Recombinant P51 As Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1

Overview

Journal Clin Diagn Lab Immunol

Publisher American Society for Microbiology

Specialty Allergy & Immunology

Date 2000 Nov 4

PMID 11063508

Authors

K Hashinaka

S Hashida

I Nishikata

A Adachi

S Oka

E Ishikawa

Affiliations

Soon will be listed here.

Abstract

An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in E. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to beta-D-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 log X + 0.23; r = 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.

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