Development of an Immunoassay for the Solid Phase Deteciton of HIV-1 Binding to Susceptible Cells
Afsari, Amelia G.
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The CD4 surface antigen on the T cells of the immune system is an important receptor for HIV-1 binding via the glycoprotein 120 (gp120) envelope gene. In this study, a solid phase immunoassay was developed to detect HIV-1 binding to susceptible cells. The HeLa-T4+ cell line, which is susceptible to infection, and the HeLa cell line, which is resistant to infection, were used to develop the immunoassay. Two additional cell lines, SK-N-MC and HepG2, were also used; these lines do not express surface CD4, yet they are susceptible to HIV-1 infection via a CD4 independent mechanism. The immunoassay is performed upon crude membrane fractions dot blotted to nitrocellulose membrane. Immunodetection involved applications with either culture supernatants (A3.0l, a non-producing cell line or 8E5 , a virus producing line) or immunoglobulins (OKT4 or H6a/1). This was followed by a detection system that consisted of incubations with an HIV immunoglobulin standard, biotinylated anti-immunoglobulin antibodies, and enzyme-conjugated streptavidin. Use of a chromogenic substrate indicated the presence of binding. Thus far, the results indicate this immunoassay is effective for cells that express CD4+. Further studies should be completed before we can determine whether the assay has sufficient sensitivity to detect virus binding to CD4 cells.