Comparison of Western blot (immunoblot) based on recombinant-derived p41 with conventional tests for serodiagnosis of human immunodeficiency virus infections

Abstract

To evaluate the performance of a serological test for human immunodeficiency virus type 1 (HIV-1) infections based on the use of a recombinant envelope gene-derived protein as the antigen, we caused expression of a 1.4-kilobase fragment of HIV DNA that codes for the complete gp4l transmembrane protein in an Escherichia coli expression vector and used Western blots (WB; immunoblots) prepared with recombinant material (pEX-41) to detect antibodies to HIV-1. This test detected all 339 sera which were positive by a combination of conventional serodiagnostic assays and produced no false-positive results with 311 negative samples. Also no false-positive results were obtained with 20 sera from systemic lupus erythematosus patients which had high titers of cross-reactive autoantibodies. In six cases, the pEX-41 WB proved to be more sensitive than individual assays applied on their own, and in five cases it was even more sensitive than a combination of conventional assays. We tested 221 sera in both our pEX-41 WB and a commercially available recombinant enzyme immunoassay (EIA [Abbott]). The results were identical in 188 cases. A total of 27 sera containing antibodies to gp4l as demonstrated in the pEX-41 WB, as weIl as the Abbott recombinant EIA, had no antibodies to the recombinant core antigen as measured in the Abbott EIA. However, 25 of these sera did stain the 24-kilodalton band on a WB with purified virus. Six sera that were positive in all of the conventional confirmatory assays and reacted strongly with the pEX-41 WB did not recognize the surface antigen used in the Abbott recombinant EIA. We conclude that the use of WB prepared with recombinant-derived p41 offers a very sensitive and specific method to detect antibodies to HIV.

Reliable confirmation and quantitation of human immunodeficiency virus type 1 antibody using a recombinant‐antigen immunoblot assay

“…Recombinant DNA-derived HIV-1 antigens and synthetic HIV-1 peptides have been used recently as substrates in anti-HIV-1 assays. [21][22][23][24][25][26] Because these reagents contain the immunodominant regions of selected viral antigens and are not contaminated with human cellular proteins, these antigens theoretically should provide both more sensitive and more specific assays than those based on native virus. In this report, we describe our evaluation of an investigational, recombinant, DNA-derived, HIV antigen-based immunoblot confirmatory assay (RIBA-HIV216, Chiron Corp., Emeryville, CA).…”

Quantitative western immunoblotting analysis in survey of human immunodeficiency virus-seropositive patients

“…First, it is important to standardize the techniques used when carrying out the actual WB; automated instruments for WB are therefore preferable. The use of synthetic viral antigens (prepared in vitro from recombinant DNA) to cover the supports would improve interseries specificity and reproducibility (13,21,27). Developing calibration and defining a standard unit of measurement for all users should also be envisaged.…”

Reliable confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) with an enzyme-linked immunoassay using recombinant antigens derived from the HIV-1 gag, pol, and env genes

“…Falsepositive results are often due to nonspecific reactions to nonviral cellular antigens (e.g., human leukocyte antigens and other cellular membrane antigens) which copurify with virions (22,24,26,27,31,33). Synthetic peptides and recombinant HIV proteins have been generated in an attempt to provide a pure source of HIV antigen(s) free frQm cellular protein contamination for serological testing (5,11,18,(37)(38)(39)42). Although these recombinant antigens were useful in detecting seropositive individuals, they were not as sensitive as the currently existing screening tests.…”

“…Although these recombinant antigens were useful in detecting seropositive individuals, they were not as sensitive as the currently existing screening tests. However, these studies used recombinant antigens representative of only one or two HIV gene products, and in all but one study (5), only small numbers of patient sera were analyzed (11,18,(37)(38)(39)42).…”