RESUMO
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.
Assuntos
Sequência de Bases , Primers do DNA , Anticorpos Anti-HIV/sangue , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/classificação , Humanos , Técnicas Imunoenzimáticas/métodos , Proteínas Recombinantes/imunologiaRESUMO
The antibody patterns of HIV-1 IgG3, IgG and IgA and of HIV-1 p24 antigen were investigated in Thai infants born to mothers infected with HIV-1. In the 17 HIV-1 infected infants, anti-HIV antibodies were detected continuously over a period of 15-18 months and a high level of specific IgG3 subclass was observed. Anti-HIV IgA could be detected at 6 months of age whereas p24Ag was detected at 2 months. In 79 uninfected infants, maternal anti-HIV IgG gradually decreased over 9 months whilst specific IgG3 decayed rapidly during the first 6 months. Both p24Ag and anti-HIV IgA were not found in these uninfected infants. Thus, the disappearance of anti-IgG3 subclass antibodies within 6 months can predict whether infants are uninfected whereas the persistence of anti-HIV IgG and IgG3 subclass antibodies, the production of anti-HIV IgA antibody and the presence of p24Ag appear as an adjunct to the diagnosis of HIV vertical transmission. The necessary assays are relatively simple and could be performed individually.