Evaluation of viral-lysate enzyme-linked immunosorbent assay kits for detecting human immunodeficiency virus (type 1) infections using human sera standardized by quantitative western blotting.
The first generation of proprietary reagents for detecting antibodies to the Human Immunodeficiency Virus Type 1 (HIV-1) by enzyme-linked immunosorbent assay (ELISA) used as antigen partially purified virus from cell culture lysates. These tests, which are still in use, may vary in their antibody measurement capabilities if different proportions of the viral polypeptides are present in the viral lysate mixtures. We determined the quantities of antibodies in the serum of persons infected with HIV-1 by dilution analysis using 3 ELISA kits: Abbott [A], Du Pont [D], Genetic Systems [G]. The proportionate antibody titres of each serum to p24gag and gp160env/120env were established by quantitative Western blotting. Serum antibody titres were high, frequently over 1:10,000, a result observed both by ELISA and Western blot. For Kit D, sera with high proportions of antibody to p24gag produced antibody titration curves with steep slopes whereas shallower slopes were found in sera with high proportions of antibody to gp160env. In contrast, Kit A gave steeper slopes with sera enriched for gp160env antibodies. Kit G gave results with slopes intermediate between Kits A and D. Serum antibody titres differed between kits depending upon the proportion and concentration of antibodies in a given serum to gp160env and p24gag. The findings that both the concentration and proportion of antibodies to specific viral polypeptides in human sera markedly affect the signal intensity produced by proprietary ELISAs suggest the need for several control sera which reflect the diversity of human serum responses. Standardization of human reference sera by quantitative Western blotting will assist in evaluation and quality control of ELISA tests.
Hardy
C T
CT
Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle 98195.
Damrow
T A
TA
Villareal
D B
DB
Kenny
G E
GE
D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation*; D005069:Evaluation Studies as Topic; D015686:Gene Products, env / Q000276:immunology; D015483:HIV Antibodies / Q000097:blood*; D016655:HIV Core Protein p24 / Q000276:immunology; D019302:HIV Envelope Protein gp160; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D011498:Protein Precursors / Q000276:immunology; D012194:RNA-Directed DNA Polymerase / Q000276:immunology; D011933:Reagent Kits, Diagnostic
D015686:Gene Products, env; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D019302:HIV Envelope Protein gp160; D011498:Protein Precursors; D011933:Reagent Kits, Diagnostic; D012194:RNA-Directed DNA Polymerase