Assessment of three methods of evaluating soluble class I HLA molecules in cell culture supernatants and serum samples from the second international workshop on soluble human leukocyte antigens.
Three methodologies were compared in assessing sHLA specificities in cell culture supernatants and serum specimens from the Second International Workshop on sHLA: CDC inhibition, FC inhibition, and cellular ELISA inhibition. Initially, the CDC inhibition assay used polyclonal antisera in commercial HLA-phenotyping trays to confirm known specificities and screen for unknown specificities in 31 specimens. Although partly successful, critical limits were imposed by the variable antiserum titers. Thus, using pools of these same antisera and renal transplant recipient antisera, the FC inhibition assay was employed to determine the endpoint serum titers before confirming the known sHLA specificities. Of 25 specimens, four were not confirmed and five gave weak inhibitory reactions. The cellular ELISA inhibition assay, incorporating patient sera and mAbs toward three HLA, successfully confirmed all three known specificities in eight selected workshop specimens. Each methodology had advantages and disadvantages, but all three methods were successful in detecting and identifying sHLA class I specificities. Success, however, was dependent on the initial characterization (specificity and titer) and titration to end point (appropriate for each method's sensitivity) of each antibody preparation.
Immunology Center, Loma Linda University Medical Center, CA 92354-2870.
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Nehlsen-Cannarella
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Buckert
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Fagoaga
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Folz
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Grinde
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Hisey
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Schmitt
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Zappia
D002478:Cells, Cultured; D003245:Consensus Statements as Topic; D003470:Culture Media; D003601:Cytotoxicity Tests, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D006680:HLA Antigens / Q000097:blood; D015395:Histocompatibility Antigens Class I / Q000097:blood*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007158:Immunologic Techniques; D016130:Immunophenotyping; D007391:International Cooperation; D012995:Solubility
D003470:Culture Media; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G