Recombinant p51 as antigen in an immune complex transfer enzyme immunoassay of immunoglobulin G antibody to human immunodeficiency virus type 1.
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.
Hashinaka
K
K
Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki, Japan.
Hashida
S
S
Nishikata
I
I
Adachi
A
A
Oka
S
S
Ishikawa
E
E
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015483:HIV Antibodies / Q000097:blood*; D015488:HIV Antigens / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000276:immunology; D006679:HIV Seropositivity / Q000276:immunology; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007074:Immunoglobulin G / Q000097:blood*; D008297:Male; D008875:Middle Aged; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000936:Antigen-Antibody Complex; D017931:DNA Primers; D015483:HIV Antibodies; D015488:HIV Antigens; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase