monoclonal antibodies to ESAT-6 and CFP-10 antigens for ELISA-based diagnosis of pleural tuberculosis.

    Science.gov (United States)

    Feng, T-T; Shou, C-M; Shen, L; Qian, Y; Wu, Z-G; Fan, J; Zhang, Y-Z; Tang, Y-W; Wu, N-P; Lu, H-Z; Yao, H-P

    2011-06-01

    To elucidate the potential of monoclonal antibodies (mAbs) of culture filtrate protein 10 (CFP-10) and early secretory antigenic target 6 (ESAT-6) in tuberculosis (TB) diagnosis. We generated and characterised monoclonal and polyclonal antibodies against Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 by immunising BALB/c mice with an ESAT-6/CFP-10 fusion protein. Stable hybridoma cell lines were established and mAbs were specifically identified by immunoblotting and immunoprecipitation. The mouse mAbs were used to coat plates, and biotin-labelled polyclonal antibodies were used to detect the antigens. One hundred and seventy-three samples of sputum culture supernatants and pleural effusion aspirates have been tested. The ESAT-6 enzyme-linked immunosorbent assay (ELISA) detected the culture supernatants and pleural effusion specimens that were positive for M. tuberculosis, but failed to identify M. tuberculosis-positive specimens in the non-M. tuberculosis culture supernatants or control specimens. This yielded a sensitivity of 95.4% and a specificity of 100% for the ESAT-6-specific ELISA. The CFP-10 ELISA presented less satisfactory sensitivity and specificity, of respectively 81.6% and 92.2%. Results showed positive detection rates of ESAT-6 and CFP-10 of 86.8% (33/38) and 76.3% (29/38) for the diagnosis of tuberculous pleural effusion in patients bacteriologically negative for M. tuberculosis culture. The ESAT-6 and CFP-10 ELISAs incorporating mAbs generated in this study serve as potential tools in the laboratory diagnosis of TB.

  19. Comparative Evaluation of Native Antigens for the Development of Brucellosis Antibody Detection System

    Directory of Open Access Journals (Sweden)

    Yasmin Bano

    2015-09-01

    Full Text Available Brucellosis is a highly infectious zoonotic disease and an economically important infection of humans and livestock with a worldwide distribution. The main mode of transmission of this disease to humans is through the consumption of infected milk, milk products, and uncooked or raw meat. The present study was designed to prepare few native antigens, that is, sonicated antigen (SA, cell envelope (CE antigen, and freeze and thaw (FT antigen from Brucella abortus S99 culture and to test them in a highly sensitive and specific indirect enzyme-linked immunosorbent assay (I-ELISA in both a microtiter plate and a dot-blot format for the development of field-based diagnosis. All 50 suspected bovine samples were tested by plate as well as in dot ELISA formats for all the three antigens prepared. The CE antigen was found to be more suitable as it had the maximum agreement with the Rose Bengal plate agglutination test results followed by the SA and the least agreement was found with that of the FT antigen. This detection system in microtiter plates and a dot-blot format will be useful for the rapid screening of samples for the disease surveillance and routine diagnosis.

  20. Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

    Directory of Open Access Journals (Sweden)

    Gema Álvarez García

    2006-08-01

    Full Text Available A Neospora caninum 17 kDa protein fraction (p17 has been described as an immunodominant antigen (IDA under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17 was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86.

  1. Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies.

    Science.gov (United States)

    Gong, K; Wen, D Y; Ouyang, T; Rao, A T; Herzberg, M C

    1995-09-01

    Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane

  2. Evaluation of the fully automated Cobas Core enzyme immunoassay for the quantitation of antibodies against hepatitis B virus surface antigen.

    Science.gov (United States)

    Doche, C; Thomé, M; Dimet, I; Bienvenu, J

    1996-04-01

    The Roche Cobas Core automated enzyme immunoassay of antibodies against hepatitis B virus surface antigen (anti-HBs) was evaluated using a protocol complying with the Société Française de Biologie Clinique guidelines. Results showed good precision (CV below 6 and 9% for intra- and inter-assay precision) and accuracy (according to the Paul-Ehrlich-Institute standard); the detection limit of the test was 2 IU/l. The manufacturer's specifications were confirmed and a satisfactory correlation (r = 0.901) was found with an automated method (IMx, Abbott) used for comparison.

  3. THE OCCURRENCE OF NATURAL ANTIBODIES AGAINST DOG ERYTHROCYTE ANTIGENS IN DOGS FROM SINOP AND SORRISO, MATO GROSSO, BRAZIL.

    Directory of Open Access Journals (Sweden)

    Dayanne Dallabona

    2013-07-01

    Full Text Available AbstractThe goal of this research was to verify the occurrence of natural antibodies against blood group antigens in dogs from Sinop and Sorriso/MT, Brazil. For this purpose, blood samples from 93 dogs were collected (20 mixed breed dogs and 73 pure breed dogs - Pitbull, Labrador, Lhasa Apso, Brazilian, Bernese Mountain Dog, Doberman Pinscher, Poodle, Shih Tzu, Boxer, Chow Chow, Yorkshire Terrier, Rottweiler, German Shepherd, Dachshund, Dalmatian, Australian Cattle Dog, Golden Retriever, Cocker Spaniel, to be tested using the cross(-matching test in three different temperatures (30C, 37C and 4C. The obtained results showed the occurrence of natural antibodies in 17.7 % of tested dogs.

  4. Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Kumar Sukhdeo

    Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

  5. 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 Impact factor: 5.720, year: 2016

  6. Impact of acute malaria on pre-existing antibodies to viral and vaccine antigens in mice and humans.

    Directory of Open Access Journals (Sweden)

    Simran Banga

    Full Text Available Vaccine-induced immunity depends on long-lived plasma cells (LLPCs that maintain antibody levels. A recent mouse study showed that Plasmodium chaubaudi infection reduced pre-existing influenza-specific antibodies--raising concerns that malaria may compromise pre-existing vaccine responses. We extended these findings to P. yoelii infection, observing decreases in antibodies to model antigens in inbred mice and to influenza in outbred mice, associated with LLPC depletion and increased susceptibility to influenza rechallenge. We investigated the implications of these findings in Malian children by measuring vaccine-specific IgG (tetanus, measles, hepatitis B before and after the malaria-free 6-month dry season, 10 days after the first malaria episode of the malaria season, and after the subsequent dry season. On average, vaccine-specific IgG did not decrease following acute malaria. However, in some children malaria was associated with an accelerated decline in vaccine-specific IgG, underscoring the need to further investigate the impact of malaria on pre-existing vaccine-specific antibodies.

  7. An ultrasensitive squamous cell carcinoma antigen biosensing platform utilizing double-antibody single-channel amplification strategy.

    Science.gov (United States)

    Ren, Xiang; Wu, Dan; Wang, Yuhuan; Zhang, Yunhui; Fan, Dawei; Pang, Xuehui; Li, Yueyun; Du, Bin; Wei, Qin

    2015-10-15

    A novel electrochemical immunosensor was developed for ultrasensitive detection of squamous cell carcinoma antigen (SCCA), which was based on the double-antibody single-channel amplification strategy. For the first time, human immunoglobulin antibody (anti-HIgG) was used as the supporting framework to amplify the loading quantity of SCCA antibody (anti-SCCA). In this strategy, SCCA can be detected without using mesoporous nanometers to amplify the signal. In addition, Pd icosahedrons were first used as the connecter to immobilize the antibodies and strengthen the sensitivity. Only one touch point exists under the limited condition between a sphere and another shape in geometry, thus the Pd icosahedron is an excellent candidate as the role of connecter. Gold nanoparticles (Au NPs) decorated with mercapto-functionalized graphene sheets (Au@GS) were synthesized as the transducing materials. The fabricated immunosensor exhibited an excellent detection limit of 2.8 pg/mL and wide linear range of 0.01-5 ng/mL. This kind of immunosensor would provide a potential application in clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. [Hepatitis B virus surface antigen reactivity in the absence of antibodies to core antigen: an atypical serological pattern having diverse significance].

    Science.gov (United States)

    Echevarría, José María; León, Pilar; Pozo, Francisco

    2004-01-01

    Reactivity for hepatitis B virus (HBV) surface antigen (HBsAg) in samples lacking antibody to the HBV core antigen (anti-HBc) may be due to either non-specific reactions or sample contamination, but it can also reflect other situations that may present clinical relevance. In Spain, the frequency described for this atypical pattern of HBV markers ranged from 0.05 to 1.3% in different series, so that it can be considered as moderately frequent. Confirmatory assays for HBsAg and tests for detecting total and IgM anti-HBc, HBV "e" antigen and viral DNA were performed on serum samples taken from 96 patients who showed reactivity for HBsAg in absence of total anti-HBc on routine studies. These samples were collected between January, 2001 and May, 2002 and were sent for study from different Spanish laboratories. Follow-up samples were also studied from selected cases. Presence of HBsAg was excluded in 70 cases (72.9%) and total anti-HBc was detected in two additional patients, so that the original results were confirmed in 24 patients (25.0%). After further investigations, two early phases of the window period of the acute HBV infection were identified, as well as a further case of chronic HBV carriage in absence of antibody response. A single case could be confirmed as due to a contamination of the aliquot of sample studied. The pattern of HBV markers and its evolution on the follow-up were the characteristic of the phenomenon known as "hepatitis B virus type 2 infection" in eight patients. No conclusions could be drawn from the remainder 12 cases, since no follow-up samples were available for study. Besides most cases respond to non-specific reactions or reflect situations of low clinical relevance, the reactivity for HBsAg in samples lacking anti-HBc should be taken into consideration and routinely investigated, since this atypical pattern can also reflect unusual, but clinically relevant facts of the HBV infection that must be noticed.

  9. Antigenic Fingerprinting following Primary RSV Infection in Young Children Identifies Novel Antigenic Sites and Reveals Unlinked Evolution of Human Antibody Repertoires to Fusion and Attachment Glycoproteins.

    Directory of Open Access Journals (Sweden)

    Sandra Fuentes

    2016-04-01

    Full Text Available Respiratory Syncytial Virus (RSV is the major cause of pneumonia among infants. Here we elucidated the antibody repertoire following primary RSV infection and traced its evolution through adolescence and adulthood. Whole genome-fragment phage display libraries (GFPDL expressing linear and conformational epitopes in the RSV fusion protein (F and attachment protein (G were used for unbiased epitope profiling of infant sera prior to and following RSV infection. F-GFPDL analyses demonstrated modest changes in the anti-F epitope repertoires post-RSV infection, while G-GFPDL analyses revealed 100-fold increase in number of bound phages. The G-reactive epitopes spanned the N- and C-terminus of the G ectodomain, along with increased reactivity to the central conserved domain (CCD. Panels of F and G antigenic sites were synthesized to evaluate sera from young children (<2 yr, adolescents (14-18 yr and adults (30-45 yr in SPR real-time kinetics assays. A steady increase in RSV-F epitope repertoires from young children to adults was observed using peptides and F proteins. Importantly, several novel epitopes were identified in pre-fusion F and an immunodominant epitope in the F-p27. In all age groups, antibody binding to pre-fusion F was 2-3 folds higher than to post-fusion form. For RSV-G, antibody responses were high following early RSV infection in children, but declined significantly in adults, using either G proteins or peptides. This study identified unlinked evolution of anti-F and anti G responses and supportive evidence for immune pressure driven evolution of RSV-G. These findings could help development of effective countermeasures including vaccines.

  10. Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies.

    Science.gov (United States)

    Apps, Richard; Murphy, Shawn P; Fernando, Raymond; Gardner, Lucy; Ahad, Tashmeeta; Moffett, Ashley

    2009-05-01

    Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class-I (HLA-I) loci has made it difficult to generate locus-specific monoclonal antibodies (mAbs). The problem of defining an antibody's reactivity against the thousands of existing HLA-I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA-I to characterize experimentally the reactivity of nine mAb against 96 common HLA-I allotypes. In conjunction with donor HLA-I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG-3 and JAR; and the placental cell lines HTR-8/SVneo, Swan-71 and TEV-1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA-C, HLA-G and HLA-E, but not HLA-A, HLA-B or HLA-DR molecules in normal pregnancy. Tumour-derived JEG-3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.

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

    Science.gov (United States)

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

    2011-12-01

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

  12. Unique recognition of a low molecular weight Onchocerca volvulus antigen by IgG3 antibodies in chronic hyper-reactive oncho-dermatitis (Sowda).

    Science.gov (United States)

    Cabrera, Z; Buttner, D W; Parkhouse, R M

    1988-01-01

    Individual human Ig class responses to Onchocerca volvulus antigens have been evaluated by Western blotting using sera from cases of generalized onchocerciasis and chronic hyper-reactive onchocerciasis (Sowda). in all cases except IgG3 the patterns of recognition by human antibody classes were similar in Sowda and generalized onchocerciasis. Weak or undetectable responses were seen with IgG1, IgG2 and IgM. The total profiles of antigens recognized by the other Ig classes were different, although in some cases certain bands were commonly identified. The result with IgG3, however, was striking. Here, two major antigens (9 kD and 72kD) were recognized by IgG3 antibodies in Sowda sera but not generalized onchocerciasis sera. Furthermore, these two antigens were not recognised by any other Ig class, either in generalized or Sowda onchocerciasis, nor were they detected by antibodies of any class present in a collection of sera representative of other nematode infections. This difference in the IgG3 response was so pronounced that Sowda sera could be distinguished from generalized onchocerciasis sera by an IgG3-specific ELISA assay with a PBS parasite extract as the antigen. Thus, a correlation has been established between one particular clinical condition of onchocerciasis (Sowda) and a serological response, defined in terms of both the parasite antigens and an immunoglobulin class restricted antibody response. Images Fig. 1 Fig. 2 Fig. 5 PMID:3224441

  13. A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas.

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    Haolin Liu

    Full Text Available B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

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

  15. Human cysticercosis and Indian scenario: a review

    Indian Academy of Sciences (India)

    2008-10-15

    Oct 15, 2008 ... Keywords. Active epilepsy; cysticercosis, neurocysticercosis; seizure; taeniasis; Taenia solium ... Hence it is important to know the epidemiology, pathogenesis and diagnostic criteria so as to assess the disease burden and adopt interventional strategies for its control. Literature search was done for this ...

  16. Case report: intraocular cysticercosis | Adegbehingbe | West African ...

    African Journals Online (AJOL)

    A case of intravitreal cysticercosis causing left uniocular cataract and eventual left visual loss in a healthy female Nigerian is presented. The diagnosis of cysticercus celulosae was not made until the patient had left cataract extraction done. The cysticercus larva found its way into the anterior chamber and this stimulated ...

  17. Bovine cysticercosis in the European Union

    DEFF Research Database (Denmark)

    Blagojevic, Bojan; Robertson, Lucy J.; Vieira-Pinto, Madalena

    2017-01-01

    Bovine cysticercosis is caused by the larval stage of Taenia saginata and has a global distribution. This zoonosis usually causes only mild disease in humans, but has an important economic impact on the meat sector as bovine carcasses that are found to be infected are either condemned or undergo ...

  18. Primary Sonographic Diagnosis of Subcutaneous Cysticercosis

    Directory of Open Access Journals (Sweden)

    M E Shivu

    2011-01-01

    Full Text Available We present the case of a 40-year-old woman with a small diffuse swelling on the left side of her face. She was diagnosed with intramuscular cysticercosis in the masseter muscle (case of disseminated cysticercosis involving the muscular system and subcutaneous tissues with surrounding phlegmon on high-resolution ultrasound and managed conservatively. To our knowledge, the imaging findings of disseminated muscular cysUcercosis have been reported before only a few numbers of times. In this case, the correct diagnosis was made on the basis of high-resolution sonography of the subcutaneous tissue and muscles. It showed multiple oval to circular, predominantly anechoic lesions, which were around 1 cm in diameter. Most of these cystic lesions showed a hyperechoic focus within suggestive of a scolex. There was no increased vascularity surrounding the lesions. Thus, sonography can primarily make the correct diagnosis of disseminated muscular cysticercosis if such lesions are seen. In endemic areas, cysticercosis should be considered one of the differential diagnosis of the subcutaneous swellings.

  19. Case Report - Cysticercosis presenting as cervical ...

    African Journals Online (AJOL)

    Lymphadenopathy is a rare mode of presentation of cysticercus infestation. Hence, in endemic areas, cysticercosis must be included in the differential diagnosis of superficial palpable swellings in the neck region. We report two cases of cervical lymphadenopathy which were clinically suspected to be of tuberculous etiology ...

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

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    Kwadwo A Kusi

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

  1. The human leucocyte-common (LC) molecule: dissection of leukaemias using monoclonal antibodies directed against framework and restricted antigenic determinants.

    Science.gov (United States)

    Ritter, M A; Sauvage, C A; Pegram, S M; Myers, C D; Dalchau, R; Fabre, J W

    1985-01-01

    Monoclonal antibodies have previously been raised against two separate antigenic determinants on the human LC molecule. One, F10.89.4, recognizes a 'framework' epitope on all LC molecules; these are found on the majority of leucocytes. The other, F8.11.13, recognizes only a 'restricted' epitope present on a subset of these molecules; this subset is found on B lymphocytes and a subpopulation of T lymphocytes. LC molecules on myeloid cells do not carry the 'restricted' antigenic determinant. We have investigated the differential expression of these LC epitopes on human leukaemias, using immunofluorescence on fresh leukaemic blasts and established cell lines. Our study shows that, as on normal haemopoietic cells, LC molecules on B leukaemias bear both 'framework' and 'restricted' epitopes, while the majority of T leukaemias bear only the 'framework' determinant. The small proportion of T cells that are F8.11.13+ ('restricted' epitope) are relatively mature, being of either OKT4+ or OKT8+ phenotype, and may be in an activated state (HLA-DR+). However, in contrast to normal haemopoietic cells, some myeloid leukaemias carry both 'framework' and 'restricted' epitopes (30% AML and AMML samples are F10.89.4+, F8.11.13+), and it is within this group that all TdT+ AML and AMML cases lie. Thus, these monoclonal antibodies should be useful for studying haemopoiesis in man and for analyzing human haemopoietic malignancies.

  2. MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies.

    Science.gov (United States)

    Semchenko, Evgeny A; Day, Christopher J; Seib, Kate L

    2017-02-01

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea. Copyright © 2017 Semchenko et al.

  3. Evaluation of Serum Specific Antibody against Recombinant ESAT-6 Antigen in Patients with Tuberculosis and Comparing to Normal Controls

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    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.

  4. Taeniasis and cysticercosis prevalence in a small village from Northeastern Brazil Prevalência de teníase e sorologia positiva para cisticercose em Mulungu do Morro, Bahia

    Directory of Open Access Journals (Sweden)

    Irenio Gomes

    2002-06-01

    Full Text Available Although not considered as an endemic region, the Northeast of Brazil has the necessary conditions for the development of taeniasis-cysticercosis complex. In a previous paper, we demonstrated that Mulungu do Morro municipality, in the State of Bahia, has a high seroprevalence to cysticercosis in epileptic patients. OBJECTIVE: to determine the prevalence of taeniasis and positive cysticercosis serology in the population of Mulungu do Morro. METHOD: blood and stool samples were collected from a random sampling of the population, by family. The identification of antibodies against T. solium cysticerci was made by EITB and T. solium antigens were identified using a polyclonal antibody-capture ELISA. RESULTS: the cysticercosis seroprevalence was 1.6% (C.I. = 0.8 to 2.8% and the taeniasis prevalence 4.5% (C.I. = 3.0 to 6.5%. Seropositivity to cysticercosis was higher among those who lived in a house of a person testing positive for coproantigen, p=0.017. CONCLUSION: our results demonstrate that the taeniasis-cysticercosis complex is endemic in Mulungu do Morro. We believe that all areas in the world with the same socio-economic and sanitary characteristics are likely to have high prevalence of this parasite.Embora alguns autores não a considerem uma região endêmica, existem no Nordeste do Brasil as condições necessárias para o desenvolvimento do complexo teníase/ cisticercose. Em uma publicação prévia demosntramos no município de Mulungu do Morro, Bahia, alta soroprevalência para cisticercose em pacientes epilépticos. OBJETIVO: determinar a prevalência de teníase e sorologia positiva para cisticercose na população de Mulungu do Morro. MÉTODO: foram coletadas amostras de sangue e fezes em 175 famílias definidas aleatoriamente. A identificação de anticorpos séricos anti-cisticerco foi feita através do método de EITB e a presença de teníase foi verificada através de ELISA de captura para identificação de antígenos do

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

    Directory of Open Access Journals (Sweden)

    Lihan Chen

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

  6. Development and field evaluation of a new serological test for Taenia saginata cysticercosis.

    Science.gov (United States)

    Ogunremi, Oladele; Benjamin, Jane

    2010-04-19

    Cattle infected with the tapeworm cyst, Taenia saginata metacestode (synonym: Cysticercus bovis) are a source of human infection if affected beef is eaten raw or undercooked. Control measures targeted at individual cattle rather than all animals in a T. saginata-exposed herd should help reduce costs and alleviate current constraints associated with managing an outbreak. To that end, we have developed a reliable diagnostic test for use in live animals that would enable veterinary regulators to focus disease control strategies. The test detects bovine anti-T. saginata immunoglobulin G1 antibodies using an enzyme-linked immunosorbent assay (ELISA) which relies on the excretory-secretory antigens of T. saginata. Animals were inoculated with 10, 100 or 1000 viable T. saginata eggs in order to simulate the parasite burden of field-infected animals (parasite load=1-86; n=28). By testing sera obtained from the inoculated animals 84 days post-inoculation, test sensitivity was estimated to be 92.9% (95% confidence interval or CI=83.4-100.0%). Another 17 animals inoculated with 5000 or 10,000 viable eggs of T. saginata and shown to harbour metacestodes at post-mortem, all tested positive in the ELISA. Test specificity estimated from a herd of field animals with no historical, epidemiological, or post-mortem evidence of infection was 90.6% (95% CI=87.0-94.2%; n=256 field cattle). Using the test on samples (n=347) from a T. saginata-infected feedlot, the Bayesian approach estimate of seroprevalence was 4.6% (95% probability intervals=0.5-10.3%). The test performance characteristics of the ELISA suggest that it will be adequate for field application in bovine cysticercosis outbreaks.

  7. Specific in situ visualization of plasma cells producing antibodies against Porphyromonas gingivalis in gingival radicular cyst: application of the enzyme-labeled antigen method.

    Science.gov (United States)

    Tsuge, Shinya; Mizutani, Yasuyoshi; Matsuoka, Kazuhiro; Sawasaki, Tatsuya; Endo, Yaeta; Naruishi, Koji; Maeda, Hiroshi; Takashiba, Shogo; Shiogama, Kazuya; Inada, Ken-Ichi; Tsutsumi, Yutaka

    2011-07-01

    The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.

  8. Antigen-Specific Monoclonal Antibodies Isolated from B Cells Expressing Constitutively Active STAT5

    NARCIS (Netherlands)

    Scheeren, F.A.; van Geelen, C.M.M.; Yasuda, E.; Spits, H.; Beaumont, T.

    2011-01-01

    Background: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. Methodology/Principal Findings: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer

  9. Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

    Science.gov (United States)

    Korec, E; Dostálová, V; Korcová, J; Mancal, P; König, J; Borisova, G; Cibinogen, V; Pumpen, P; Gren, E; Hlozánek, I

    1990-05-01

    Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-09-10

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

  11. Establishing China's national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines.

    Science.gov (United States)

    Liang, Zhenglun; Mao, Qunying; Gao, Qiang; Li, Xiuling; Dong, Chenghong; Yu, Xiang; Yao, Xin; Li, Fengxiang; Yin, Weidong; Li, Qihan; Shen, Xinliang; Wang, Junzhi

    2011-12-06

    Enterovirus 71 (EV71) is a highly infectious agent that causes hand-foot-mouth disease (HFMD) in humans. Effective vaccination against EV71 infection is critically important, given the recent outbreak of HFMD in the Asia-Pacific region, where it has shown significant mortality and morbidity. There is currently no approved anti-viral therapy available to treat the disease. While several vaccine manufacturers are actively developing EV71 vaccines, there are no international reference standards available to conduct quality control on EV71 vaccines or to assess the effectiveness of EV71 vaccines in immunized populations. In the current report, antigen reference standard based on the C4 subtype of the EV71 vaccine strain was developed. In addition, neutralizing antibody (NTAb) reference panels were analyzed and standards with various neutralizing titers were selected. These reference antigens were used to calibrate vaccine samples from several producers and found that five EV71 antigens and the national reference standards showed good linearity and parallelism. Moreover, mice immunized with various vaccines at doses standardized by these national references showed comparable NTAb responses. Finally, the national NTAb reference panels were found to effectively reduce assay discrepancy between different labs. Taken together, these national reference standards are highly valuable for the standardization and evaluation of EV71 vaccines. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Prevalence, Antigenic Specificity, and Bactericidal Activity of Poultry Anti-Campylobacter Maternal Antibodies

    OpenAIRE

    Sahin, Orhan; Zhang, Qijing; Meitzler, Jerrel C.; Harr, Brian S.; Morishita, Teresa Y.; Mohan, R

    2001-01-01

    Poultry are considered the major reservoir for Campylobacter jejuni, a leading bacterial cause of human food-borne diarrhea. To understand the ecology of C. jejuni and develop strategies to control C. jejuni infection in the animal reservoir, we initiated studies to examine the potential role of anti-Campylobacter maternal antibodies in protecting young broiler chickens from infection by C. jejuni. Using an enzyme-linked immunosorbent assay (ELISA), the prevalence of anti-C. jejuni antibodies...

  13. 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.

  14. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants.

    Science.gov (United States)

    Chen, Qiang; He, Junyun; Phoolcharoen, Waranyoo; Mason, Hugh S

    2011-03-01

    Expression of recombinant vaccine antigens and monoclonal antibodies using plant viral vectors has developed extensively during the past several years. The approach benefits from high yields of recombinant protein obtained within days after transient delivery of viral vectors to leaves of Nicotiana benthamiana, a tobacco relative. Modified viral genomes of both RNA and DNA viruses have been created. Geminiviruses such as bean yellow dwarf virus (BeYDV) have a small, single stranded DNA genome that replicates in the nucleus of an infected plant cell, using the cellular DNA synthesis apparatus and a virus-encoded replication initiator protein (Rep). BeYDV-derived expression vectors contain deletions of the viral genes encoding coat and movement proteins and insertion of an expression cassette for a protein of interest. Delivery of the geminiviral vector to leaf cells via Agrobacterium-mediated delivery produces very high levels of recombinant DNA that can act as a transcription template, yielding high levels of mRNA for the protein of interest. Several vaccine antigens, including Norwalk virus capsid protein and hepatitis B core antigen, were expressed using the BeYDV vector at levels up to 1 mg per g of leaf mass. BeYDV replicons can be stacked in the same vector molecule by linking them in tandem, which enables production of multi-subunit proteins like monoclonal antibody (mAb) heavy and light chains. The protective mAb 6D8 against Ebola virus was produced at 0.5 mg per g of leaf mass. Multi-replicon vectors could be conveniently used to produce protein complexes, e.g. virus-like particles that require two or more subunits.

  15. Trichinella britovi human infection in Spain : antibody response to surface, excretory/secretory and somatic antigens

    Directory of Open Access Journals (Sweden)

    Rodríguez-Osorio M.

    2003-06-01

    Full Text Available A third outbreak of Trichinella britovi with 140 people involved, occurred in Granada Spain (December 1998. The source of infection was sausage made from uninspected wild boar meat. Fifty-two patients agreed to participated in this study. An elevated eosinophil level (> 5 % was detected in 59.6 % of patients, and persisted in most of these cases for two months. A moderate IgG response was observed. At the onset of symptoms, Western blot (WB test detected more positive cases than Enzyme linked immunosorbent assay (ELISA and indirect immunofluorescence (IIF. Six months from infection, ELISA revealed fewer positive cases than the other two tests. It would appear that the response to somatic antigens starts earlier than those to cuticular and excretory/secretory (ES antigens and that the response to ES antigens is the first to decrease.

  16. Serum pneumolysin antibody and urinary pneumococcal antigens (Binax level in children with upper respiratory tract infection versus normal controls

    Directory of Open Access Journals (Sweden)

    Noorbakhsh S

    2010-11-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Streptococcus pneumoniae is a common cause of respiratory infection. Pneumococcal upper respiratory tract infection (URTI in children is seldom bacteremic. Determination the prevalence of S.pneumoniae infections in children with URTI using rapid urinary antigen test (BINAX now and titration of serum pneumolysin antibody (added to conventional culture was the object of this study."n"n Methods: A cross sectional, case-control study done in ENT & pediatric departments of Rasoul Hospital in Tehran, Iran, (2008 -2010 upon 133 cases with upper respiratory tract infection (otitis media, sinusitis and tracheitis. The nosocomial infection omitted in first step. 60 remaining cases followed for S.pneumoniae infection by culture and rapid urinary antigen test (Binax Now. Serum pneumolysin antibody titers compared between 45 cases and 66 controls. "n"n Results: Positive culture (S.pneumoniae, H.influenza obtained in 4/60 URTI cases. Positive urinary S.pneumoniae antigen detected in 50% (30/60 of cases and 6% (4/66 of controls (p=0.01. The pneumolysin antibody level with cut-off level 525pg/ml was higher in URTI cases than controls (982±441 Vs. 525±42, p<0.0001. Area under the ROC curve for pneumolysin antibody was 0

  17. Dog erythrocyte antigens (DEA) 1, 4, 7 and suspected naturally occurring anti-DEA 7 antibodies in Italian Corso dogs.

    Science.gov (United States)

    Spada, E; Proverbio, D; Priolo, V; Ippolito, D; Baggiani, L; Perego, R; Pennisi, M G

    2017-04-01

    We sought to determine the prevalence of dog erythrocyte antigen (DEA) 1, 4 and 7 and naturally occurring anti-DEA7 antibodies in Italian Corso dogs. In addition, we correlated DEAs with different epidemiologic variables, compared the prevalence of DEAs against other canine populations and assessed the risk of sensitisation and transfusion reactions (TRs) following unmatched transfusion. Blood samples from 100 Corso dogs were evaluated for DEA 1, 4, 7 and naturally occurring anti-DEA 7 antibodies. Seventy-one percent of samples were DEA 1-negative, 100% tested DEA 4-positive, and 95% tested DEA 7-negative. Suspected anti-DEA7 antibodies were found in 32% dogs. The DEA 1 and 7-negative phenotypes were significantly more common than in most canine populations. When a previously tested Italian canine population was considered as blood donors for Corso dogs, the risk of DEA 1 sensitisation using DEA 1 untyped blood was 29%, and of acute haemolytic TRs after a second untyped DEA 1-incompatible transfusion was 8%. The potential for delayed TRs between DEA 7-negative Corso dogs with suspected naturally occurring anti-DEA 7 antibodies receiving untyped DEA 7-positive blood was 11%. Conversely, when Corso dogs were blood donors for the same population, the risk of DEA 1 sensitisation was 17% and the risk of an acute haemolytic TR after a second DEA 1-incompatible blood transfusion was 3%. Corso dogs can be suitable blood donors. Additional studies are needed to clarify whether the high prevalence of naturally occurring anti-DEA 7 antibodies in this breed could increase their risk of delayed TRs when they are blood recipients. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. LDL Receptor-Related Protein 2 (Megalin) as a Target Antigen in Human Kidney Anti-Brush Border Antibody Disease.

    Science.gov (United States)

    Larsen, Christopher P; Trivin-Avillach, Claire; Coles, Paige; Collins, A Bernard; Merchant, Michael; Ma, Hong; Wilkey, Daniel W; Ambruzs, Josephine M; Messias, Nidia C; Cossey, L Nicholas; Rosales, Ivy A; Wooldridge, Thomas; Walker, Patrick D; Colvin, Robert B; Klein, Jon; Salant, David J; Beck, Laurence H

    2017-10-26

    Primary renal tubulointerstitial disease resulting from proximal tubule antigen-specific antibodies and immune complex formation has not been well characterized in humans. We report a cohort of patients with a distinct, underappreciated kidney disease characterized by kidney antibrush border antibodies and renal failure (ABBA disease). We identified ten patients with ABBA disease who had a combination of proximal tubule damage, IgG-positive immune deposits in the tubular basement membrane, and circulating antibodies reactive with normal human kidney proximal tubular brush border. All but one of the patients also had segmental glomerular deposits on renal biopsy specimen. Patients with ABBA disease were elderly and presented with AKI and subnephrotic proteinuria. Serum from all patients but not controls recognized a high molecular weight protein in renal tubular protein extracts that we identified as LDL receptor-related protein 2 (LRP2), also known as megalin, by immunoprecipitation and mass spectrometry. Immunostaining revealed that LRP2 specifically colocalized with IgG in the tubular immune deposits on the ABBA biopsy specimen but not the control specimen analyzed. Finally, ABBA serum samples but not control samples showed reactivity against recombinantly expressed N-terminal LRP2 fragments on Western blots and immunoprecipitated the recombinantly expressed N-terminal region of LRP2. This case series details the clinicopathologic findings of patients with ABBA disease and shows that the antigenic target of these autoantibodies is LRP2. Future studies are needed to determine the disease prevalence, stimulus for ABBA, and optimal treatment. Copyright © 2017 by the American Society of Nephrology.

  19. Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

    Directory of Open Access Journals (Sweden)

    M Rodero

    2005-05-01

    Full Text Available An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG. However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

  20. Anti-idiotype antibodies to Marek's disease-associated tumour surface antigen in protection against Marek's disease.

    Science.gov (United States)

    Dandapat, S; Pradhan, H K; Mohanty, G C

    1994-04-01

    Marek's disease-associated tumour surface antigen (MATSA) removed by enzymatic (papain) digestion of Marek's disease tumour cells was fractionated by gel filtration chromatography. The first peak (F1) was used to raise antibody in rabbits. Monoclonal antibody (RPH-6) directed against MATSA and the anti-F1 IgG were used as idiotypic antibodies to raise polyclonal anti-idiotype serum in heterologous hosts; rabbit and goat, respectively. The anti-idiotypes (anti-Id) were purified by affinity chromatography and characterized by competitive binding assay using immunofluorescent (IF) tests. Day-old white Leghorn chicks were immunized with anti-Id to MATSA (Group 1) or anti-Id to F1 (Group 3) and challenged with virulent Marek's disease virus (MDV) on the tenth day post immunization. In positive control groups, the day-old chicks were inoculated with anti-BALB/c mouse globulin (Group 2) and anti-rabbit globulin (Group 4) and challenged with virulent MDV on the tenth day post inoculation. As compared with positive control groups, the vaccinated groups (1 and 3) had considerably lower level of MATSA positive cells during the post challenge observation period. The protection level against MD in the immunized groups was 66.6% (Group 1) and 86.6% (Group 3).

  1. Rational monoclonal antibody development to emerging pathogens, biothreat agents and agents of foreign animal disease: The antigen scale.

    Science.gov (United States)

    Berry, Jody D

    2005-09-01

    Many factors influence the choice of methods used to develop antibody to infectious agents. In this paper, we review the current status of the main technologies used to produce monoclonal antibodies (mAbs) from the B cells of antigen-sensitized animals. While companies are adopting advanced high-throughput methods, the major technologies used by veterinary and medical research laboratories are classical hybridoma fusion and recombinant library selection techniques. These methods have inherent advantages and limitations but have many common aspects when using immunized rodents. Laboratories with expertise in both methods of antibody development have a distinct advantage in their ability to advance mAb technology. New and re-emerging infectious threats in today's world emphasize the need for quality immunoreagents and the need to maintain expertise in mAb development. We provide examples of some common applications for mAb reagents used to identify pathogens such as the SARS-coronavirus (SARS-CoV), Bacillus anthracis, and foot-and-mouth disease (FMD) virus. We also outline a framework for investigators to make rational decisions concerning which method to use to develop mAbs based upon characteristics of the pathogen under study and the intended downstream application. Lastly, we provide parameters for the immunisation of mice and a classification system which describes the expected outcome for mAb development strategies when using classes of immunogens to generate mAbs with desired activities.

  2. Prevalence of Serum IgG Antibodies to Cystic Echinococcus Antigen among Patients in an Uzbekistan Emergency Hospital.

    Science.gov (United States)

    Park, Se Jin; Han, Sung Sik; Anvarov, Khikmat; Khajibaev, Abdukhakim; Choi, Min-Ho; Hong, Sung-Tae

    2015-12-01

    Cystic echinococcosis (CE) is one of the most widespread zoonotic helminthiases, which can last an asymptomatic infection for several years. The purpose of this study was to demonstrate serum antibody prevalence of CE among asymptomatic people in Uzbekistan using ELISA. A total of 2,547 serum samples were collected, 66 from confirmed CE patients and 2,481 of patients with other diseases than CE at a hospital in Tashkent, Uzbekistan. The serum samples were screened for CE specific IgG antibodies by ELISA using cystic fluid antigen obtained from sheep. The serum antibody positive rate was 89.4% (59/66) in CE and 3.6% (89/2,481) in other disease patients. The present ELISA recognized 89.4% sensitivity and 96.4% specificity. The ELISA absorbance of positive samples was distributed 0.271-0.971 for CE and 0.273-0.887 for other disease patients. The other disease patients with high absorbance over 0.3 were 50 (2.0%) who were presumed to be active CE patients. The patients in their 40s showed the highest positive rate of 5.2% (P=0.181), and women were 4.4% while men were 3.1% positive (P=0.136). The data confirmed that there are many asymptomatic patients of CE in Tashkent. It is indicated that CE is an endemic disease of public health importance in Uzbekistan.

  3. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    Science.gov (United States)

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-05

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera.

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

    African Journals Online (AJOL)

    Failure to detect the two samples with viral loads considered above threshold of detection for antigen proteins suggested a lack of sensitivity by this assay to discover viral capsid protein in patient samples. Genotyping of these samples revealed genotype 1b, a HCV-subtype which is widespread and should thus be easily ...

  5. Refining the LPS-Antigen in Salmonella Antibody Elisa for Poultry Enhanced Specificity without Impairing Sensitivity

    DEFF Research Database (Denmark)

    Lauritsen, Klara Tølbøl; Lind, Peter; Klausen, Joan

    2014-01-01

    In the Danish serological surveillance for Salmonella in poultry (serum and egg yolk) a mix-ELISA is used, based on S. typhimurium and S. enteritidis antigens (Feld et al., 2000). When we evaluated results of the test retrospectively, over the years an unacceptably large fraction of seropositive...

  6. Production and characterization of a monoclonal antibody specific to 16 kDa antigen of Paramphistomum gracile.

    Science.gov (United States)

    Anuracpreeda, Panat; Watthanadirek, Amaya; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2017-01-01

    A number of monoclonal antibodies (MoAbs) against the 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg) were produced in vitro by hybridoma technique. Reactivity and specificity of these MoAbs were evaluated by ELISA and immunoblotting assays. Seven MoAb clones were selected from the stable hybridoma clones, namely 1D10, 2D7, 3B10, 3D9, 4F1, 4G4, and 5G12. It was found to be IgM and kappa light chain isotypes. By immunoblotting and ELISA, all MoAbs reacted with purified 16 kDaAgPg at molecular weight (MW) of 16 kDa and with the native 16 kDa antigen at MW of 16 kDa in the whole body (WB) and excretory-secretory (ES) fractions, but not with tegumental antigens (TA) of adult fluke. All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants, including Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Moniezia benedeni, Avitellina centripunctata, Haemonchus placei, Trichuris sp., and Setaria labiato-papillosa. Localization and distribution of the native 16 kDaAg in adult P. gracile by immunohistochemistry, using MoAbs as probes, showed that the native 16 kDaAg was present in high concentration in the cytoplasm of vitelline cells, eggshell globules, and the shells of eggs, but not in the tegument, muscle, parenchymal cells, and cecum of adult fluke. This finding indicated that the 16 kDaAg is a copiously expressed parasite protein that is released into the ES; thus, 16 kDaAg and its MoAb could be a good candidate for immunodiagnosis of paramphistomosis in ruminants.

  7. PEMBUATAN DAN STANDARISASI ANTIGEN AI H5N1 KOMERSIAL UNTUK MONITORING TITER ANTIBODI HASIL VAKSINASI AI DI INDUSTRI PETERNAKAN AYAM

    Directory of Open Access Journals (Sweden)

    Retno D. Soejoedono

    2012-04-01

    Full Text Available Vaccination is one of the chosen strategy for controling AI H5N1 in Indonesia. Vaccination able to induce protective antibodies against AI but unable to inhibit viral infection. Determination of antibody titers in the serum from bird vaccinated with AI-H5N1 vaccine consisting of 2 or 3 different AI virus isolates difficult to be meassured if the antigen for HI test is uncalibrated yet. Furthermore, the determination of a minimum protective antibody titer against the challenge of AI virus circulating in the field at this time needs to be done. This study aims to determine the H5N1 AI virus antigen for standart HI test and the minimum titre of antibodies that able neutralize virus infection. As much as 55 chickens were divided into 11 groups, 10 groups vaccinated with commercial AI vaccine and AI H5N1 field isolat antigen. Four types of commercial vaccines were veccinated to one group and seven other groups vaccinated with the antigen AI Legok 2004, Nagrak Ag 2009, Ag Lawang 2010, as well as polyvalent Ag combination of these three types of antigen. After third vaccinations, the presence of antibodieswere meassured by HI test. Serum with a titer test 26-28 were tested for the capability of virus neutralizationin using virus neutralization test against three different H5N1 AI virus field isolates. The test results showed that the H5N1 subtype AI virus antigen representative as standart antigen for HI test is antigen Legok 2004 and the minimum titer which able neutralize H5N1 AI virus field isolates 28

  8. Structures of a pan-specific antagonist antibody complexed to different isoforms of TGFβ reveal structural plasticity of antibody-antigen interactions.

    Science.gov (United States)

    Moulin, Aaron; Mathieu, Magali; Lawrence, Catherine; Bigelow, Russell; Levine, Mark; Hamel, Christine; Marquette, Jean-Piere; Le Parc, Josiane; Loux, Christophe; Ferrari, Paul; Capdevila, Cecile; Dumas, Jacques; Dumas, Bruno; Rak, Alexey; Bird, Julie; Qiu, Huawei; Pan, Clark Q; Edmunds, Tim; Wei, Ronnie R

    2014-12-01

    Various important biological pathways are modulated by TGFβ isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFβ neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFβ3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFβ1 and TGFβ2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFβ1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFβ2 to 2.83 Å. The structures provide further insight into the details of TGFβ recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoform-specific antibodies using a fresolimumab scaffold. © 2014 The Protein Society.

  9. SEVA TB ELISA - Multi antigen and antibody assays for serodiagnosis of suspected cases of pulmonary and extra pulmonary tuberculosis in tertiary care hospital -A retrospective study

    Directory of Open Access Journals (Sweden)

    Pranita J Waghmare

    2012-10-01

    Full Text Available Objective: To assess the usefulness of in-house developed multiantigen and antibody assays, in diagnosis of both pulmonary and extra-pulmonary tuberculosis. Method: Clinically suspected cases of 31 pulmonary and 171 extra-pulmonary tuberculosis (TB were screened by ELISA using cocktail (ES-31 + EST-6 antigen and their specific antibodies (anti ES-31 + anti EST-6 IgG for detection of antibody and and antigen (circulating antigen and immune complexed antigen respectively and correlated with antituberculosis therapy in retrospective study. Results: Out of 31 cases of pulmonary TB screened, 15 patients showed ELISA positivity out of which five cases were given antituberculosis therapy. Out of 171 cases of EPTB screened, 76 cases showed ELISA positivity out of which 18 were given antituberculosis therapy. Further 4 EPTB cases which showed AFB negativity were given ATT. The data was further analyzed based on PTB & EPTB, adults and children, OPD and IPD patients to understand false positivity in clinically suspected PTB and EPTB cases. There was significant correlation (108/202 cases with ELISA negativity and no ATT advised in clinically suspected PTB and EPTB patients. Conclusions: In house developed multi antigen and antibody assays have been observed to be quite useful as adjunct test in serodiagnosis of suspected cases of tuberculosis in particular extrapulmonary tuberculosis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-31

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

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

    Science.gov (United States)

    Starkie, Dale O; Compson, Joanne E; Rapecki, Stephen; Lightwood, Daniel J

    2016-01-01

    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 antibody from mice

  12. 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

  13. Cysticercosis in chronic psychiatric inpatients from a Venezuelan community.

    Science.gov (United States)

    Meza, Néstor W; Rossi, Nineth E; Galeazzi, Tatiana N; Sánchez, Nora M; Colmenares, Francisco I; Medina, Oscar D; Uzcategui, Néstor L; Alfonzo, Nacarid; Arango, Celso; Urdaneta, Haideé

    2005-09-01

    Cysticercosis due to Taenia solium infection is endemic in developing countries of the Americas, Asia, and Africa. This study was designed to establish the prevalence of cysticercosis in 158 inpatients of a psychiatric institution in the state of Tachira (Venezuela) and in 127 healthy control subjects. Positive blood tests for cysticercosis by Western blotting were recorded in 18.35% of the patients and in 1.57% of the controls. Individuals with mental retardation were found to carry an increased risk of cysticercosis (RR: 2.92; 1.22 7.0; P health care system.

  14. Detection of antibodies against hepatitis B core antigen using the avidin-biotin system.

    Science.gov (United States)

    Korec, E; Korcová, J; König, J; Hlozánek, I

    1989-06-01

    An enzyme avidin-biotin assay for the detection of anti-HBcAg antibody in human sera was developed. The assay uses genetically engineered HBcAg. HBcAg is immobilized on the surface of the wells of microtitre plates and the test serum sample, biotin-labelled HBcAg and streptavidin-labelled horseradish peroxidase are added. The assay was found to be specific and was compared with a commercial radioimmunoassay kit for sensitivity by testing 96 human clinical sera for anti-HBcAg antibody. Both assays gave identical results.

  15. High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on Staphylococcus aureus Antigen MntC.

    OpenAIRE

    Alexey V Gribenko; Kevin Parris; Lidia Mosyak; Sheng Li; Luke Handke; Julio C Hawkins; Elena Severina; Yury V Matsuka; Annaliesa S Anderson

    2016-01-01

    The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three i...

  16. Expression of class 5 antigens by meningococcal strains obtained from patients in Brazil and evaluation of two new monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Elizabeth N. De Gaspari

    Full Text Available Determining the profile of antigen expression among meningococci is important for epidemiologic surveillance and vaccine development. To this end, two new mouse monoclonal antibodies (MAbs have been derived against Neisseria meningitidis proteins (class 5. The MAbs were reactive against outer membrane antigens and were bactericidal. Selected anti-class 5 MAbs [(5.1-3E6-2; (5.3-3BH4-C7; (5.4-1BG11-C7; (5.5-3DH-F5G9 also 5F1F4-T3(5.c], and the two new monoclonal antibodies C14F10Br2 (5.8 and 7F11B5Br3 (5.9, were then tested against different meningococcal strains, (63 strains of serogroup A, 60 strains of serogroup C (from 1972 to 1974; and 136 strains of serogroup B (from 1992 meningococci. Our results demonstrated that the expression of class 5 proteins in the N. meningitidis B Brazilian strains studied is highly heterogeneous. The serotypes and subtypes of B:4:P1.15, B:4:P1.9, B:4:P1.7, B:4:P1.3, B:4:P1.14, B:4:P1.16, B:4:NT, and B:NT:NT were detected in N. meningitidis B serogroups.The strains C:2a:P1.2 and A:4.21:P1.9 were dominant in the C and A serogroups, respectively. Serogroup B organisms expressed the class 5 epitopes 5.4 (18%, 5.5 (22%, 5.8 (3.6%, 5.9 (8.0% and 5c (38%. Serogroup C expressed class 5 epitopes 5.1 (81%, 5.4 (35%, 5.5 (33% and 5.9 (5.0%; and serogroup A showed reactivity directed at the class 5 protein 5c (47%; and reactivity was present with the new monoclonal antibody, 5.9 (5.5%. We conclude that the two new MAbs are useful in detecting important group B, class 5 antigens, and that a broad selection of serogroup B, class 5 proteins would be required for an effective vaccine based on the class 5 proteins.

  17. Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 antigens as markers for early and late infection in dogs.

    Science.gov (United States)

    Wagner, Bettina; Freer, Heather; Rollins, Alicia; Garcia-Tapia, David; Erb, Hollis N; Earnhart, Christopher; Marconi, Richard; Meeus, Patrick

    2012-04-01

    Lyme disease in the United States is caused by Borrelia burgdorferi sensu stricto, which is transmitted to mammals by infected ticks. Borrelia spirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses to B. burgdorferi Osp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

  18. Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

    Science.gov (United States)

    Freer, Heather; Rollins, Alicia; Garcia-Tapia, David; Erb, Hollis N.; Earnhart, Christopher; Marconi, Richard; Meeus, Patrick

    2012-01-01

    Lyme disease in the United States is caused by Borrelia burgdorferi sensu stricto, which is transmitted to mammals by infected ticks. Borrelia spirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses to B. burgdorferi Osp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs. PMID:22336289

  19. Antibody response to a T-cell-independent antigen is preserved after splenic artery embolization for trauma.

    Science.gov (United States)

    Olthof, D C; Lammers, A J J; van Leeuwen, E M M; Hoekstra, J B L; ten Berge, I J M; Goslings, J C

    2014-11-01

    Splenic artery embolization (SAE) is increasingly being used as a nonoperative management strategy for patients with blunt splenic injury following trauma. The aim of this study was to assess the splenic function of patients who were embolized. A clinical study was performed, with splenic function assessed by examining the antibody response to polysaccharide antigens (pneumococcal 23-valent polysaccharide vaccine), B-cell subsets, and the presence of Howell-Jolly bodies (HJB). The data were compared to those obtained from splenectomized patients and healthy controls (HC) who had been included in a previously conducted study. A total of 30 patients were studied: 5 who had proximal SAE, 7 who had distal SAE, 8 who had a splenectomy, and 10 HC. The median vaccine-specific antibody response of the SAE patients (fold increase, 3.97) did not differ significantly from that of the HC (5.29; P = 0.90); however, the median response of the splenectomized patients (2.30) did differ (P = 0.003). In 2 of the proximally embolized patients and none of the distally embolized patients, the ratio of the IgG antibody level postvaccination compared to that prevaccination was <2. There were no significant differences in the absolute numbers of lymphocytes or B-cell subsets between the SAE patients and the HC. HJB were not observed in the SAE patients. The splenic immune function of embolized patients was preserved, and therefore routine vaccination appears not to be indicated. Although the median antibody responses did not differ between the patients who underwent proximal SAE and those who underwent distal SAE, 2 of the 5 proximally embolized patients had insufficient responses to vaccination, whereas none of the distally embolized patients exhibited an insufficient response. Further research should be done to confirm this finding. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

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

    2017-03-01

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

  1. The kinetics of antibody binding to Plasmodium falciparum VAR2CSA PfEMP1 antigen and modelling of PfEMP1 antigen packing on the membrane knobs

    Directory of Open Access Journals (Sweden)

    Arnot David E

    2010-04-01

    Full Text Available Abstract Background Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target. Methods A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob. Results Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparum-infected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the knobs and the possible densities of PfEMP1 packing on the knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the knobs and the large size of PfEMP1 molecules. Conclusions High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18 nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable

  2. High-resolution characterization of antibody fragment/antigen interactions using Biacore T100.

    Science.gov (United States)

    Papalia, Giuseppe A; Baer, Mark; Luehrsen, Kenneth; Nordin, Helena; Flynn, Peter; Myszka, David G

    2006-12-01

    A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.

  3. Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis Toxin

    Science.gov (United States)

    1988-01-21

    characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin. Infect. Immun. 50:442-448. 32. Wright, 6. 6., and 6. L...and inhibits growth of certain cell lines (15). No enzymatic activity has been identified for LF. Both the lethal and edema toxins inhibit the

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    Hens have a tremendous capacity for producing polyclonal antibodies that can subsequently be isolated in high concentrations from their eggs. An approach for further maximizing their potential is to produce multiple antisera in the same individual through multiplexed (multiple simultaneous) immun...

  5. Autoimmune response of IgE antibodies to cellular self-antigens in systemic Lupus Erythematosus.

    Science.gov (United States)

    Atta, Ajax Mercês; Santiago, Mittermayer Barreto; Guerra, Fernanda Garcia; Pereira, Mariana Menezes; Sousa Atta, Maria Luiza B

    2010-01-01

    Systemic lupus erythematosus (SLE) patients may exhibit high total IgE and antinuclear IgE antibodies (ANA-IgE). Here, we investigated the specificity of ANA-IgE in SLE patients and the involvement of cytokines in this immune response. Sera from 92 SLE patients and 68 healthy controls were evaluated for the presence of antinuclear IgE antibodies by immunoperoxidase with HEp-2,000(R) cells and immunoblotting with IgG-depleted sera. Total IgE, IgE specific to allergens, and serum cytokine levels were determined by ELISA. Antinuclear IgE antibodies were detected only in SLE patients (29/92, 31.5%). High total IgE was associated with ANA-IgE (p seronegative patients (p seronegative patients (p < 0.05) or healthy controls (p = 0.001). Controls displayed higher IL-5/IFN-gamma ratios than either SLE patients with ANA-IgE (p < 0.05) or patients without these immunoglobulins (p < 0.01). We conclude that IgE antibodies against cell autoantigens involved in protein expression, cellular proliferation, and cell death are present in patients with SLE. Interleukin-10 seems to down-regulate this IgE autoimmune response in SLE. Copyright 2010 S. Karger AG, Basel.

  6. Evaluation of recombinant HP6-Tsag, an 18 kDa Taenia saginata oncospheral adhesion protein, for the diagnosis of cysticercosis.

    Science.gov (United States)

    Ferrer, Elizabeth; González, Luís Miguel; Martínez-Escribano, José Angel; González-Barderas, María Eugenia; Cortéz, María Milagros; Dávila, Iris; Harrison, Leslie J S; Parkhouse, R Michael E; Gárate, Teresa

    2007-08-01

    With the objective of providing inexpensive and reproducible assays for the detection of antibodies indicating exposure to Taenia saginata and Taenia solium, we have evaluated the diagnostic utility of the T. saginata oncosphere adhesion protein (HP6-Tsag), expressed in baculovirus (HP6-Bac) and bacteria (HP6-GST [glutathione S-transferase]), employing enzyme-linked immunosorbent assays (ELISAs) and sera from T. saginata infected cattle, T. solium infected pigs and serum and cerebrospinal fluid (CSF) samples from clinically defined T. solium neurocysticercosis (NCC) patients. The two recombinant proteins were antigenic in all three systems, with the signal to background ratio of the HP6-Bac ELISA slightly higher than that for the HP6-GST ELISA. Assay performance in cattle was similar to previously described peptide-based ELISA assays, although NCC sample sensitivity/specificity was marginally better. The sensitivity of the HP6-Bac and HP6-GST ELISAs was close for active human NCC (77.4 and 80.6% for serum and 76.9 and 73.1% for CSF samples, respectively). In inactive human NCC, however, the sensitivity of the HP6-Bac ELISA was almost twice that of the HP6-GST ELISA. Because peptides are relatively expensive and recombinant proteins are simple and economical to produce, the latter may provide useful reagents for antibody detection in countries with endemic cysticercosis/NCC.

  7. Lingual palpation for porcine cysticercosis: a rapid epidemiological tool for estimating prevalence and community risk in Africa.

    Science.gov (United States)

    Guyatt, Helen L; Fèvre, Eric M

    2016-10-01

    To assess the association between the prevalence of tongue cyst-positive and antigen-positive pigs across different settings in Africa, to evaluate whether examining pigs for cysts could be used as a rapid surveillance tool for identifying geographical areas with a higher probability of high transmission of cysticercosis. Published data were collated from 26 study sites across Africa that reported the prevalence of porcine cysticercosis by both lingual and serological examinations. The study sites were located in 10 countries across Africa. Seroprevalence rates ranged from 4% to 41%. Despite the varied study sites, the relationship between the two variables was highly consistent and suggests identification of tongue cysts may be useful for cysticercosis surveillance. We found that all areas with more than 10% of pigs having cysts in their tongues had at least 30% seroprevalence (PPV of 100%), although this cut-off is less reliable at predicting that an area is of low transmission (NPV of 84%). Assessing the prevalence of tongue cyst-positive pigs is a potential rapid epidemiological tool for identifying areas at high risk of cysticercosis, although further refinement and validation is required using standardised data sets. © 2016 The Authors. Tropical Medicine & International Health Published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2017-08-21

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

  9. Cardiac-oxidized antigens are targets of immune recognition by antibodies and potential molecular determinants in chagas disease pathogenesis.

    Directory of Open Access Journals (Sweden)

    Monisha Dhiman

    Full Text Available Trypanosoma cruzi elicits reactive oxygen species (ROS of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE, malondialdehyde (MDA, carbonyl, and 3-nitrotyrosine (3-NT adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE/Western blotting (WB was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats and human patients. Human cardiomyocytes exhibited LD(50 sensitivity to 30 µM 4-HNE and 100 µM H(2O(2 at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H(2O(2 resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans. Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic

  10. Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

    Directory of Open Access Journals (Sweden)

    Rong-Hong Hua

    Full Text Available Japanese encephalitis virus (JEV non-structural protein 1 (NS1 contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA, five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5AIDITRK(11, (72RDELNVL(78, (251KSKHNRREGY(260, (269DENGIVLD(276, and (341DETTLVRS(348. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

  11. Preparasi Imunoglobulin G Kelinci sebagai Antigen Penginduksi Antibodi Spesifik Terhadap Virus Avian Influenza H5N1 Strain Legok

    Directory of Open Access Journals (Sweden)

    Ketut Karuni Nyanakumari Natih

    2010-06-01

    Full Text Available The aim of this research was to prepare rabbit Immunoglobulin G as anti-idiotype antibody (Ab2 ofAvian Influenza Virus (AIV H5N1. A polyclonal antibody was collected from guinea pigs immunized withinactivated AI vaccine H5N1of Legok strain. Antibody of H5N1 AI in serum was detected by Agar gelprecipitation test (AGPT and an Inhibition Hemmaglutination test (IHT. The highest titre of antibodywas obtained one week after the third immunization. Serum of guinea pigs containing IgG was purifiedusing the Montage Antibody purification kit & spin column with Prosep A media (Millipore. The AI H5N1IgG concentration was 8 mg/ml. AI H5N1 IgG, was then digested with pepsin to obtain F(ab2 fraction andwas called Ab1. The concentration of IgG and F(ab2 and purity of IgG were determined by UVspectrophotometer which showed Ab1 concentration 1 mg/ml. Molecular weight was estimated by sodiumdodecyl sulfate- polyacrilamide gel electrophoresis (SDS-PAGE. Ab2 was produced by immunization ofrabbit with Ab1. The first immunization was carried out by subcutaneous injection with 500 ?g of Ab1emulsified in Complete Freund Adjuvant. The immunization was repeated with the same dose of Ab1emulsified in Incomplete Freund Adjuvan at 1 week intervals. One week after the second immunization,rabbit’s serum was harvested and IgG was purified using the Montage Antibody purification kit & spincolumn with Prosep A media (Millipore. The rabbit IgG, called Ab2, was an anti-idiotypic antibody againstAIV-H5N1. In AGPT, a precipitation line appeared between Ab1 and Ab2. A partial reaction appearedbetween Ab2 and the AI H5N1 antigen was also detected. The results indicated that Ab2 is a possiblecandidate of imunogen for protection against an AI virus H5N1 infection.

  12. Antibodies against a class II HLA-peptide complex raised by active immunization of mice with antigen mimicking peptides

    DEFF Research Database (Denmark)

    Dam-Tuxen, R; Riise, Erik Skjold

    2009-01-01

    Multiple sclerosis (MS) is an autoimmune disease linked to the human leucocyte antigen (HLA) class II genes DRB1*1501, DRB5*0101 and DQB1*0602. T cells reactive towards the DRB1*1501 in complex with various peptides derived from myelin basic protein (MBP), which is the major component of myelin......, have been found in the peripheral blood of MS patients. These autoreactive T cells are believed to play a role in the pathogenesis of MS. In this article, antibodies against the HLA complex DR2b (DRA1*0101/DRB1*1501) in complex with the MBP-derived peptide MBP(85-99) have been generated by immunization...

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    Monoclonal antibodies (MAbs) against the alpha- and beta-chain of lymphocyte-associated antigen-1 (LFA-1) were examined for inhibition of HIV-1 infection in vitro. Infection of the T cell line MT4 and the monocytic cell line U937 by isolates HTLVIIIB and SSI-002, respectively was inhibited...... in a concentration dependent manner by MAb against the beta-chain but not against the alpha-chain. No cross-reactivity was found between MAb against LFA-1 and against the CD4 receptor (MAb Leu3a). MAbs against the beta-chain and the CD4 receptor were found to act synergistically in inhibiting HIV infection....... These data indicate that the beta-chain of LFA-1 in addition to the CD4 receptor may be involved in HIV infection in vitro....

  14. A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

    Science.gov (United States)

    Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

    2014-01-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

  15. Computerized tomographic evaluation of cerebral cysticercosis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Bo Young; Lee, Mi Sook; Jeon, Doo Sung; Kim, Hong Soo; Rhee, Hak Song [Precbyterian Medical Center, Chonju (Korea, Republic of)

    1988-08-15

    Cerebral cysticercosis, unfortunately frequent in Korea, is a parastic disease in which man serve as the intermediate host of taenia solium. The larvae have a predilection for the central nervous system and can cause a variety of neurologic symptoms. The authors reviewed 19 cases of surgically proven cerebral cysticercosis and following results were obtained. 1. The most frequent age distribution was 5th and 6th decade and male to female ratio was 14:5. 2. The most frevalent involving site was cerebral parenchyme and following by ventricles. 3. Clinical manifestations were symtom and sign of increased ICP, seizure and focal neurological dificit. 4. It was assumed that computerized tomography was the procedure of choice for the diagnosis of these parasitic brain disease.

  16. Control of Taenia solium taeniasis/cysticercosis

    DEFF Research Database (Denmark)

    Gabriël, Sarah; Dorny, Pierre; Mwape, Evans Kabemba

    2017-01-01

    level, improved standards of meat inspection, pig management, treatment of individual patients and possibly human populations, and treatment and/or vaccination of porcine populations. This manuscript looks critically into currently existing control options and provides suggestions on which (combination...... of) tools would be most effective in the control of T. solium taeniasis/cysticercosis in sub-Saharan Africa. Field data and disease transmission simulations suggest that implementation of a single intervention control strategy will not lead to a satisfactory reduction of disease morbidity......Taenia solium taeniasis/cysticercosis is a neglected parasitic zoonosis with significant economic and public health impacts. Control measures can be broadly grouped into community health education, improvements in hygiene and sanitary conditions, proper meat handling at household and community...

  17. Immunochemical characterization and purification of Sm-97 a Schistosoma manosin antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine

    Energy Technology Data Exchange (ETDEWEB)

    Pearce, E.J.; James, S.L.; Dalton, J.; Barrall, A.; Ramos, C.; Strand, M.; Sher, A.

    1986-12-01

    Mice protected against Shistosoma mansoni infection by intradermal (i.d.) vaccination with nonliving schistosomula or soluble extracts of larval or adult schistosomes (SCHLARP and SWAP, respectively) produce antibodies that react by Western blot analysis with one antigen of M/sub r/ (x 10/sup -3/) 97 in SWAP prepared in the presence of protease inhibitors and two antigens of M/sub r/ (x 10/sup -3/) 95 and 78 in SWAP prepared in their absence. Vaccine antibodies also immunoprecipitated a single 97k molecule, with a pI of 5.5, from detergent extracts of (/sup 35/S)methionine-labeled schistosomes. Three hybridomas, produced from spleen cells of i.d. immunized mice, all recognized both the 95k/78k doublet and one 97k antigen, indicating that the two lower M/sub r/ components are degradation products of the same 97k molecule. /sup 125/I-concanavalin a bound weakly to purified Sm-97, indicating that this antigen is minimally glycosylated. Competitive radioimmunoassays performed with /sup 125/I-labeled monoclonal antibodies and purified antigen defined at least two distinct epitopes on Sm-97. Antibodies from i.d. vaccinated mice recognized both monoclonal antibody-defined epitopes, whereas anti-Sm-97 antibodies in chronic infection sera recognized neither. Finally, purified Sm-97 was shown to elicit delayed-type hypersensitivity in i.d. vaccinated mice, suggesting that this molecule is also capable of evoking cell-mediated responses, a finding consistent with its proposed function as a vaccine immunogen.

  18. Validation of an immunohistochemical assay for bovine cysticercosis, with comparison to a standard histological method.

    Science.gov (United States)

    Scandrett, W Brad; Haines, Deborah M; Parker, Sarah E; Robinson, Yves; Forbes, Lorry B; Brandt, Jef; Geerts, Stanny; Dorny, Pierre; Gajadhar, Alvin A

    2012-05-25

    The larval stage (syn Cysticercus bovis) of the human tapeworm Taenia saginata causes cysticercosis in cattle, which has both aesthetic and food safety implications to consumers of beef. A monoclonal antibody-based immunohistochemical (IHC) assay developed to improve postmortem diagnosis of this parasite and a standard histological method were assessed to determine their fitness for intended use. Sections from 169 known-positive specimens of T. saginata from experimentally or naturally infected cattle, and from 30 known-negative specimens and lesions of various etiologies from non-infected cattle, were tested. The IHC assay identified significantly more known positive bovine cysticerci than the histological method (91.7% and 38.5%, respectively). Positive IHC staining occurred on sections from other cestode species, but should not affect the diagnostic specificity of this assay for bovine cysticercosis, due to the different host and/or tissue preferences amongst these parasites. Use of the IHC assay should improve the reliability of diagnosing lesions caused by degenerated cysticerci, facilitating more effective and efficient control of bovine cysticercosis. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  19. Current Concepts and Future Directions for the Assessment of Autoantibodies to Cellular Antigens Referred to as Anti-Nuclear Antibodies

    Directory of Open Access Journals (Sweden)

    Michael Mahler

    2014-01-01

    Full Text Available The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA, is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD. Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. In addition, solid phase immunoassays using well characterized antigens have gained widespread adoption in high throughput laboratories due to their ease of use and open automation. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. This review provides a comprehensive overview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests an approach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges.

  20. Seroprevalence of antibodies against the excreted antigen superoxide dismutase by Trypanosoma cruzi in dogs from the Yucatan Peninsula (Mexico).

    Science.gov (United States)

    López-Cespedes, A; Longoni, S S; Sauri-Arceo, C H; Rodríguez-Vivas, R I; Villegas, N; Escobedo-Ortegón, J; Barrera-Pérez, M A; Sánchez-Moreno, M; Bolio González, M E; Marín, C

    2013-06-01

    Numerous studies have shown the role of dogs as a reservoir for the American trypanosomiasis, as the bridge connecting sylvatic and peridomestic cycles. The objective of this study was to determine the prevalence of American trypanosomiasis in the dog population (630 sera) from seven localities in the Yucatan Peninsula (city of Mérida and the towns of Molas, Playa del Carmen, Akumal, Xcalacoop, Xcalac and Xahuachol). These data are key for developing control measures for the disease. The sera were analysed to detect antibodies against Trypanosoma cruzi, using Fe-SOD excreted as the antigenic fraction by ELISA and Western blot as confirmation. The total prevalence found in the Yucatan Peninsula was some 14.76%, with 10.74% in the state of Yucatan (city of Mérida, towns of Molas and Xcalacoop) and 21.34% in the state of Quintana Roo (towns of Playa del Carmen, Akumal, Xcalac and Xahuachol). However, a more thorough epidemiological study of the dog population, both wild and urban, in the Yucatan Peninsula will be required to design a control strategy for these diseases, paying particular attention to the population affected and even broadening the study to other Mexican states as well as neighbouring countries. These results again confirm that iron-superoxide dismutase excreted by T. cruzi constitutes a good source of antigen for serodiagnosis in epidemiological studies. © 2012 Blackwell Verlag GmbH.

  1. Transient human anti-mouse antibody generated with immune enhancement in a carbohydrate antigen 19-9 immunoassay after surgical resection of recurrent cancer.

    Science.gov (United States)

    Nakano, Keiichi; Yasuda, Keiko; Shibuya, Hitoshi; Moriyama, Takanori; Kahata, Kaoru; Shimizu, Chikara

    2016-07-01

    We report a case of transient human anti-mouse antibody from a 64-year-old man in a carbohydrate antigen 19-9 immunoassay using an AIA 1800 analyser that generated immune enhancement after surgical resection of recurrent cancer. The carbohydrate antigen 19-9 concentration was measured using an AIA 1800 analyser and a UniCel Dxl 800. Size-exclusion high-performance liquid chromatography was carried out on a Superose 12 column to estimate the carbohydrate antigen 19-9 elution profile using an AIA 1800 analyser. To determine whether IgM in the patient contributed to the carbohydrate antigen 19-9 immunoassay, immunoprecipitation was performed. Furthermore, mouse immunoglobulins were added to the patient's serum to verify that the patient's IgM reacted with it. The carbohydrate antigen 19-9 concentration was >400 and 9.5 kU/L using an AIA 1800 analyser and using a UniCel Dxl 800, respectively. In the single carbohydrate antigen 19-9 peak, the molecular weight corresponded to IgM by size-exclusion high-performance liquid chromatography on a Superose 12 column. In the immunoprecipitation reaction and addition of mouse immunoglobulins, there was interference for anti-human IgM and mouse immunoglobulins whose recoveries were 3.2 and 14.2%, respectively. These results indicated that IgM in the patient's serum interfered with the carbohydrate antigen 19-9 immunoassay using an AIA 1800 analyser. A novel transient human anti-mouse antibody generated with immune activation in a carbohydrate antigen 19-9 immunoassay using an AIA 1800 analyser was identified in a patient with rectal cancer after surgical resection. These findings demonstrate the importance of monitoring tumour markers in patients after treatment with mouse monoclonal antibody. © The Author(s) 2016.

  2. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

    OpenAIRE

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-01-01

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) te...

  3. Evaluation of an immunoblot methodology for the detection of relevant Entamoeba histolytica antigens by antibodies induced in human amebiasis.

    Science.gov (United States)

    Argüello-García, R; Sánchez-Guillén, M C; Garduño, G; Valadez-Salazar, A; Martínez-García, M C; Muñoz, O; Ortega-Pierres, M G

    1990-01-01

    The complexity of the clinical spectrum in human amebiasis and the high variability in laboratory methods used to detect Entamoeba histolytica infections have impeded the collection and evaluation of reliable epidemiological data. Thus, more sensitive, specific and standardized methods are needed in order to accurately identify infections with this parasite. An important step in the development of serological diagnostic methods is the identification and isolation of specific parasite antigens which are immunogenic in the host. In this work, we have standardized an electroimmunotransfer blot technique to characterize E. histolytica antigens recognized by antibodies present during human amebic infections. An important aspect was an investigation of technical variations in the preparation of cell lysates including the use of different protease inhibitors and solubilizing agents. The highest yield of protein was achieved by homogenization of trophozoites in the presence of 10 mM p-hydroxymercuribenzoate (pHMB) as a protease inhibitor and by lysis using Triton X-100 and a mixture of protease inhibitors. Recovery of degraded vs non-degraded proteins in the cell extracts was evaluated by gradient polyacrylamide gel electrophoresis. Both quantitative and qualitative differences were noted between the different methods of preparing soluble cell extracts. A more complete set of antigenic components was obtained by homogenization and use of pHMB. Thus parasite extracts prepared by this method were selected for protein transfer. In this, the optimal protein concentration was of 120 micrograms of protein per cm of gel width and efficient transfer of proteins to nitrocellulose sheets was achieved at 100 V for 2 hrs and at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Computed tomographic findings of intracerebral cysticercosis

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Jin Kyo; Lee, Sun Wha; Kim, Ho Kyun; Ahn, Chi Yul [School of Medicine, Kyung-Hee University, Seoul (Korea, Republic of)

    1980-12-15

    Cysticercosis is a parasitic disease in which man serves as the intermediate host of Taenia Solium, the pork tapeworm. The computed tomographic findings of 25 cases of intracerebral cysticercosis proven by pathologic and/or clinical findings during past 2 years were analysed. The results were as follows; 1. The sex was 19 males and 6 females, and 56 percent of the patients were seen in fourth and fifth decades. The most common symptom was epilepsy (72%). 2. The C. T. findings in precontrast study were varied; such as ill defined low density (48%), cystic low density (20%), dilated ventricles (20%), ill defined low density with isodense nodule (18%), cystic low density with isodense mural nodule (12%) and calcification (8%). 3. The areas of involvement were 20 cases (80%) of parenchymal form, 3 cases (12%) of ventricular form and 2 cases (8%) of mixed form. 4. The contrast-enhanced 13 cases were 5 nodular, 5 ring or rim-like and 3 mixed type enhancements, while 12 cases were not enhanced. 5. C.T. scan demonstrated more precise location and extents of cerebral cysticercosis, especially in parenchymal form. It was considered to be important in determination of surgical feasibility and its approach.

  5. Antibody responses to a major Pneumocystis carinii antigen in human immunodeficiency virus-infected patients with and without P. carinii pneumonia

    DEFF Research Database (Denmark)

    Lundgren, Bettina; Lundgren, Jens Dilling; Nielsen, T

    1992-01-01

    Antibody responses to a major purified human Pneumocystis carinii surface antigen (gp95) were determined by ELISA in human immunodeficiency virus (HIV)-infected patients. Serum IgG directed against gp95 was measured in 129 consecutive HIV-infected patients who underwent bronchoscopy for evaluation...... was higher (mean optical density ratio: 0.6 vs. 0.23 and 0.2, respectively; P less than .01). Changes in antibody response were investigated in 78 patients for whom serial serum samples taken around the time of bronchoscopy were available. Of the 47 patients with verified PCP, 20 (43%) mounted an antibody...

  6. Identification of Eps15 as antigen recognized by the monoclonal antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila brain.

    Directory of Open Access Journals (Sweden)

    Partho Halder

    Full Text Available The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15 as the antigen for these two antibodies.

  7. Studies on mixed agglutination with special reference to the A antigen and the physical properties of the operative antibodies

    Science.gov (United States)

    Coombs, R. R. A.; Franks, D.; Gurner, B. W.; Polley, Margaret J.; Richards, C. B.

    1965-01-01

    Differences in reactions in the mixed agglutination test with anti-A sera from different individuals were found to be unrelated to the titre against human or pig A red cells or simply to the presence or absence of either 7S or 19S antibody. It was found that 7S anti-A was capable of producing mixed agglutination, but that 19S fractions more frequently produced mixed agglutination than the 7S fraction from the same serum. Treatment of sera with 2-mercapto-ethanol abolished the capacity to produce mixed agglutination from some sera, whilst other sera were unaffected. Some anti-A sera gave the same titre of mixed agglutination with both secretor and non-secretor buccal cells, but other sera reacted more strongly with secretor than with non-secretor cells. Production of mixed agglutination by 7S antibodies was also demonstrated with fractions of rabbit antisera against Forssman antigen and against bovine red cells. ImagesFIG. 2 PMID:14295642

  8. The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody.

    Science.gov (United States)

    Douglas, Alexander D; Williams, Andrew R; Illingworth, Joseph J; Kamuyu, Gathoni; Biswas, Sumi; Goodman, Anna L; Wyllie, David H; Crosnier, Cécile; Miura, Kazutoyo; Wright, Gavin J; Long, Carole A; Osier, Faith H; Marsh, Kevin; Turner, Alison V; Hill, Adrian V S; Draper, Simon J

    2011-12-20

    Current vaccine strategies against the asexual blood stage of Plasmodium falciparum are mostly focused on well-studied merozoite antigens that induce immune responses after natural exposure, but have yet to induce robust protection in any clinical trial. Here we compare human-compatible viral-vectored vaccines targeting ten different blood-stage antigens. We show that the full-length P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) is highly susceptible to cross-strain neutralizing vaccine-induced antibodies, out-performing all other antigens delivered by the same vaccine platform. We find that, despite being susceptible to antibody, PfRH5 is unlikely to be under substantial immune selection pressure; there is minimal acquisition of anti-PfRH5 IgG antibodies in malaria-exposed Kenyans. These data challenge the widespread beliefs that any merozoite antigen that is highly susceptible to immune attack would be subject to significant levels of antigenic polymorphism, and that erythrocyte invasion by P. falciparum is a degenerate process involving a series of parallel redundant pathways.

  9. A mucosal IgA response, but no systemic antibody response, is evoked by intranasal immunisation of dogs with Echinococcus granulosus surface antigens iscoms.

    Science.gov (United States)

    Carol, H; Nieto, A

    1998-09-16

    The search for protective antigens of intestinal parasites is conditioned by the methodology used to induce a relevant local immune response against them. The present work describes the use of immuno stimulating complexes (iscoms) from tegumental antigens from protoscoleces (PSC) of the cestode Echinococcus granulosus as immunogens in dogs by the intranasal route. It also describes the evaluation of the immune response evoked at the antibody level (systemically and at a distant mucosal location) as well as at the level of antibody secreting cells in peripheral blood. Iscoms from both E. granulosus tegumental antigens and hen ovalbumin (OVA), given at 50 microg doses by intranasal route, evoked significant secretory IgA antibody responses detected in saliva. Specific IgA secreting cells in peripheral blood also increased 10-20-fold, although transiently, after primary and secondary stimulation, whereas specific IgG secreting cells in peripheral blood were only detected in some individuals after the second antigenic exposure. Generation of immune responses at a related mucosal site provides evidence of localised immunity. No significant increase in systemic antibody titers of either IgM, IgG or IgA isotype was detected in plasma as a result of the immunisation. This fact could reflect that the nasopharyngeal mucosal associated lymphoid tissue of dogs is more strictly compartmentalised than that of other mammals.

  10. Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); N. Juntti; B. Klingeborn; J. Groen (Jan); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractAn enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of

  11. Novel Non-Histocompatibility Antigen Mismatched Variants Improve the Ability to Predict Antibody-Mediated Rejection Risk in Kidney Transplant

    Directory of Open Access Journals (Sweden)

    Silvia Pineda

    2017-12-01

    Full Text Available Transplant rejection is the critical clinical end-point limiting indefinite survival after histocompatibility antigen (HLA mismatched organ transplantation. The predominant cause of late graft loss is antibody-mediated rejection (AMR, a process whereby injury to the organ is caused by donor-specific antibodies, which bind to HLA and non-HLA (nHLA antigens. AMR is incompletely diagnosed as donor/recipient (D/R matching is only limited to the HLA locus and critical nHLA immunogenic antigens remain to be identified. We have developed an integrative computational approach leveraging D/R exome sequencing and gene expression to predict clinical post-transplant outcome. We performed a rigorous statistical analysis of 28 highly annotated D/R kidney transplant pairs with biopsy-confirmed clinical outcomes of rejection [either AMR or T-cell-mediated rejection (CMR] and no-rejection (NoRej, identifying a significantly higher number of mismatched nHLA variants in AMR (ANOVA—p-value = 0.02. Using Fisher’s exact test, we identified 123 variants associated mainly with risk of AMR (p-value < 0.001. In addition, we applied a machine-learning technique to circumvent the issue of statistical power and we found a subset of 65 variants using random forest, that are predictive of post-tx AMR showing a very low error rate. These variants are functionally relevant to the rejection process in the kidney and AMR as they relate to genes and/or expression quantitative trait loci (eQTLs that are enriched in genes expressed in kidney and vascular endothelium and underlie the immunobiology of graft rejection. In addition to current D/R HLA mismatch evaluation, additional mismatch nHLA D/R variants will enhance the stratification of post-tx AMR risk even before engraftment of the organ. This innovative study design is applicable in all solid organ transplants, where the impact of mitigating AMR on graft survival may be greater, with considerable benefits on

  12. Increased Avidity of the Sambucus nigra Lectin-Reactive Antibodies to the Thomsen-Friedenreich Antigen as a Potential Biomarker for Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Oleg Kurtenkov

    2015-01-01

    Full Text Available Aim. To determine whether the naturally occurring Thomsen-Friedenreich (TF antigen-specific antibodies differ in avidity between cancer patients and controls to find a novel biomarker for stomach cancer. Methods. Serum samples were taken from patients with cancer and controls. The level of TF-specific antibodies and their sialylation were determined using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specific Sambucus nigra agglutinin (SNA. The avidity was determined using ammonium thiocyanate as a chaotrope. Results. A significantly higher SNA lectin binding to anti-TF antibodies was found in cancer patients irrespective of disease stage. The avidity of only IgM TF-specific antibodies was significantly higher in cancer patients compared to controls. The SNA-positive anti-TF antibodies of cancer patients showed a significantly higher avidity, P<0.001. The sensitivity and specificity of this increase for gastric cancer were 73.53% and 73.08%, respectively, with a 73.2% diagnostic accuracy. The higher avidity of SNA-reactive anti-TF antibodies was associated with a benefit in survival of stage 3 cancer patients. Conclusion. The SNA-reactive TF-specific antibodies display a significantly higher avidity in gastric cancer patients compared to controls, which can be used as a potential serologic biomarker for gastric cancer. It appears that IgM is the main target responsible for the above changes.

  13. Recombinant envelope protein of HIV-1 subtype E as antigen in HIV-1 antibody detection enzyme immunoassay.

    Science.gov (United States)

    Sutthent, Ruengpung; Kanoksinsombat, Chinda; Horthongkham, Navin; Louisirirotchanakul, Suda; Auewarakul, Prasert; Kantakamalakul, Wannee

    2002-06-01

    In order to develop a reliable and inexpensive serodiagnostic method to be used for anti-HIV antibody detection in Thailand, recombinant envelope (TM or gp41 subunit) protein of HIV-1 subtype E was produced from prokaryotic cell (Escherichia coli) as the source of antigen in enzyme immunoassay (TE diagnostic EIA kit). HIV-1 gp41 subunit of subtype E was successfully expressed in E. coli in the form of polyhistidine-tagged proteins, comprising of rgp41A (601 bases N-terminal half of TM or 25kDa) and rgp41B (560 bases C-terminal half of TM or 24 kDa) by using an expression vector, pBAD/His C. The amount of protein, dilution of sera, and anti-human IgG labeled HRP used in the EIA test optimized by a checker board titration of the protein and seropositive or seronegative sera, were 5.0 microg/ml, 1:300, and 1:4,000, respectively. The blinded test evaluation of TE-diagnostic EIA in 500 seropositive and 500 seronegative sera which have been simultaneously tested by two available commercial kits and compared with our TE diagnostic EIA, gave 99.6% sensitivity and specificity. The other known genetic subtypes sera such as subtype A (n=5), B (n=9), C (n=4) and D (n=5) were also positive with this EIA. The estimated manufacturer cost per test of rgp41 based anti-HIV antibody detection EIA or TE-diagnostic EIA was about 15 baht. This recombinant envelope (gp41 or TM) protein from HIV-1, which can be produced in large quantities without any hazards from growing the virus and has lower cost to produce anti-HIV antibody serological diagnostic kit, should be considered as an HIV screening test in Thailand.

  14. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

    Science.gov (United States)

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya

    2015-06-10

    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

  15. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

    Science.gov (United States)

    Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

    2017-08-08

    Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

  16. Complement C3d conjugation to anthrax protective antigen promotes a rapid, sustained, and protective antibody response.

    Directory of Open Access Journals (Sweden)

    Ravi V Kolla

    Full Text Available B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4 of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure.

  17. DISSEMINATE CYSTICERCOSIS. One-day treatment in a case

    Directory of Open Access Journals (Sweden)

    Mashiyi MK

    2004-01-01

    Full Text Available We report a patient presenting disseminated cysticercosis characterized by neurocysticercosis, subcutaneous, muscular, and cardiac cysticercosis treated with praziquantel during one day RESUMEN: Se comenta el caso de un paciente que presentó cisticercosis diseminada, caracterizada por neurocisticercosis, y cisticercosis subcutánea, muscular y cardiaca, tratada con praziquantel durante un dia.

  18. Cysticercosis Cellulosae in Labial Mucosa: A Rare Case Report

    Directory of Open Access Journals (Sweden)

    Raghavendra M Shetty

    2010-01-01

    Full Text Available Cysticercosis is a condition in which a human acts as the intermediate host of Taenia solium, a pork tapeworm. The larvae infestation sites frequently include cerebral tissue, ocular organs and muscles. The present case report describes a rare incidence of cysticercosis cellulosae in the labial mucosa of upper lip

  19. Cysticercosis in cattle and its public health implications in Mekelle ...

    African Journals Online (AJOL)

    Analysis of data from Governmental and non-governmental health centers on7171human patients' positive for various parasitic infections revealed Taenia saginata in 23. Presence of taenaisis/cysticercosis in man and animals highlights the public health significance of this infection. Key words: Cysticercosis, Cysticercus ...

  20. Healthy pigs for healthy people. A cysticercosis advocacy information tool

    DEFF Research Database (Denmark)

    Saarnak, Christopher; Johansen, Maria Vang; Mejer, Helena

    2013-01-01

    changes in people’s day-to-day practices such as use of latrines and keeping pigs in pens would stop the life cycle of the disease and considerably reduce the risk of cysticercosis transmission. The need for political will and resources are basic requirements in order to control not only cysticercosis...

  1. Factors influencing transmission of porcine cysticercosis in Tanzania

    DEFF Research Database (Denmark)

    Braae, Uffe Christian; Wendy, Harrison; Magnussen, Pascal

    porcine cysticercosis could be associated with absence or completely open latrines (p=0.035, OR 5.98, CI: 1.33- 43.02) compared to enclosed latrines, and feeding potato peels to pigs (P=0.007, OR 3.45, CI: 1.43-8.79). Prevalence of porcine cysticercosis fluctuated throughout the seasons, and confined pigs...

  2. Sonographic Appearance of a Solitary Intramuscular Cysticercosis: A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Ju Hee; Joo, Seung Ho; Shim, Joo Eun; Kim, Yee Jeong; Oh, Hyun Cheol; Kim, Tae Hwan [NHIC Ilsan Hospital, Ilsan (Korea, Republic of)

    2009-03-15

    The development of antiparasitic drugs and public health strategies has reduced the prevalence of cysticercosis in South Korea. In contrast, the disease is still endemic in Southeast Asia. The influx of immigrants from endemic areas has been on the increase. We report the sonographic and pathological findings of cysticercosis that presented as an intramuscular solitary mass in a 27-year-old Philippine woman

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

    Science.gov (United States)

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

    2014-02-01

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

  4. Antibody responses to a major Pneumocystis carinii antigen in human immunodeficiency virus-infected patients with and without P. carinii pneumonia

    DEFF Research Database (Denmark)

    Lundgren, Bettina; Lundgren, Jens Dilling; Nielsen, T

    1992-01-01

    Antibody responses to a major purified human Pneumocystis carinii surface antigen (gp95) were determined by ELISA in human immunodeficiency virus (HIV)-infected patients. Serum IgG directed against gp95 was measured in 129 consecutive HIV-infected patients who underwent bronchoscopy for evaluation...... of pulmonary symptoms. Significantly more patients with P. carinii pneumonia (PCP) had detectable antibodies compared with HIV-infected patients without PCP and with HIV-negative controls (50 [66%] of 76 vs. 18 [34%] of 53 and 7 [35%] of 20, respectively; P less than .001), and the level of antibody response...... was higher (mean optical density ratio: 0.6 vs. 0.23 and 0.2, respectively; P less than .01). Changes in antibody response were investigated in 78 patients for whom serial serum samples taken around the time of bronchoscopy were available. Of the 47 patients with verified PCP, 20 (43%) mounted an antibody...

  5. Antibody Response to Actinomyces Antigen and Dental Caries Experience: Implications for Caries Susceptibility

    Science.gov (United States)

    Levine, Martin; Owen, Willis L.; Avery, Kevin T.

    2005-01-01

    Fluoridated dentifrices reduce dental caries in subjects who perform effective oral hygiene. Actinomyces naeslundii increases in teeth-adherent microbial biofilms (plaques) in these subjects, and a well-characterized serum immunoglobulin G (IgG) antibody response (Actinomyces antibody [A-Ab]) is also increased. Other studies suggest that a serum IgG antibody response to streptococcal d-alanyl poly(glycerophosphate) (S-Ab) may indicate caries experience associated strongly with gingival health and exposure to fluoridated water. The aim of this study was to investigate relationships between A-Ab response, oral hygiene, S-Ab response, and caries experience. Measurements were made of A-Ab and S-Ab concentrations, caries experience (number of decayed, missing, and filled teeth [DMFT], number of teeth surfaces [DMFS], and number of decayed teeth needing treated [DT]), exposure to fluoridated water (Flu), mean clinical pocket depth (PD; in millimeters), and extent of plaque (PL) and gingival bleeding on probing (BOP). A-Ab concentration, the dependent variable in a multiple regression analysis, increased with S-Ab concentration and decreased with PL and DMFT adjusted for Flu (R2 = 0.51, P < 0.002). Residual associations with age, DMFS, DT, and BOP were not significant. In addition, an elevated A-Ab response, defined from immunoprecipitation and immunoassay measurements, indicated a significant, 30% reduction in DMFT after adjustment for significant age and Flu covariance (analysis of variance with covariance F statistic = 10.6, P < 0.003; S-Ab response and interactions not significant). Thus, an elevated A-Ab response indicates less caries in subjects performing effective oral hygiene using fluoridated dentifrices. Conversely, a low A-Ab response is suggestive of decreased A. naeslundii binding to saliva-coated apatite and greater caries experience, as reported by others. PMID:15939752

  6. Neutralizing