Computational prediction of MHC class I epitopes for most common viral diseases in cattle (Bos taurus).

Viral diseases like foot-and-mouth disease (FMD), calf scour (CS), bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR) etc. affect the growth and milk production of cattle (Bos taurus) causing severe economic loss. Epitope-based vaccine designing have been evolved to provide a new strategy for therapeutic application of pathogen-specific immunity in animals. Therefore, identification of major histocompatibility complex (MHC) binding peptides as potential T-cell epitopes is widely applied in peptide vaccine designing and immunotherapy. In this study, MetaMHCI tool was used with seven different algorithms to predict the potential T-cell epitopes for FMD, BVD, IBR and CS in cattle. A total of 54 protein sequences were filtered out from a total set of 6351 sequences of the pathogens causing the said diseases using bioinformatics approaches. These selected protein sequences were used as the key inputs for MetaMHCI tool to predict the epitopes for the BoLA-A11 MHC class I allele of B. taurus. Further, the epitopes were ranked based on a proposed principal component analysis based epitope score (PbES). The best epitope for each disease based on its predictability through maximum number of predictors and low PbES was modeled in PEP-FOLD server and docked with the BoLA-A11 protein for understanding the MHC-epitope interaction. Finally, a total of 78 epitopes were predicted, out of which 27 were for FMD, 25 for BVD, 12 for CS and 14 for IBR. These epitopes could be artificially synthesized and recommended to vaccinate the cattle for the considered diseases. Besides, the methodology adapted here could also be used to predict and analyze the epitopes for other microbial diseases of important animal species.

Aspectos clínicos, patológicos, imuno-histoquímicos e virológicos em cinco bezerros persistentemente infectados com o vírus da diarreia viral bovina em uma propriedade do Rio Grande do Sul / Clinical, pathological, immunohistochemical and viral aspects of five calves persistently infected with bovine viral diarrhea virus in a farm of Rio Grande do Sul, Brazil

O vírus da diarreia viral bovina (BVDV) é responsável por diferentes síndromes que afetam bovinos em todo o mundo, causando grandes perdas econômicas. O presente trabalho analisou as características clínicas, patológicas e imuno-histoquímicas e virais de cinco bovinos persistentemente infectados pelo BVDV de uma mesma propriedade, localizada no Município de Viamão, Rio Grande do Sul. Dentre os sinais clínicos verificados destacaram-se subdesenvolvimento, secreções nasais e oculares, além de catarata congênita unilateral em dois bovinos. As principais lesões observadas durante a necropsia consistiram de aumento dos linfonodos mesentéricos, evidenciação das placas de Peyer e pododermatite e lesões crostosas no plano nasal e na região periocular em um animal. Os achados microscópicos caracterizavam-se, principalmente, por infiltrado mononuclear na lâmina do intestino delgado e rarefação linfoide com infiltrado histiocitário nos centrofoliculares de linfonodos e nas placas de Peyer. Antígenos virais foram detectados por imuno-histoquímica principalmente em queratinócitos da epiderme, no epitélio de folículos pilosos e células mononucleares da derme de orelhas e pele; histiócitos e em linfócitos dos linfonodos; células foliculares da tireoide; no citoplasma de neurônios e, em menor escala, em células da micróglia no córtex cerebral e no hipocampo. O isolamento viral de amostras de sangue e órgãos dos animais confirmou a presença de BVDV não citopático. Também foi possível detectar a presença do genoma viral por RT-PCR no soro dos animais. A análise filogenética do fragmento parcial da região 5' não traduzida do genoma viral permitiu a classificação da amostra viral como BVDV tipo 2b. O presente estudo reforça a necessidade de investigar e caracterizar surtos de BVD e descrever suas diferentes for-mas de apresentação.

Bovine viral diarrhea virus (BVDV) is responsible for different syndromes that affect cattle worldwide causing important economic losses. This study analyzed the clinical, pathological, immunohistochemical and viral aspects of persistent infection by BVDV in five animals of a farm located in the county of Viamão, Rio Grande do Sul, southern Brazil. The clinical signs included growth impairment, nasal and ocular discharge and, in two animals, congenital cataract. The main gross lesions observed at the necropsy were enlargement of mesenteric lymph nodes and Peyer's patches, and in one case, pododermatitis and crusted lesions on nasal planum and periocular region. Microscopic findings were characterized mostly by mononuclear infiltrate in the lamina propria, primarily in the small intestine and lymphoid depletion with histiocytic infiltrate in follicular centers of lymph nodes and Peyer's patches. Viral antigens were more frequently demonstrated in epidermal keratinocytes, epithelium of hair follicles and dendritic cells of the dermis of the ears and skin, histiocytes and lymphocytes in lymph nodes, thyroid follicular cells, in the cytoplasm of neurons and to a lesser extent, in glial cells in the cerebral cortex and hippocampus. Viral isolation from blood samples and organs confirmed the presence of non-cytopathic BVDV. Moreover, viral RNA was detected by RT-PCR in serum samples. Phylogenetic analysis of a partial fragment of the5' non-translated region of the viral genome allowed the classification of the sample as BVDV type 2b. The present study strengthens the need to investigate and to characterize BVD outbreaks and to describe its different clinic-pathological presentations.

Análisis de concordancia de cuatro pruebas rápidas para la detección de influenza A en Bogotá / Analysis of concordance of four rapid diagnostic tests for influenza A detection in Bogota-Colombia

La rapidez, eficacia y oportunidad del diagnóstico de influenza facilita el manejo de casos confirmados a nivel terapéutico más aun considerándose el estado actual del virus pandémico H1N1/2009. Objetivo: Analizar la concordancia de cuatro pruebas rápidas para la detección de Influenza A en Bogotá. Métodos: En este estudio descriptivo de corte transversal fueron comparadas cuatro pruebas rápidas para la detección de Influenza A (Quick Vue Influenza A+B; Directigen Ez Flu A+B®; SD Bioline Influenza Antigen® y Clearview Exact Influenza A and B®) en un grupo de 57 hisopados nasofaríngeos de pacientes sospechosos del virus pandémico H1N1/2009, los cuales fueron analizados previamente por PCR en tiempo real y clasificados como positivos o negativos para Influenza A. Resultados: El comportamiento de las pruebas rápidas valorado por su concordancia global con la prueba de referencia fluctuó entre 68,19% y 74,47%, sin evidencias de diferencias estadísticamente significativa entre ellas (c2=0,35; p=0,95). Tampoco se encontró un comportamiento diferencial estadísticamente significativo al valorar la proporción de concordancia entre los positivos de cada una de las pruebas rápidas con la prueba de referencia. Conclusión: Existen varios trabajos a favor y en contra de las pruebas rápidas para influenza, y algunos destacan su baja sensibilidad y especificidad comparadas con otras metodologías, como inmunofluorescencia directa, cultivo viral y RT-PCR. Sin embargo,ante la emergencia que actualmente se vive por la pandemia viral H1N1/2009, diferentes estrategias de vigilancia en salud pública, incluidas los estudios centinela y de conglomerados, pudieran ser implementados y las pruebas rápidas influenza (sin importar la casa comercial) serían útiles por sus características operativas, bajo costo y la posibilidad de lograr mayores coberturas de identificación de casos nuevos, descongestión de servicio y ajuste rápido de medidas en salud pública.

Rapidity, effectiveness and opportunity of influenza diagnosis make easy confirmed cases handling at therapeutic level most of all considering actual state of pandemic virus A H1N1/2009. Objective: To analyze agreement of four quick tests for Influenza A detection of in Bogotá. Methods: In a cross sectional fashion it was compared four quick tests designed for influenza detection (Quick Vue Influenza A + B; Directigen Ez Flu A + B®; SD Bioline Influenza Antigen® and Clearview Exact Influenza A and B®) in 57 nasopharynxeal hyssoped examples of suspicious patients of pandemic A H1N1/2009 virus, all of them were previously analyzed by real-time PCR and classified as positive for that virus. Results: Behavior of fast tests valued by its global agreement against test of reference fluctuated between 68.19% and 74,47%, without significant statistical differences among them (p=0,95). Neither significant statistical differences were found upon valuing proportion of agreement among positives each one of fast tests with reference test. Conclusions: There are some works on behalf or against of influenza rapid tests, and some of them emphasizes their low sensibility and specificity against other techniques, like direct inmunofluorescence inmunofluorescence, viral culture and RT-PCR. Nevertheless, in order to face the actual viral AH1N1/2009 pandemic, different strategies of public health surviellance, (including sentinel and conglomerate studies), could be implemented. Influenze Rapid tests (making exclusion of trade mark) would be serviceable by their operative characteristics, low cost and high possibility of identify new cases and by that way, it would possible emergence services decongestion and fast adjustment of measures in Public Health.

Indirect immunofluorescence to demonstrate lichen planus specific antigen (LPSA) in lichen planus.

BACKGROUND: Current evidence suggests that lichen planus is an immunological disease. Cytotoxic CD8+ cells in the lesional epidermis recognize a unique antigen called lichen planus specific antigen. This antigen could be demonstrated by indirect immunofluorescence using the patient's serum and autologous lesional skin. AIM: To study indirect immunofluorescence pattern in lichen planus, among Indian patients. METHODS: Twenty-five consecutive patients with the clinical diagnosis of lichen planus were enrolled in the study. Direct immunofluorescence was done in all patients. Indirect immunofluorescence using lesional skin as substrate was done in all 25 patients and five patients with other dermatoses. RESULTS: A specific fluorescence pattern corresponding to the distribution of lichen planus specific antigen was observed in the stratum spinosum and granulosum in 22 (88%) patients. It was absent from other parts of the epidermis, dermis and in patients with other dermatoses. CONCLUSION: Indirect immunofluorescence is a useful adjuvant test in lichen planus, particularly in atypical cases.

Identification and antigenic site analysis of foot-and-mouth disease virus from pigs and cattle in Korea

From May to June 2002, a total of 16 foot-and mouth disease (FMD) outbreaks due to the serotype O virus, Pan Asia strain, were recorded in Korea. The viruses were identified by antigen ELISA, RT-PCR and sequence analysis. The overall nucleotide sequence divergence of the VP1 region among the 4 isolates in 2002 was 0 to 1.4%, but between O/SKR/2002 and O/SKR/2000 isolates was 1.9-4.9%. Phylogenetic analysis with the some known strains from East Asian countries showed that the 4 Korean isolates in 2002 formed one distinct cluster, which different from clusters of Korean isolates in 2000, with in the same lineage of the ME-SA topotype strains. Deduced amino acid sequences around neutralizable antigenic site on VP1 site of O/SKR/2002 isolates were aligned and compared with other strains. At the antigenic site 1, the replacements of the critical amino acid residues at position 144 from V to L and at position 152 from A to T were observed in O/SKR/2002 viruses. For antigenic site 2 and 4, there were not significant variations in general. At the antigenic site 3, the substitutions of amino acid residues were present at positions 54 and 56 in O/SKR/2002 isolates and an alternative residue I at position 54 are observed only at the sequence of O/SKR/AS/2002 (cow) virus. And the substitution (L-->P) of significant residue at position 144 was detected at the amino acid sequence of the O/SKR/2002 (cow) virus.

Enzymatic N-glycan analysis of 31 kDa molecule in plerocercoid of Spirometra mansoni (sparganum) and its antigenicity after chemical oxidation

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

Two-dimensional gel electrophoresis and immunoblot analysis of Neospora caninum tachyzoites

Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.

Rotavirus serotypes: classification and importance in epidemiology, immunity, and vaccine development.

The development and implementation of safe and effective vaccines to prevent the enormous health burden of rotavirus-associated disease is a global public health goal. Human rotaviruses, the major aetiological agents of severe infantile diarrhoea worldwide, display surprisingly diverse and complex serotypic specificities. Ten VP7 serotypes and 7 VP4 serotypes have so far been detected. An increasing number of observations, obtained from analyses of (i) natural rotavirus infections in infants and young children, (ii) experimental rotavirus infections in laboratory animals, and (iii) extensive rotavirus vaccine field trials performed in different populations of various parts of the world, appears to support the concept that serotype-specific antibodies to rotaviruses play an important role in protection against rotavirus-associated illnesses. Thus, the first licensed rotavirus vaccine (RRV-based quadrivalent vaccine) was designed to cover the epidemiologically important VP7 serotype 1, 2, 3, and 4.

An IgM monoclonal antibody to Japanese encephalitis virus recognizing a cross-reactive epitope on nuclear histones.

An IgM class of monoclonal antibody (MAb) raised against 'envelope' (E) glycoprotein of Japanese encephalitis (JE) virus, cross reacted with nuclear histones, in addition to recognizing the viral antigen present in the cytoplasm of infected cells by indirect fluorescent antibody (FA) technique. The experiments on histone depletion by the acid treatment of uninfected PS (porcine kidney) cells, revealed the loss of nuclear immunofluorescence (IF) which was regained after the reconstitution of acid treated cells with histones, prior-to reacting with MAb NHA-2. The IgM MAb recognized specifically the viral antigens expressed on the surface of JE virus infected PS cells by a modified indirect FA. The adsorption of MAb NHA-2 with calf thymus histones (type II-AS) showed a comparative higher drop in the reactivity to JE virus (54.2% reduction) as compared to that against uncomplexed histones (33.3%) by ELISA, thus indicating a higher MAb affinity to the former. In contrast, the adsorption of MAb with chicken RBC nuclei resulted in comparatively more reduction in the reactivity to the uncomplexed histones (52.4% reduction) as against JE virus (37.5%), suggesting that DNA plays some role in modifying and presenting these epitopes. The cross-linkage of epitopes by glutaraldehyde treatment of JE virus antigen and histones showed a 2-fold and higher rise in the MAb reactivity as against those with unfixed or methanol fixed antigens (no cross-linkage), suggesting that the epitope is conformation dependent. Thus, histones seem to share a partial conformational homology with 'E' glycoprotein of JE virus and immune reaction with histones might lead to an autoimmune disorder.

Trypanosoma cruzi: amastigote polymorphism defined by monoclonal antibodies

We have raised monoclonal antibodies (mAbs) directed towards amastigote forms of Trypanosoma cruzi, and shown that mAbs 1D9 and 4B9 are carbohydrate while mAb 4B5 activity is resistant to periodate oxidation of the antigen. Here we used an ELISA to quantitate and compare the expression of surface epitopes on fixed parasites among different parasite isolates. The expression of markers varied among T. cruzi amastigotes isolated from infected cells or after extracellular differentiation of trypomastigotes. Moreover, we also observed an extensive polymorphic expression of these epitopes among amastigotes derived from different strains and clones. For instance, mAb 2C2 strongly and evenly reacted with 9 strains and clones (G, Y, CL, Tulahuen, MD, and F, and clones Sylvio X-10/4, D11, and CL.B), with absorbance at 492 nm (A492 nm) from 0.6 to 0.8. By contrast, mAb 4B5 had a higher expression in Tulahuen amastigotes (around 0.9 at 492 nm) whereas its reactivity with amastigotes from clones CL.B, Sylvio X-10/4 and D11 was much lower (around 0.4). mAb 1D9 displayed an interesting pattern of reactivity with amastigotes of the different strains and clones (A492 nm of G>D11ÝSylvio X-10/4 = MD>Tulahuen = F = Y>CL>CL.B). Finally, we observed that mAb 4B9 had the lowest reaction with the parasites studied, with higher values of A492 nm with Y strain (around 0.6) and lower values with Tulahuen, F and CL.B strains (around 0.2). Immunoblotting analysis also showed extensive variations among amastigotes of the various parasite isolates and mAbs 4B9, 1D9 and 4B5 revealed significant differences in expression between clones and parental strains. These data describe a previously uncharacterized polymorphism of T. cruzi amastigote surface components

Comparative studies on the antigenic structures of five subspecies of Oncomelania snails by immunoelectrophoresis.

To approach the biochemical relationships of five subspecies of Oncomelania snails, antigenic structures among the subspecies were compared using immunoelectrophoresis. The results obtained are summarized as follows: 1) For five subspecies of Oncomelania hupensis snails (Oncomelania hupensis hupensis, O.h.nosophora, O.h. formosana, O.h. chiui and O.h. quadrasi), 23-24 precipitin bands were observed between the antigens and their homologous antisera, while 18-22 bands were observed in the heterologous reactions. 2) For each subspecies, residual bands observed after absorption procedure demonstrated the presence of antigens unique to each subspecies except O.h. chiui. Based on the immunological antigenic structures among the Oncomelania subspecies, it is suggested that O.h. nosophora and O.h. hupensis forms are closely related group, while O.h. formosana, O.h. chiui and O.h. quadrasi forms are another group.

Expresión de epitopos alfa-galactosil en lesiones de VPH en vulva / Expression of alfa-galactosil in lesions of HPV in vulva

En humanos, la alta expresión de epitopos alfa-galactosil está asociada con respuestas a situaciones de peligro como infecciones, enfermedades autoinmunes y senescencia. Al virus papiloma humano (VPH) se le asignado un papel iniciador en el cáncer cervical y lesiones precursoras. Las verrugas venereas o condilomas acuminados se localizan en genitales femeninos, pene y región anal. En el presente trabajo se realizó la caracterízación celular en el epitelio de lesiones en vulva con VPH (n=10) utilizando anticuerpos monoclonales específicos contra Gal-alfa 1-3 y PCNA (control positivo) con el método de la avidina-biotina inmunoperoxidasa. Demostramos que la infección por VPH incide la expresión del epitopo alfa-galactosil, el cual nos manifiesta en condiciones normales

A conserved epitope on nuclear pore phosphoproteins reflects cell division status.

The nuclear pore complexes mediate the selective nuclear import of proteins in a signal- and energy-dependent process. We have earlier reported the characterization of a monoclonal antibody, Mab E2, that recognizes a novel class of nuclear pore phosphoproteins involved in signal-binding and protein transport. In the present study, we have analyzed the pattern of immunoreactivity of Mab E2 in cultured rat fibroblasts and have observed significant differences in the expression of epitopes in proliferating and quiescent cells. Furthermore, the common epitope recognized by Mab E2 is conserved across species, consistent with its essential role in nuclear protein import.

Lysine- and threonine-sensitive aspartokinase isoenzymes from spinach leaves share common antigenic determinants.

The lysine- and threonine-sensitive isoenzymes of aspartate kinase were purified to homogeneity from spinach leaves and polyclonal antibodies were raised in rabbits. The antibodies were characterized by various immunological tests like Ouchterlonys-double-diffusion, titrations of the inhibition of enzyme activity and ELISA. The antibodies against the lysine-sensitive isoenzyme could recognise as little as 50 ng of the pure antigen protein and that against the threonine-sensitive form could recognise 200 ng of the protein in the ELISA tests. The immunological tests have also shown that the lysine and threonine sensitive isoenzymes of aspartate kinase share some common antigenic determinants and differ in others.

Improved presentation of antigenic sites in enzyme-linked immunosorbent assay: antigenicity of reduced and carboxymethylated riboflavin carrier protein.

Disulphide reduced and carboxymethylated riboflavin carrier protein (RCM-RCP), an unfolded derivative of chicken RCP, does not bind riboflavin and there is a drastic reduction in its ability to interact with antiserum to cRCP. Antibodies to RCM-RCP are directed against sequential epitopes(s). On radioiodination of RCM-RCP, a maximum of 30-50% binding at dilution of 1:500 was obtained with rabbit antibodies RCM-RCP [n = 5]. However, high titer antibodies was obtained when radioiodinated RCM-RCP was immobilized on ELISA microtiter plates suggesting that immobilization of RCM-RCP leads to either preservation of antigenic sites or improved presentation of the antigenic determinants. An avidin-biotin system was utilized to develop an ELISA.

Characterization of poly- and monoclonal antibodies to physalis mottle (tymo) virus.

Polyclonal antibodies were raised against the Physalis mottle virus (PhMV) and its denatured coat protein (PhMV-P). Analysis of the reactivity of the polyclonal antibodies with tryptic peptides of PhMV-P in dot-blot assays revealed that many of the epitopes were common to intact virus and denatured coat protein. Five monoclonal antibodies to the intact virus were obtained using hybridoma technology. These monoclonal antibodies reacted well with the denatured coat protein. Epitope analysis suggested that probably these monoclonal antibodies recognize overlapping epitopes. This was substantiated by epitope mapping using the CNBr digest of PhMV-P in western blots. All the five monoclonals recognized the N-terminal 15 K fragment. Attempts to further delineate the specific region recognized by the monoclonals by various enzymatic cleavages resulted in the loss of reactivity in all the cases. The results indicate that these monoclonals probably recognize epitopes within the N-terminal 15 K fragment of the coat protein.

Desenvolvimento de um ensaio imunofluorométrico para a medida da molécula inteira da gonaatrofina coriônica (hCG) no soro / Development of an immunofluorometric assay for serum measurement of the whole molecule of human chorionic gonatropin (hCG)

Tendo como base dois anticorpos monoclonais produzidos em nosso laboratório, um específico contra a subunidade beta de hCG/LH (E2P1) e outro específico contra um epítopo descontínuo presente na molécula inteira da hCG (F1P16), desenvolvemos um ensaio imunométrico específico para a molécula inteira de hCG. O monoclonal E2P1 foi adsorvido a placas de microtitulaçåo e o monoclonal F1P16 foi marcado com Európio; o ensaio consta de duas incubaçöes sequenciais de 1h cada e leitura final de fluorescência em fluorômetro tempo-sincronizado. A sensibilidade calculada foi de 1,8 UI/L; o perfil de imprecisåo mostrou erro inferior a 19 por cento entre os valores de 5 a 3000 UI/L; o coeficiente de variaçåo interensaio mostrou-se de 8 por cento para uma amostra de valor médio de 1065 UI/L (n=15) e de 13,9 por cento para outra de valor médio de 28 UI/L (n=15). A comparaçåo com método equivalente (Delphia hCG) nåo mostrou nenhuma discordância em 300 amostras dosadas sequencialmente, sendo que em 1388 amostras onde os valores eram iguais ou superiores a 20 UI/L pelo Delfia, obtivemos alto índice de correlaçåo (r=0,988). Os valores obtidos pelo nosso método mostraram-se discreta, porém, significativamente mais elevados, com medianas de 4610 e 4370 UI/L, respectivamente

A search for immunocontraceptive agent from marine sources--role of antispermatheca globulin of Telescopium telescopium on fertility regulation in male rat.

Polyclonal antisera were developed in rabbits against 'spermatheca', the reproductive gland of T. telescopium, a marine mollusc. The gland contains spermatozoa. Antisera and its IgG fraction (ASTG) indicated common antigenic determinants by immunodiffusion and had titer values 81920 and 1280 against crude antigen extract. Cycling female rats when exposed to passively immunized male rats with different doses of ASTG, had reduction in implantation sites and litter size. Females had pseudopregnancy when exposed to higher doses of immunized males and had normal cycle after 20 days in average. ASTG in male rats caused decrease in weight of the reproductive glands, alteration in sperm concentration, motility and morphology, formation of multinucleated giant cells and vacuoles leading to arrest of spermatogenesis and reduction in seminiferous tubular diameter. The effects were dose dependent with reversible infertility. The results indicate presence of a common antigenic determinants which cross-react with vertebrates and existence of common relation through phylogenetic evolution and their immune responses.

Enzyme-linked immunosorbent assay for riboflavin carrier protein and modified protein: application for epitope analysis.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards the conformational epitopes and antibodies to disulphide bond reduced carboxymethylated riboflavin carrier protein (RCM-RCP) to the sequential epitopes. Taking advantage of this premise and in order to map the epitopes of RCP recognized by the antibodies, enzyme-linked immunosorbent assays were validated for RCP and RCM-RCP using the Avidin-Biotin system. The usefulness of these assays were illustrated when antigenicity of peptides derived from RCM-RCP following trypsinization were examined. Two major (T1,T2) and one minor peptide (T3) fractions were obtained when the tryptic peptides were fractionated on DEAE-cellulose. RCP has a blocked N-terminal. Tryptic peptides (T1 and T2) on microsequencing revealed the absence of an N-terminal amino acid, indicating that these fragments emanate from the N-terminal region of RCP. In support of this observation is the finding that antipeptide antibody to cRCP (10-24) of cRCP interacted with T1 as well as T2 indicating the presence of the sequential epitope (10-24) of cRCP in these fragments. In RCP-ELISA, only T2 displaced RCP and peptides T1 and T2 displaced RCM-RCP in RCM-RCP ELISA. Differences in the ability of these fragments (T1 and T2) to displace RCP and RCM-RCP reflect the subtle changes in the spatial structures of these epitopes in RCP and RCM-RCP.

Determinant analysis and interaction studies on monoclonal antibodies to bovine IgG1 and IgG2.

Two mouse monoclonal antibodies SKb1 and SKb6 were prepared by fusion of myeloma cells with spleen cells of female Balb/c mouse immunized with a mixture of bovine IgG1 and IgG2. In radioimmunoassay, SKb1 bound specifically to IgG2 but SKb6 reacted with both IgG1 and IgG2 molecules. In the competition experiments, heavy chain isolated from bovine IgG could inhibit the binding of 125I-IgG1 and 125I-IgG2 to SKb6, while it failed to inhibit the binding of 125I-IgG2 to SKb1. The epitope reacting with SKb1 was found to be present not only on bovine IgG2 but also on goat IgG and was not present on IgG molecules isolated from the serum of rabbit, rat, sheep, horse, human and monkey. Similarly, the epitope reacting to SKb6 was found to be present on bovine IgG1 and IgG2 and also on IgG molecules isolated from goat and sheep serum but was absent in the IgG molecules isolated from the serum of rabbit, rat, horse, human and monkey. The association constants of the interactions of SKb1 with 125I-IgG2 and of SKb6 with 125I-IgG1 and 125I-IgG2, determined by Scatchard analysis, Steward-Petty plot and Sips plot, were found to be in the order of 10(8)-10(10) L/M. The association constants were determined at varying temperatures to obtain the thermodynamic parameters. The enthalpy (delta H0) and entropy (delta S0) values for the above antigen-antibody interactions were in the range of 9.15-15.96 kcal/mole and 36.96-41.15 eu/mole respectively. The heterogeneity indices for similar interactions determined by Sips equation were consistent with the expected values for binding of monoclonal antibodies with homogeneous protein determinants.