Detection of Early Anti-p24 HIV Responses in EIA- and Immunoblot-Negative Individuals

Abstract

A sensitive and specific radioimmunoprécipitation assay was developed for the detection and analysis of anti-HIV antibody response in human sera with the use of 125I-labelled purified HIV proteins with subsequent sodium-dodecylsulfate gel electrophoresis (125I-RIPA). The 125I-RIPA was shown to be as specific but at least 1 log more sensitive with respect to the detection of gp41^env and p24^gag than the immunoblot analysis as tested in serum samples from several risk groups. Sequential sera were obtained from 9 individuals who seroconverted for HIV antibodies. In 4 individuals, antibody to p24^gag was detected in earlier serum samples by the 125I-RIPA than by EIA or immunoblot; in the other 5 individuals, the detection of p24^gag concorded in enzyme-linked immunosorbent assay (EIA), immunoblot and 125I-RIPA. Moreover, in one of 78 randomly chosen EIA-negative sera from individuals at high risk, antibodies to p24^gag could be detected by the l25I-RIPA. This early seroconversion was confirmed 3 months later by means of immunoblotting and EIA. The specificity of the 125I-RIPA was further demonstrated by analyzing sequential EIA-negative serum samples from 10 individuals at risk for AIDS, collected during 2 years at 3-monthly intervals. All 80 serum samples were found to be negative in the 125I-RIPA and the individuals revealed no signs of HIV infection. The 125I-RIPA technique may be a valuable confirmatory assay in the serology of HIV infections. The sensitivity of this test provides a reliable measure of effective sensitivity when new-generation screening tests are evaluated. In addition, this technique appears to be a specific and sensitive assay to detect antibodies to p24^gag at very early stages of HIV infection.

Increased Neutralization Sensitivity and Reduced Replicative Capacity of Human Immunodeficiency Virus Type 1 after Short-Term In Vivo or In Vitro Passage through Chimpanzees

“…In brief, 50-l solutions of phosphate-buffered saline (PBS) containing either 20 g of peptides or 5 g of protein was used for labeling with Na-125 I using chloramine T for 30 s (27). The radiolabeled preparations were purified from free iodine by dialysis and subsequently aliquoted in the presence of protease inhibitor phenylmethylsulfonyl fluoride and bovine serum albumin (BSA; fraction V; Sigma) in a final concentration of 0.1% and stored until use at Ϫ20°C.…”

Wegener's granulomatosis autoantibodies identify a novel diisopropylfluorophosphate-binding protein in the lysosomes of normal human neutrophils.

“…After dialysis against PBS the extracts and membrane preparations from subcellular neutrophil fractions (pools a, fl, and y) were supplemented with NaCI, SDS, and Triton X-100 to final concentrations of 0.5 M, 0.5% (wt/vol), and 1% (wt/vol), respectively. The samples thus obtained were radiolabeled with 1251I by the iodogen method (31) and assayed by immunoprecipitation with the protein A method described by Huisman et al (32). Immunoprecipitation with sheep anti-cathepsin G was performed with antibodies linked covalently to CNBr-activated Sepharose 4B.…”

Blood Donors with Indeterminate Anti-p24^gag Reactivity in HIV-1 Western Blot: Absence of Infectivity to Transfused Patients and in Virus Culture

“…We believe that the WB assay in these cases of atypical anti-p248a® reactivity recognizes crossreacting antibodies against a common epitope or epitopes, which may be enhanced by the changes in structure of the viral proteins during the WB procedures, and therefore are hardly found with 125I-RIPA, which in our hands is as sensitive as WB in detecting true anti-p24gag antibodies [13,15]. This is supported by the specific inhibition of the reactivity with infected H9 cells.…”

“…Additional HIV-1 antibody testing was performed with HIV-1 125I radioimmunoprécipitation assay (RIPA) and HIV-1 WB devel oped by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands (WB-CLB) [12,15] and HIV-1 WB by Diagnostic Pasteur (Mames-la-Coquette, France) (WB-Pasteur).…”