Open AccessPreparations of recombinant HIV‐1 p66 antigen to improve the specificity of immune complex transfer enzyme immunoassay of antibody IgG to HIV‐1 reverse transcriptase
Author(s) -
Hashinaka Kazuya,
Nishikata Ichiro,
Hashida Seiichi,
Adachi Akio,
Oka Shinichi,
Ishikawa Eiji
Publication year - 2000
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/1098-2825(2000)14:4<169::aid-jcla6>3.0.co;2-e
Subject(s) - maltose binding protein , fusion protein , microbiology and biotechnology , immunoassay , recombinant dna , antigen , antibody , reverse transcriptase , protein subunit , chemistry , protease , integrase , enzyme , protein g , biology , biochemistry , dna , rna , genetics , immunology , gene
Recombinant HIV‐1 p66 (rp66, a subunit of reverse transcriptase (RT), a heterodimer of p66 and p51) was produced in Escherichia coli in three different ways. First, rp66 was produced as a part of the fusion protein of lacZ protein and HIV‐1 pol protein consisting of three components: protease (p10), RT (p51/p66), and integrase (p31), and was released from the fusion protein by the protease ( pol –rp66). Second, rp66 with Ser–Ser at the N ‐terminus was produced as a fusion protein with maltose‐binding protein containing a factor Xa site between the two proteins (MBP–Ser–Ser–rp66) and was released from the fusion protein by factor Xa (Ser–Ser–rp66). Third, rp66 with Met–Gly at the N ‐terminus was produced in transformed cells (Met–Gly–rp66). The recombinant proteins were purified from sonic extracts of transformed cells by ammonium sulfate fractionation and various column chromatographies. MBP–Ser–Ser–rp66 and Met–Gly–rp66 were readily purified in sufficient amounts for labeling with 2,4‐dinitrophenyl groups and β‐ D ‐galactosidase from E. coli , but pol –rp66 and Ser–Ser–rp66 were not for enzyme‐labeling. Ser–Ser–rp66 was not only polymerized but also degraded to considerable extents. The purified preparations were labeled with 2,4‐dinitrophenyl groups and β‐ D ‐galactosidase and were tested in immune complex transfer enzyme immunoassay of antibody IgG to HIV‐1 RT using serum samples from 600 HIV‐1 seronegative and 30 HIV‐1 seropositive subjects. Among various combined uses of the two labeled preparations, the uses of 2,4‐dinitrophenylated MBP–Ser–Ser–rp66 and pol –rp66 with β‐D‐galactosidase‐labeled Met–Gly–rp66 showed the highest (99.8%) and the second highest (99.5%) specificities, which were higher than that with the labeled preparations used in the previous study (98.0%). J. Clin. Lab. Anal. 14:169–179, 2000. © 2000 Wiley‐Liss, Inc.