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COMPARISON OF REACTIONS OF ANTIBODIES TO RAT COLLAGEN AND TO RAT KIDNEY IN THE BASEMENT MEMBRANES OF RAT RENAL GLOMERULI* BY SIDNEY ROTHBARD, M.D., AND ROBERT F. WATSON, M.D. (From the Department of Medicine of The New York Hospital--Cornell Medical Center and The Hospital for Special Surgery a~liated ugtk The New York Hospital- Cornell Medical College, New York 10021) (Received for publication 6 January 1969) The pattern of immunofluorescence in the basement membranes of the kidneys appeared to be similar after antibody either to rat tail collagen (1) or to rat kidney (2) had been injected into the circulation of normal rats. Yet these antibodies reacted by complement fixation with different antigens and induced different microscopic renal lesions in rats (3). In attempts to define the antigens with which these antibodies react, appli- cation of the antisera to frozen normal rat kidney sections was found to give satisfactory immunofinorescence. This in vitro method allowed pretreatment of the kidney sections with various enzymes to determine whether selective destruction would permit identification of the antigens. In addition, compari- sons of the two antibodies were made by cross-absorption studies with subse- quent immunofluorescence in normal rat kidney sections and in kidneys of rats injected with the antisera. By comparing the antisera to rat kidney and to rat collagen, we have at- tempted to clarify their relationship to each other and to identify the antigens in the renal basement membranes with which each of these antisera reacts. Materials and Metkods The preparation of purified acid-soluble rat tail collagen, rabbit anti-rat collagen sera, and the technique of the complement fixation tests have been described in detail (4). The anti- collagen serum had complement fixation titers of 1:256 to 1:512. Rabbit anti-rat kidney sera were prepared by intradermal and subcutaneous immunization of rabbits with a suspension of cortices from saline-pedused rat kidneys. The suspension was mixed, 2:1, with complete Freund adjuvant. Each rabbit was given 4 ml of the mixture in divided doses each week for 3 wk; blood was taken 5 days after the last injection. These antisera promptly induced severe proteinurea and nepbrotoxic nephritis in normal rats. For absorption of antisera with collagen, three parts of sennn were mixed with one part of packed, washed, finely minced native rat tail collagen. For absorption with rat kidney, serum * This investigation was supported by the National Heart Institute, Research Grant H- 01138 (C 16), Public Health Service, and The Arthritis Foundation, New York Chapter. 1145 on March 23, 2016 jem.rupress.org Downloaded from Published June 1, 1969