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Related post: ;i; (a) HUMAt, SUBJECTS n (al) MINORS D Order Skelaxin Online (a2) INTERVIEWS □ (b) HUMAN TISSUES (c) NEITHER SUMMARY OF WORK (200 words or less - underline keywords) During malaria infection the host responds to a variety of parasite Ags, but the location, number and specificity of the Ags involved have been difficult to determine. Thus during the last year we have used monoclonal antibodies (Abs) to study the antigenic nature of the rodent malaria parasite P^. yoel i i . The 18 hybridomas which have been studied in detail were produced by fusing P3-X63-NS/I myeloma cells with spleen cells from mice infected for ^i or 6 days with the lethal or nonlethal strain of P^. yoe 1 i i . Results show that the host responds to a number of Ags located either on the surface or within the cyto- plasm of Buy Skelaxin the parasite. The majority of monoclonal Abs identify Ags possessed by all erythrocytic stages of the parasite, but three bind preferentially to merozoites, and one is completely stage-specific for merozoi tes . Two of the l8 monoclonal Abs detect Ags shared by the sporozoi te and erythrocytic stages of P. yoel i i . Approximately ^5% of the Abs detect species-specific Ags; 10^ denote ftgs confined to related rodent malaria parasites (P. berghei , FT vinckei , P^. chabaudi ) ; whereas, k5% detect Ags shared among rodent, primate (P^. knowlesi , P^. falciparum ) and avian (P^. gallinaceum) malarias. The 18 hybrids identify a Tiinimum of 12 distinct plasmodial Ags. 2^-35 PHS-6040 (Rev. 10-75) Project No. ZOl Al 00l4l-06 LMI Project Description : During malarial infection the host responds to a variety of parasite antigens (Ag) , but the location, number, and specificity of the Ags involved have been difficult to determine. Initially a biochemical approach was undertaken to identify the number of polypeptides present in extracts of the rodent malarial parasite Plasmodium yoel i i . Two-dimensional analysis of parasite proteins using isoelectric focusing in the first dimension and SDS-poly- acrylamide gel electrophoresis in the second, identified a minimum of 125 polypeptides. The separation and immunologic analysis of 125 polypeptides, using conventional biochemical methods, would be time Purchase Skelaxin consuming and probably require a prohibitively vast amount of starting meterial. Thus we decided to continue making use of hybridoma technology Generic Skelaxin to study the immunologic nature of malaria parasites. Monoclonal antibodies (Abs) Buy Skelaxin Online have provided us not only with reagents for isolating plasmodial Ags but also have allowed us to identify by immunofluorescence: (1) the location of Ags, i.e., Ags within or on the surface of parasites; (2) stage specific Ags, i.e., Ags unique to rings, trophozoites, schizonts and merozoites; (3) species specific Ags and (4) those Ags which are common to various members of the Plasmodial spp. i.e., crossreactive Ags. In addition, monoclonal Abs have been used to isolate malarial Ags. These Ags have then been tested in in vitro proliferation assays for effectiveness in stimulating various lymphocyte subsets from sensitized and normal animals. Production of Anti-Plasmodial Secreting Hybridomas : BALB/c pice were primed to malarial Ags in the following ways: (l) infected with 10 lyXL parasites A days prior to fusion; (2) infected with 10 17XNL parasites k, 6, and 11 days prior to fusion; (3) infected Order Skelaxin with the 17XNL strains, allowed to recover, and then inoculated with 17XL parasites k days prior to fusion (hyperimmune); (^) inoculated intravenously once or 3 times with irradiated P. yoelii sporozoites, and Skelaxin Online (5) vaccinated twice with an Ag preparation enriched for merozoites. Also BALB/c mice made congenital ly athymic (BALB/c'Nu) infected with 17XL malaria k days earlier were used. Fusions were performed using the procedure of Margulies, et. al., 1976 Cold Spring Harbor Symposium on Quantatati ve Biology 91:78l. In brief, BALB/c spleen cells were fused with the P3-X63-NS/1 cell line using polyethylene glycol. Cells were distributed to microtiter wells and cultured in hypoxanthine-aminopterin-thymidine selection medium for lA days. Culture supernatants were screened for anti-plasmodial activity using a solid-phase radioimmunoassay (RIA) developed by Kim, Taylor, Evans and Asofsky (manuscript submitted). RIA positive wells were verified by the indirect fluorescent antibody (IFA) technique. Hybrids, cloned by limiting dilution on a thymo- cyte feeder layer, were maintained in vivo in BALB/c mice. Although positive hybrids have Cheap Skelaxin been produced using spleen cells from all primed animals, only those produced in fusions using spleen cells from mice infected h days with the 17XL and k and 6 days with 17XNL have been studied 24-36 Project No. ZOl Al 001i|1-06 LMI in detail. To date approximately 18 cell lines have been established. Hybrids produced in more recent ipusions are in various stages of cloning and study. The isotype of the ant i -malarial hybrid cell lines Purchase Skelaxin Online was determined by Ouchterlony analysis of culture supernatants using class specific reagents, by Immunoelectrophoresis (lEP) of ascites fluid using the above reagents, and by I FA analysis using f luoresceinated heavy chain specific goat anti- mouse IgM, IgG., and lgG_. Monoclonal Abs of IgM, yZa and y3 classes have been found. Ammonium-sulfate precipitated globulins from ascites fluids were Buy Cheap Skelaxin used in most of the studies. However, some of the results have been confirmed recently using monoclonal Abs purified by protein A chromatography. Identification of Stage Specific, Species Specific and Serologic Cross- reactive Ags Using Monoclonal Abs : At least k distinct patterns of fluorescence are produced by monoclonal Abs on fixed smears of P . yoe 1 i i . The patterns we have observed are similar, though not identical, to those reported. by Freeman, et. al. (Nature 28A : 366, 1980). Abs from II/I8 hybrid lines produce a fluorescence pattern identical to that observed using hyperimmune serum, 3/l8 bind to Ags located in the membrane of either the parasite or infected RBC, 3/1 8 bind to all erythrocytic stages but when titered to 100 yg/ml bind only to a specific region within merozoites, and I/I8 is stage specific for merozoites.
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