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 antibodies was developed and compared to complement-dependent microlymphocytotoxicity. ELISA plates were coated with a panel of sHLA class I antigens isolated from the culture supernatants of 46 different EBV-transformed phenotyped B-cell lines. After the incubation of the coated plates with test serum, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Absorbance was read using an ELISA plate reader and assay results were analyzed by computer. Antibody specificities were determined by Fisher's exact test tail analysis. The reproducibility of ELISA assay results was evaluated in a blinded, controlled multicenter study. A total of 102 serum specimens from patients on waiting lists to receive kidney transplants were tested five times by ELISA in five different laboratories. The correlation coefficients (r) of %PRA values determined by ELISA ranged from 0.89 to 0.96, and the average agreement on qualitative assay results (antibody positive vs antibody negative) was 98%. Endpoint titration of several serum specimens demonstrated equivalent sensitivity of ELISA and microlymphocytotoxicity (using the anti-globulin antibody protocol). Most of the antibody specificities determined by ELISA were in agreement with specificities determined by microlymphocytotoxicity. To evaluate the correlation of ELISA and microlymphocytotoxicity (CDC) assay results the same 102 specimens were tested six times by CDC in five different laboratories. The interlaboratory correlation coefficient (r) of %PRA values determined by microlymphocytotoxicity ranged from 0.57 to 0.94, and the average agreement on qualitative assay results was 85%. A comparison of ELISA with microlymphocytotoxicity was performed using consensus microlymphocytotoxicity results. This showed a high correlation (r = 0.81) of %PRA values determined by ELISA and microlymphocytotoxicity. This demonstrates that the detection of anti-HLA class I antibodies by soluble HLA ELISA is a reliable alternative to microlymphocytotoxicity testing.
Buelow
R
R
SangStat Medical Corporation, Menlo Park, CA 94025, USA.
Mercier
I
I
Glanville
L
L
Regan
J
J
Ellingson
L
L
Janda
G
G
Claas
F
F
Colombe
B
B
Gelder
F
F
Grosse-Wilde
H
H
D000918:Antibody Specificity; D002461:Cell Line, Transformed; D003601:Cytotoxicity Tests, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D015234:HLA-A Antigens / Q000276:immunology*; D015235:HLA-B Antigens / Q000276:immunology*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000276:immunology; D007518:Isoantibodies / Q000032:analysis* / Q000276:immunology; D007753:Laboratories / Q000592:standards; D015203:Reproducibility of Results; D016037:Single-Blind Method
D015234:HLA-A Antigens; D015235:HLA-B Antigens; D007074:Immunoglobulin G; D007518:Isoantibodies