14th International AIDS Conference


Barcelona, Spain - July 7-12, 2002

DonateNow
Print this article

A new cell line-based neutralisation assay for primary HIV-1 isolates.

Int Conf AIDS 2002 Jul 7-12; 14:(abstract no. A10076)

Shi Y, Albert J, Francis G, Holmes H, Fenyo EM
Dept of Clinical Virology, Huddinge University Hospital, Karolinska Institute and Dept. of Virology, Lund University, Stockholm and Lund, Sweden

BACKGROUND: Simple and standardized assays for detection and quantification of neutralising antibodies to primary HIV-1 isolates are needed in research on HIV-1 vaccines and pathogenesis.

METHOD: HIV-1 infected cells such as U87.CD4-CCR5 or U87.CD4-CXCR4 cells can form syncytia, i.e. plaques, which can be stained with hematoxylin or monoclonal antibody to HIV-1 p24 antigen and can be enumerated by light microscopy. Neutralisation is determined by the ability of a serum to reduce the number of plaque forming units (PFU) relative to controls exposed to medium or negative serum.

RESULT: Inter- and intra-assay variation was evaluated using a panel of virus isolates and sera representing different HIV-1 subtypes and showed good reproducibility. Neutralisation titre was found to be dependent on virus dose and therefore a dose of 10-100 PFU/well was chosen for these experiments. Based on the results of intra-assay variation, cut-off for neutralisation (i.e. plaque reduction) was set to 30% (3.3 standard deviations). The reproducibility of the new neutralisation assay was tested using four primary viruses and nine sera for a total of 20 virus/serum combinations. The mean difference in neutralization between days was 11%. None of ten Swedish sera and one Ugandan serum from HIV-1 uninfected subjects was positive for neutralisation indicating that the assay has high specificity.

CONCLUSION: The new U87.CD4 cell line based neutralisation assay for primary HIV-1 isolates is a highly reproducible, sensitive and high-throughput assay, which is well suited for large-scale HIV-1 neutralisation studies.

Keywords: AEGIS, HIV-1, Cell Line, Antigens, CD4, AIDS Vaccines, CD4-Positive T-Lymphocytes, Giant Cells, Antibodies, Monoclonal, immunologyKWDaegis,hiv-1,cellline,antigens,cd4,aidsvaccines,cd4-positivet-lymphocytes,giantcells,antibodies,monoclonal,immunology

020707
A10076

Copyright © 2002 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.