Development of an Immunoassay for the Solid Phase Deteciton of HIV-1 Binding to Susceptible Cells
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Authors
Afsari, Amelia G.
Issue Date
1990
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
vii, 64 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.