A novel enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HIV-1 envelope glycoproteins based on immobilization of viral glycoproteins in microtiter wells coated with concanavalin A.

We have developed a novel method that greatly simplifies the preparation of solid-phase HIV-1 envelope glycoproteins for use in an ELISA that detects serum antibodies to HIV envelope antigens. This method utilizes concanavalin A absorbed to wells of microtiter plates to affinity immobilize detergent-solubilized viral glycoproteins released in culture fluids of HIV-1 infected cell lines grown in serum free medium. Antibodies binding to ConA-immobilized viral antigens are detected by peroxidase-conjugated antibodies and appropriate enzyme substrates. Unlike most commercial HIV ELISAs, which utilize gp120 depleted-purified virus as the source of antigens and thus favor detection of antibodies to core antigens, the ConA envELISA is highly sensitive for detecting antibodies to native gp120, as evidenced by the strong reactivity of gp120-specific human monoclonal antibodies. Our results also suggest that representation of gp41 in the assay varies and depends on which virus infected cell lines are used for antigen production. Since this assay accurately identified 14 HIV-1 antibody positive patient sera and no false positives were detected among 16 HIV-1 negative sera, the ConA envELISA shows promise as an inexpensive assay for the serologic diagnosis of HIV infections.

Identification of conserved and variant epitopes of human immunodeficiency virus type 1 (HIV-1) gp120 by human monoclonal antibodies produced by EBV-transformed cell lines.

Using Epstein-Barr virus (EBV) transformation of B cells isolated from peripheral blood of two asymptomatic human immunodeficiency virus type 1-(HIV-1) infected subjects, we have produced four IgG1 human monoclonal antibodies (HMAbs) that bind to HIV-1 gp120, as determined by Western blot analysis. Two of these HMAbs, designated N70-1.5e and N70-2.3a, react with epitopes of gp120 expressed by all strains tested thus far, and therefore, appear to identify conserved epitopes. The other two HMAbs, K24-3b and N70-1.9b, identify variant epitopes; K24-3b binds to an epitope which is absent from two strains but heterogeneously expressed in eight other strains; N70-1.9b binds to an epitope that is found in relatively few strains. We also describe a novel immunoassay in which viral glycoproteins, produced by HIV-1-infected cells grown in serum-free medium, are affinity immobilized in Concanavalin A-coated wells of enzyme-linked immunosorbent assay (ELISA) plates. This method greatly facilitates the preparation of solid-phase HIV envelope glycoproteins from multiple virus strains and screening immunoassays based on this method are highly sensitive and effective in detecting antibodies to gp120.