Isolation and characterization of rat cell membranes carrying histocompatibility antigens.

Three 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.

This Dissertation for the Doctor of Philosophy Degree by John Henry McDonald III has been approved July 1970 Supervisory Committee Supervisory Committee Without the love, patience, understanding, consideration and encouragement of my wife, Sue, this thesis would not have been possible. I wish to thank my parents, John H. and Mae r1cDonald for the many years of financial and moral suppor t and encouragements and especially for the example th ey set for me to follow at home. The author is deeply grateful for the moral and educational values taught to him by his grandparents, Mr. and Mrs. C.A. Sundberg. The author wishes to thank Dr. C.W. DeWitt for the academically stimulating environment that he provided during the progress of the research here-in contained. The author wishes to thank Dr. Ivan M. Lytle for the advise and council that he has so generously given over the past five years and Dr. N. Strickland and Dr. James Lords who read and made constructive criticism of this thesis. Grateful acknowledgement is made of the effort on the part of Dr. DeWitt Hunter to provide for me the opportunity for this research. The author wishes to thank Dr. A.P. Crane and his staff at Cottonwood Hospital for their assistance in the preparation of this manuscript. Dr. Crane's patience wi th the author during these last two years surely must rival that of Job's. The assistance of Dr. Alfred Linker and his staff is gratefully acknow 1 edged. This research was supported, in part, by the Public Health Ser­ vice, Allied Health Profession Advanced Traineeship Grant AHT-68-043. iii