The immunodominant surface antigen of Plasmodium gallinaceum is present in both the salivary gland and oocyst sporozoites.
Eliana Maria Maurício da RochaRodrigo Corrêa‐OliveiraCélia R. Whitaker CarneiroJosé Daniel LopesA U Krettli
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Abstract:
1. Monoclonal antibodies (MAbs) against surface antigens of Plasmodium gallinaceum sporozoites, an avian malaria parasite, were produced using spleen cells from mice immunized with sporozoites from mosquito salivary glands (SGS) or from midguts containing oocysts (OoS). 2. All of the 15 MAbs tested (11 anti-SGS and 4 anti-OoS) reacted with SGS and OoS by indirect immunofluorescence and circumsporozoite precipitation reactions. Fourteen of these MAbs (11 anti-SGS and 3 anti-OoS) produced a Western blot (WB) pattern identical to that produced with serum from mice hyperimmunized with viable intact sporozoites. 3. All MAbs and the immune sera recognized only two polypeptide bands of approximate molecular weight 76 and 64 kDa. 4. No difference in the WB pattern was observed when 9- or 12-day SGS or OoS extracts were used as antigens in WB. This antigenic similarity was confirmed when the total protein extracts were visualized on silver-stained SDS-PAGE gel.Keywords:
Plasmodium gallinaceum
Immunofluorescence
Plasmodium (life cycle)
Circumsporozoite protein
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To define Babesia bigemina-specific antigens on the surface of infected erythrocytes, monoclonal antibodies (MAbs) were identified by live-cell immunofluorescence. As determined by live-cell immunofluorescence, two MAbs made to the Mexico strain reacted with the Mexico strain and three Kenya strains, while three MAbs made to the Kenya-Ngong strain reacted with the Kenya strains but not the Mexico strain. Binding of MAb 44.18 (made to the Mexico strain) to a strain-common epitope was confirmed by immunoelectron microscopy and by surface-specific immunoprecipitation of [35S]methionine-labeled proteins (200, 28, and 16 kDa in size), which also demonstrated that the MAb recognized an epitope on proteins encoded by B. bigemina. In immunoblots, the MAb bound to predominant antigens with sizes of 200 and 220 kDa in erythrocyte lysates infected with strains from Puerto Rico, St. Croix, Texcoco (Mexico), Kenya, and Mexico. Major antigens with sizes of 200 and 220 kDa were isolated from a MAb 44.18 affinity matrix. Calf serum antibodies to these isolated antigens bound to erythrocytes infected with either the Mexico or Kenya strains as determined by live-cell immunofluorescence, allowing the conclusion that at least one conserved surface epitope was recognized. Calf serum antibodies identified major labeled proteins with sizes of 200 and 72 kDa by surface-specific immunoprecipitation, and infected erythrocytes sensitized with these antibodies were phagocytized by cultured bovine peripheral blood monocytes. These results provide a rationale for evaluating antigens identified by MAb 44.18 individually and as components of subunit vaccines.
Babesia bigemina
Immunofluorescence
Immunoelectron microscopy
Immunoprecipitation
Antigenic variation
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Eight monoclonal antibodies directed against Squamous Cell Carcinoma Antigens (A1 and A2) were collected and evaluated by three working groups. Recombinant antigens, fusion proteins and native antigens from normal tissue were used to evaluate antibody specificity. Five antibodies reacted with both A1 and A2. Two of these antibodies (K123 and K131) showed related binding characteristics, whereas SCC140, K182 and SCC111 demonstrated unique epitope specificity and were not related to the reference antibodies included (F1H3, F2H7 and SCC107). SCC111 reacted particularly well with antigen on Western blot, indicating that the epitope was partly hidden when the antigen was in solution. Two antibodies (SCC103 and SCC109) reacted only with A2 and the fusion protein A1/A2, indicating that they recognized an A2 epitope in exon 8. The A2-specific antibodies are unique in their binding to A2 and are different from the reference antibodies included (SCC104 and K122). SCC103 is probably the best A2-specific antibody available. One antibody, K136, was A1-specific and is related to reference antibody K135. The new antibodies can be used to establish immunometric assays for specific measurement of A1, A2 or both A1 and A2 together.
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Collodion
Blocking antibody
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Paratope
Isotype
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Summary Monoclonal antibodies directed against the 51 kD merozoite surface antigen of Plasmodium falciparum also bind to other antigens within the infected cell. The sizes of these cross–reacting antigens have been characterized. Immunofluorescence due to the reaction of one of the monoclonal antibodies with these cross–reacting antigens was localized in the intra–erythrocytic parasite and in granules in the infected red cell cytoplasm. This immunofluorescence could be distinguished from the merozoite surface antigen in parasite lines with a variant serotype of the merozoite surface antigen which fails to react with the monoclonal antibodies. It was found that the in–vitro growth inhibition caused by the presence of one of the monoclonal antibodies, 8G10/48, was dependent on the expression of the corresponding serotype of merozoite surface antigen, a finding consistent with the inhibitory effect of this antibody being primarily directed against the merozoite surface antigen and not the cross–reacting antigens. Analysis of the frequency at which epitopes occur suggests that such cross–reacting proteins will be commonly seen in malaria, without the need to postulate a selective advantage for such cross–reacting specificities.
Immunofluorescence
Merozoite surface protein
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The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.
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Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes. The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo. Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines. In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive. Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants. With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants. Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo. This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice. Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays.
Immunofluorescence
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Immunochemical assays are based on the fact that the mammalian immune system forms protein molecules, which bind with high affinity to an antigen molecule. All kinds of molecules can be antigen molecules, and the specificity of these proteins, called antibodies, is rather high. When an animal is injected (immunized) with an antigen, the resulting immune response leads to the generation of a variety of antibody molecules against the antigen, a serum. The composition of the serum depends on the history of the animal. Therefore it is not always easy to reproduce the exact antibody mixture of a serum in different immunizations. These antibody mixtures are very specific, because they recognize a number of different sites or epitopes on the antigen molecule. Each of these antibody molecules is made by one B-cell type. These B-cells can be isolated and grown. A colony which is generated from a single B-cell creates one kind of antibody molecule, a monoclonal antibody. Monoclonal antibodies bind or recognize one epitope on an antigen molecule. As a consequence, monoclonal antibodies bind to any molecule which shows this epitope or molecular geometry. Once a B-cell culture is established, monoclonal antibodies can be reproduced in large quantities.
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Objective To characterize antigens on uninfected T lymphocytes reactive with monoclonal antibodies (MAb) directed against the HIV-1 Nef protein, and to search for antibodies directed against this epitope in HIV-1-infected individuals. Design Murine MAb directed against an epitope of Nef defined by amino acids 60–73 reacted with cell surface antigens of normal peripheral blood lymphocytes and permanent human T-cell lines. Methods The specificity of the MAb reaction was investigated by flow cytometry and immunofluorescence. The antigen was precipitated from lysates or uninfected cells using MAb or sera from HIV-1-infected individuals and analysed by Western blot and isoelectrofocusing. Results An antigen with an apparent relative molecular mass of 137000 and an isoelectric point of 8.45 was immunoprecipitated with the cross-reactive MAb from uninfected human T cells. Sera from HIV-positive individuals recognizing a Nef epitope partially overlapping with the binding site of the cross-reactive MAb stained the 137 kD protein precipitated with the MAb in Western blot analysis, while HIV-positive sera without antibodies to this Nef region and sera from uninfected individuals were negative. Conclusion The induction of autoantibodies cross-reactive with cellular surface proteins may play a role in the pathogenesis of AIDS.
Surface protein
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