Abstracts ~77

DETECTION OF CELL SURFACE A~"~IGENS BY THE CELLUIARENZY~-LZT~KED IHMUNO~OHBI~TA~A¥ (CELISA) R. Morris t P. Thomas I & R. Hong, Depts. of Surg. & PedSo UnJvo of WIS.

Two novel techniques that exploit the invention of the enzyme Immunoasss¥ ~ave been de s igned to d e t e c t c e l l s u r f a c e a n t l g ~ n s . I n l t x a l l y a suspenslo~- phase mzeroCELISA was used t o measure the bzndtn~ o f p o l y - and monn~peelrJe ~ H L A s e r a (Sul ) to human lymphocytes (~0,000 c e l l s ) in V - b o t t o m ~ i e r o - plates. A goat~human IgG eovalently lln~,ed to alkaline phosphatase (CAH- AP) was used as an Jndlrect Indicator of hound ~HLAant~body° Cells~ere washed ~ree ofurtouniantzbodzes by repea ted centrz£~atlonandresuspen- s t o n . The co lo r ed enzymat ic p roduc t (formed by t he a c t i o n o f t h e enzyme on t he added c o l o r l e s s s u b s t r a t e ) xn t he w e l l s was ~easured s p e e t r o p h o t o ~ e t r i e - a l l y . Us ing s t a n d a r d t yp ln~ s e r a and c e l l s , t h e CEL!SA was s p e c i f i c and more s e n s i t i v e than t h e mzcrodrople t cy to toxzc~ ty t e s t (MCT), e . g . f o r a po ly specz fxe ~ H I A a n t x b o d y MCT t z t e r = ~ , C E L I S A t l t e r • 1/ lO00; f o r a monospeezfze ~HLA an t i body MCT t x t e r = 1 / 1 , CELISA t i t e r ~ 1/200. A f a l t e r - p h a s e CELISA was developed t h a t u s e s f l a t bot tom m l e r o t i t e r p l a t e s i n which a f i l t e r ~orms t h e bot tom o f each w e l l . Th is a s s a y r e q u i r e s a s f ev a s 12,500 c e l l s and a s l i t t l e as 5 u l o f an t ibody . Pas~xng wash b u f f e r p a s t t he c e l l s on t h e £11tez e f f e c t i v e l y washes ou t u n b o ~ d a n t i b o d i e s . Bindin~ o f homologous and h e t e r o l o g o u s (mouse monoclonal) eHIA a n t i b o d i e s t o lympho- e y t e s has been d e t e c t e d In a d d i t i o n , ~xndzng o~ a l~ to txny la t edmonoe lona l an t ibody to a human T - c e l l d i f f e r e n t i a t i o n a n t i g e n has been observed wi th av ldzn - AP. The CE~,ISA i s a s u n p l e , r a p i d , s e n s l t x v e , s p e c i f i e m ic ro - t e c h n i q u e fo r o b j e c t i v e l y d e t e c t i n g c e l l s u r f a c e a n t z g e n s . T h e r e f o r e , t h e CELISA may have broad research and cl/nzeal application.

IN SEARCH OF THE H[CHANISH BY HHICH BLOOD TRANSFUSIONS IHPGOVE KIDNEY GRAFT SUR- VIVAL. D.J. Norman and J.M. Barr~, Departments of Medtctn¢ and Surgery, University of Oregon Health Sciences center, Portland, Oregon.

To Improve our understanding of the mechanisms by which blood transfusions Improve renal a11ograft survival, we began a prospective study tn December 1979 using HLA typed blood donors. Prospective recipients of f i r s t cadaver grafts who had not received blood products were transfused with one unit of packed cells from each of f ive HLA typed donors. 91ood donors who were mismatched for as many HLA-A and -B antigens as posszble were selected and the blood was always transfused within four days Of donation. Fifteen pa- t ients have completed the protocol and 10 have been transplanted. Lympho- cytotoxtc antibodies developed transiently or pemanently tn 11 patients (735}. Only one patient (7~) doveloped antibodies whtch ki l led >15~ of a lymphocyte panel. This patient kf11$ >99~ of the panel and since being placed on the Cadaver l i s t has been crossmatch (+) with six prospective do- nors. All of the other 10 patients who have been put on the cadaver Hs t have received kidneys and, of the Z5 crossmatches perfumed on prospective donors, only one was positive. Actuartal graft survival is lOO~at 10 months. Seven of the 10 patients received only HLA mismatched blood~hfle three required 2, 3 and 5 additional units, respectively. Nine patients .ere transrlanted from donors who shared at least one HLA-A and -B antigen with their blood donors. One shared four antigens, four shared two and three shared one. Because of: I) the low incidence of broad|y reactive 3ymphocytotoxtns, 2) low Incidence of positive cro;matches with prospec- t ive donors, and 3) exce]lent graft survival, our data suggest that fresh HtA m|smatched transfusions induce a fom of Immunologic unresponsiveness.

THREE KINDS UP B CELL ANTIBODIES IN KIDNEY TRANSPLANT RECIPIENTS. Cabr ie l Nunez 9 ~ Pe t e r Sta~tny~ Department of I n t e r n a l Mediczne. The U n i v e r s i t y of Texas Heal th Science Center a t D a l l a s . Texas .

An t ibod ies a g a i n s t B c e l l s in r e c i p i e n t s of k~doey a l l o g r a f t s a re known to be "~eterogeneous. Some a r e d i r e c t e d aaazns t HLA-DR a n t i g e n s , o t h e r s have been shown to r e a c t wi th non-DR s u r f a c e de te rminan t s such a s i~munoglobulln. We have developed a procedure fo r i d e n t i f y i n g B c e l l a n t i b o d i e s by b lock ing t h e i r r e a c t i o n s wl th non-complement - f lx ing r e a g e n t s p r i o r to c y t o t o x i c l t y t e s t i n g . A monoclonal ant i -DR (~DR) was used to i d e n t i f y ant i-DR a u t i -