Brigido LF, Gilbert M, Culp J, Mills J; International Conference on AIDS.
Int Conf AIDS. 1991 Jun 16-21; 7: 97 (abstract no. M.A.1023).
SFGH-University of California, San Francisco, CA
OBJECTIVE: To develop a simple, rapid and economical system to measure the binding of variant gp120 molecules to rsCD4, and to screen for inhibitors of binding potentially suitable as antivirals. METHODS: An ELISA previously described (Gilbert et al, J. Clin. Micro 29:142-147;1991) was modified for these purposes. 10 ng/ml of recombinant or native gp120, with or without preincubation with inhibitors were added to wells coated with 1 ug of rsCD4 and binding was detected with sheep anti-gp120-biotin-avidin-phosphatase system; with results expressed as percent of control wells. RESULTS: Up to 80% of enzymatic deglycosylation with Endo F/Endo H did not modify the binding to CD4 and recognition by the antiserum, although a non-glycosylated gp120 tested, produced in yeast (NIH 388), gave an absorbance 10x less than that obtained with glycosylated material. The presence of gp120 partially blocked the binding of leu3a, not decreasing the binding of other anti-CD4 monoclonal antibodies (mAb) unrelated to the T4a epitope. On the other hand, leu3a blocked sequential binding of gp (50% inhibition, I50% = 250 ng/ml) whereas other mAbs, including OKT4 did not RsCD4 also inhibited (I50% = 30 ng/ml) binding, and several sulphonated polysaccharides, as dextran sulfate and polyanetholsulfonic acid blocked binding at 5 ug to 500 ng/ml, as did aurintricarboxylic acid (I50% = 1.5 ug/ml). At higher concentrations, 1 mg to 10 ug/ml, these inhibitors were shown to bind to CD4, as preincubation of plaques blocked binding of gp added sequentially. Several lectins blocked binding (ConA, Sweet pea, lentil, human mannose binding protein), presumably through attachment to gp120 sugar residues. CONCLUSION: This assay system can be used to determine easily and simply the relative binding of various gp120 molecules to CD4, and to quantify inhibitors of that binding. It may be useful as a screen for inhibitors of binding with potential antiviral activity.
Publication Types:
Keywords:
- Antibodies, Monoclonal
- Antigens, CD4
- Dextran Sulfate
- Enzyme-Linked Immunosorbent Assay
- Epitopes
- HIV Envelope Protein gp120
- Humans
- Ligands
- Polysaccharides
- Research Design
- antagonists & inhibitors
- immunology
- metabolism
- pharmacokinetics
Other ID:
UI: 102182587
From Meeting Abstracts