DETECTION OF PANEL-REACTIVE ANTI-HLA CLASS-I ANTIBODIES BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY OR LYMPHOCYTOTOXICITY - RESULTS OF A BLINDED, CONTROLLED MULTICENTER STUDY

A soluble HLA ELISA for the detection of anti-HLA class I IgG antibodi es was developed and compared to complement-dependent microlymphocytot oxicity, ELISA plates were coated with a panel of sHLA class I antigen s isolated from the culture supernatants of 46 different EBV-transform ed phenotyped B-cell lines. After the incubation of the coated plates with test serum, bound antibodies were detected using a peroxidase-con jugated anti-human IgG antibody. Absorbance was read using an ELISA pl ate reader and assay results were analyzed by computer. Antibody speci ficities were determined by Fisher's exact test tail analysis. The rep roducibility of ELISA assay results was evaluated in a blinded, contro lled multicenter study. A total of 102 serum specimens from patients o n waiting lists to receive kidney transplants were tested five times b y ELISA in five different laboratories. The correlation coefficients ( r) of %PRA values determined by ELISA ranged from 0.89 to 0.96, and th e average agreement on qualitative assay results (antibody positive vs antibody negative) was 98%. Endpoint titration of several serum speci mens demonstrated equivalent sensitivity of ELISA and microlymphocytot oxicity (using the anti-globulin antibody protocol). Most of the antib ody specificities determined by ELISA were in agreement with specifici ties determined by microlymphocytotoxicity. To evaluate the correlatio n of ELISA and microlymphocytotoxicity (CDC) assay results the same 10 2 specimens were tested six times by CDC in five different laboratorie s. The interlaboratory correlation coefficient (r) of %PRA values dete rmined by microlymphocytotoxicity ranged from 0.57 to 0.94, and the av erage agreement on qualitative assay results was 85%. A comparison of ELISA with microlymphocytotoxicity was performed using consensus micro lymphocytotoxicity results. This showed a high correlation (r = 0.81) of %PRA values determined by ELISA and microlymphocytotoxicity. This d emonstrates that the detection of anti-HLA class I antibodies by solub le HLA ELISA is a reliable alternative to microlymphocytotoxicity test ing.