Recombinant envelope protein of HIV-1 subtype E as antigen in HIV-1 antibody detection enzyme immunoassay.
In order to develop a reliable and inexpensive serodiagnostic method to be used for anti-HIV antibody detection in Thailand, recombinant envelope (TM or gp41 subunit) protein of HIV-1 subtype E was produced from prokaryotic cell (Escherichia coli) as the source of antigen in enzyme immunoassay (TE diagnostic EIA kit). HIV-1 gp41 subunit of subtype E was successfully expressed in E. coli in the form of polyhistidine-tagged proteins, comprising of rgp41A (601 bases N-terminal half of TM or 25kDa) and rgp41B (560 bases C-terminal half of TM or 24 kDa) by using an expression vector, pBAD/His C. The amount of protein, dilution of sera, and anti-human IgG labeled HRP used in the EIA test optimized by a checker board titration of the protein and seropositive or seronegative sera, were 5.0 microg/ml, 1:300, and 1:4,000, respectively. The blinded test evaluation of TE-diagnostic EIA in 500 seropositive and 500 seronegative sera which have been simultaneously tested by two available commercial kits and compared with our TE diagnostic EIA, gave 99.6% sensitivity and specificity. The other known genetic subtypes sera such as subtype A (n=5), B (n=9), C (n=4) and D (n=5) were also positive with this EIA. The estimated manufacturer cost per test of rgp41 based anti-HIV antibody detection EIA or TE-diagnostic EIA was about 15 baht. This recombinant envelope (gp41 or TM) protein from HIV-1, which can be produced in large quantities without any hazards from growing the virus and has lower cost to produce anti-HIV antibody serological diagnostic kit, should be considered as an HIV screening test in Thailand.
Sutthent
Ruengpung
R
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. sirst@mahidol.ac.th
Kanoksinsombat
Chinda
C
Horthongkham
Navin
N
Louisirirotchanakul
Suda
S
Auewarakul
Prasert
P
Kantakamalakul
Wannee
W
D001483:Base Sequence; D017931:DNA Primers; D015483:HIV Antibodies / Q000097:blood*; D015700:HIV Envelope Protein gp41 / Q000276:immunology*; D015497:HIV-1 / Q000145:classification / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D011994:Recombinant Proteins / Q000276:immunology
D017931:DNA Primers; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D011994:Recombinant Proteins