NATIVE PARTICLE SUSPENSION ELISA (NPSE): A NOVEL METHOD FOR STUDYING THE IMMUNOCHEMISTRY OF HIV-1 SURFACE GLYCOPROTEINS

Abstract

A novel approach termed native particle suspension ELISA (NPSE) was developed to study more thoroughly the immunochemistry and physiochemical properties of immunoglobulin's interaction with the intact, native, oligomeric surface glycoproteins (gp120/gp41) on infectious HIV - 1 particles. This approach takes advantage of a rather simple series of sensitive and sophisticated technical methodologies. By combining suspension equilibrium-dissociation ultra-centrifugation, solid phase enzyme-linked immunosorbent assays, and utilizing accurately quantitated mono-specific immunoglobulin, a direct measurement of the binding behaviors can be made on the intact, native virus particle. For the initial studies, a neutralizing monoclonal (0.5β) directed to the major immunodominant neutralizing epitope of gp120 (V3) was chosen because the V3 epitope has been shown to be involved in neutralization, cell to cell fusion and tropism. Following the calibration of necessary standardization and control assays, equilibrium-binding studies were designed to measure the binding of 0.5β to native virus particles during their spontaneous shedding of the viral envelope, a recognized natural property of the virus. The function(s) of this "shed" envelope glycoprotein are currently unknown. However, results of this study suggest several functional roles may exist. NPSE analysis demonstrated that the monoclonal 0.513, derived from detergent-disrupted whole virus, recognized, in a preferential manner, the shed viral envelope glycoproteins. On the intact virion, as little as 0.2 to 1.1% of the available 0.5β bound, whereas the earliest shed form(s) of the viral envelope bound 168% more antibody. A intermediate form of 0.5β binding was recognized for soluble gp120 present in the supernate after 24 to 48 hours, or following detergent disruption coupled to a freeze/thaw cycle. Time course NPSE experiments using two closely related strains of HIV -1 demonstrated shedding rates could differ by as much as 1/3 over a 24 hour period. A good correlation could be made between binding of 0.5β to the virion and neutralization. Overall, NPSE provided a reliable, quantitative and sensitive method for assessing the immunochemistry of the viral envelope of HIV -1 and should play an important role in the dissection and understanding of humoral immunity to HIV -1 and other viral agents of man and animal.