PhD

dissertationThree antisera were prepared containing antibodies against (1) a major allospecificity of rat (Fischer anti-BN) (2) a minor allospecificity of rat (Lewis anti-Fischer) and (3) xenospecificity (rabbit anti-rat). A crude membrane fraction, carrying all three antigenic complexes, was prepared by mechanical disruption and differential centrifugation in weak sucrose. Antigens were assayed by inhibition of the appropriate specific cytotoxic antiserum. It was found that crude BN membrane antigen carried all three specificities (major and minor allospecificities and species specificities) in various concentrations. The membrane preparation was subjected to the enzymatic action of alph-glucosidase, beta-glucosidase, beta-glucosidase, beta-galactosidase, hyaluronidase, beta-glucuronidase, neuraminidase, chitinase and crude testicular extract. Changes in antigenicity were tested for by observing any alteration in its semi-quantitative cytotoxicity inhibition pattern. The supernatant of the treated antigen was assayed for release of carbohydrate by paper chromatography. With the exception of beta-glucosidase (sweet almond emulsion) the enzymes had no effect on any of the three antigenic complexes. Treatment with beta-glucosidase caused a loss of only the BN antigen. Beta-glucosidase was also the only enzyme that released significant levels of sugars from the membranes. The tow sugars release from the membranes with beta-glucosidase were shown not to be glucose, mannose, galactose, fucose or neuraminic acid (sugars generally associated with mammalian cell membrane). This destruction of Ag8-3, 30 antigenicity by specific carbohydrase treatment with concomitant release of sugars in presumptive evidence that carbohydrate is an essential portion of the immunodominant tip in this rat major alloantigen