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The specificity of A9 antiserum produced in rabbits immunized with high density lipoprotein-associated HLA-A9 from serum was investigated by using serologic and immunochemical approaches. After suitable absorption, the xenoantiserum No. 2958 is monospecific for A9 antigens in direct cytotoxic test. Data from lysostrip experiments and from blocking of cytotoxicity with Fab2 fragments indicate that the A9 xenoantiserum No. 2958 react with the same or closely associated antigenic structures recognized by A9 alloantisera. Immunoprecipitates obtained with the specific xenoantiserum No. 2958 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis contained only proteins of the size of detergent-solubilized HLA and beta2-microglobulin (45,000 and 12,000 m.w.). Immunodepletion experiments indicated that the precipitation was mediated by antibodies directed to the heavy chain, and not the light chain of the complex. These results indicate that monospecific HLA-A9 xenoantisera have the same discriminatory capacity of alloantisera and can be useful for the serologic and immunochemical characterization of HLA antigens.

Serologic and immunochemical techniques were used to characterize plasma membrane antigens of the murine lymphoma 6C3HED. Syngeneic C3H/HeJ anti-6C3HED antisera were prepared and were shown by cytotoxicity, absorption, and immunoprecipitation tests to be specifically reactive with the lymphoma cells. Alloantisera to H-2.23 were found to be unreactive with intact lymphoma cells by cytotoxicity and absorption assays. However, H-2.23 antigens were detectable in tumor cell lysates by inhibition of cytotoxicity and immunoprecipitation. SDS-PAGE electropherograms of immunoprecipitates of radiolabeled tumor cell extracts obtained with anti-6C3HED and anti-H2.23 antisera were essentially identical; proteins with Mr of approximately 70,000, 45,000, and 12,000 were apparent in both. An immuno-adsorbent of insolubilized anti-6C3HED removed H-2.23 antigens detectable in lymphoma extracts, but had no effect on H-2.23 antigens in normal splenocyte extracts. These data indicate that H-2.23 and TAA are physically associated on 6C3HED cells.

One out of four rabbits injected with partially purified soluble HL-A antigen from serum produced cytotoxic antibodies to human lymphocytes. These antibodies appear to be directed to the specificity W24 as determined by correlation studies with HL-A alloantisera and by absorption-inhibition experiments with human platelets and with serum soluble HL-A antigens. In the sera from the other three immunized rabbits, no antibodies to human lymphoid cells could be detected by the cytotoxic test, blocking of T cell rosette formation, stimulation of DNA synthesis and inhibition of mixed lymphocyte reaction.

Xenoantisera to human melanoma cells and to partially purified melanoma-associated antigens were coupled to protein A-bearing Staphylococcus Aureus or protein A-Sepharose and used as immunoadsorbents for the indirect immunoprecipitation of intrinsically radiolabeled proteins released into culture medium from various cultured human tumor and nontumor cell lines. These radiolabeled immunoprecipitates when analyzed by SDS-PAGE revealed highly reproducible molecular profiles of proteins and glycoproteins released by various cultured tumor lines and control cells into their spent culture media. A comparison of molecular profiles together with data indicating the binding specificity of known xenoantisera produced against human melanoma cells or their extracts led to the discovery of 2 macromolecules that are associated with human melanoma cells; a glycoprotein with a subunit m.w. of 240,000 (240K) and a single-chain glycoprotein of 94,000 (94K) m.w. also found in association with human carcinoma cells are described in this report.

Rabbits immunized with killed type III pneumococci respond to anamnestic challenge by type specific polysaccharide (S III) with the synthesis of anti-S III antibody if a long interval is allowed to elapse between primary and secondary immunization. A study of the anti-S III antibody produced early and late in the immune response revealed no change in molecular class, banding pattern of dissociated light chains, or S III binding characteristics as measured under equilibrium conditions or by study of dissociation kinetics utilizing radioiodinated p-OH-benzyl-S III. Sequential booster injections of S III into rabbits primarily immunized with whole organisms 8 or 9 months earlier led to a progressive decrease in the number of animals showing successful anamnestic responses and in the magnitude of those responses. It is concluded that S III depletes the antigen sensitive cell population in the secondary response largely because of its limited ability to stimulate sustained proliferation by such cells.