Improved detection of serum HIV p24 antigen after acid dissociation of immune complexes

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Abstract
Objective To evaluate an acid pretreatment method designed to dissociate HIV p24 antigen from immune complexes in serum. Design Patient sera and sera containing experimental immune complexes were quantified for p24 antigen before and after immune complex dissociation (ICD). The clinical application of ICD was assessed in 1328 serum and plasma samples collected from HIV-infected patients. Methods Immune complexes were created artificially by mixing purified p24 antigen with antibody-positive sera or a standardized concentration of human antibody to p24. ICD was achieved by incubation of samples with an equal volume of Clycine HCI for 90min at 37$$C followed by neutralization with Tris NaOH. Samples were quantified for p24 antigen using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Results ICD resulted in significant release of purified antigen from simulated immune complexes in antibody-positive sera. Variation in antigen sequestration and dissociation was related to anti-gag antibody titers. ICD resulted in complete recovery of 500 pg of antigen complexed with human anti-p24 antibody at concentrations up to 2.5 U/ml. In seropositive patients, the mean level of serum antigen was 3.5-fold higher after ICD, and an additional 21% were antigen-positive. Conclusions Pretreatment greatly improved antigen detection in HIV-antibody-positive sera by effectively dissociating immune complexes without compromising reactivity of the antigen itself. The treatment also facilitated routine monitoring of patients by revealing fluctuations in serum antigen that were indistinguishable or poorly defined in untreated sera.

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