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A new cell line-based neutralization assay for primary HIV type 1 isolates

Shi, Y; Albert, J; Francis, G; Holmes, H and Fenyö, Eva Maria LU (2002) In AIDS Research and Human Retroviruses 18(13). p.957-967
Abstract
Simple and standardized assays for detection and quantification of neutralizing antibodies to primary HIV-1 isolates are needed in research on HIV-1 vaccines and pathogenesis. Here we describe a new HIV-1 neutralization assay that is based on plaque formation in U87.CD4-CCR5 and U87.CD4-CXCR4 cells, which is an attractive alternative to peripheral blood mononuclear cell-based assays. Infected cells form syncytia, that is, plaques, that can be stained with hematoxylin and enumerated by light microscopy. Neutralization 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. The intraassay variation of the plaque-forming unit determinations was tested... (More)
Simple and standardized assays for detection and quantification of neutralizing antibodies to primary HIV-1 isolates are needed in research on HIV-1 vaccines and pathogenesis. Here we describe a new HIV-1 neutralization assay that is based on plaque formation in U87.CD4-CCR5 and U87.CD4-CXCR4 cells, which is an attractive alternative to peripheral blood mononuclear cell-based assays. Infected cells form syncytia, that is, plaques, that can be stained with hematoxylin and enumerated by light microscopy. Neutralization 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. The intraassay variation of the plaque-forming unit determinations was tested with 15 serum-virus combinations and showed good reproducibility. The differences ranged from -19 to +27% and had a standard deviation of +/-9.1%. On the basis of these data the cutoff for neutralization (i.e., plaque reduction) was set to 30% (3.3 standard deviations). Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10-100 PFU/well. The reproducibility of the new neutralization assay was tested with 4 primary viruses and 9 sera for a total of 20 virus-serum combinations. The mean difference in neutralization (i.e. plaque reduction) determinations performed on different days was as small as 11%. None of 10 Swedish sera and I Ugandan plasma pool from HIV-1-uninfected subjects were positive for neutralization, indicating that the assay has high specificity. In summary, the new U87.CD4 cell line-based neutralization assay for primary HIV-1 isolates is a highly reproducible, sensitive, and high-throughput assay that is well suited for large-scale HIV-1 neutralization studies. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
AIDS Research and Human Retroviruses
volume
18
issue
13
pages
957 - 967
publisher
Mary Ann Liebert, Inc.
external identifiers
  • pmid:12230938
  • wos:000177814500007
  • scopus:0036382602
ISSN
1931-8405
DOI
10.1089/088922202760265623
language
English
LU publication?
yes
id
40aec790-eb0a-425a-a522-c41a98cdd64b (old id 329936)
date added to LUP
2007-11-13 15:02:08
date last changed
2017-01-01 05:06:49
@article{40aec790-eb0a-425a-a522-c41a98cdd64b,
  abstract     = {Simple and standardized assays for detection and quantification of neutralizing antibodies to primary HIV-1 isolates are needed in research on HIV-1 vaccines and pathogenesis. Here we describe a new HIV-1 neutralization assay that is based on plaque formation in U87.CD4-CCR5 and U87.CD4-CXCR4 cells, which is an attractive alternative to peripheral blood mononuclear cell-based assays. Infected cells form syncytia, that is, plaques, that can be stained with hematoxylin and enumerated by light microscopy. Neutralization 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. The intraassay variation of the plaque-forming unit determinations was tested with 15 serum-virus combinations and showed good reproducibility. The differences ranged from -19 to +27% and had a standard deviation of +/-9.1%. On the basis of these data the cutoff for neutralization (i.e., plaque reduction) was set to 30% (3.3 standard deviations). Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10-100 PFU/well. The reproducibility of the new neutralization assay was tested with 4 primary viruses and 9 sera for a total of 20 virus-serum combinations. The mean difference in neutralization (i.e. plaque reduction) determinations performed on different days was as small as 11%. None of 10 Swedish sera and I Ugandan plasma pool from HIV-1-uninfected subjects were positive for neutralization, indicating that the assay has high specificity. In summary, the new U87.CD4 cell line-based neutralization assay for primary HIV-1 isolates is a highly reproducible, sensitive, and high-throughput assay that is well suited for large-scale HIV-1 neutralization studies.},
  author       = {Shi, Y and Albert, J and Francis, G and Holmes, H and Fenyö, Eva Maria},
  issn         = {1931-8405},
  language     = {eng},
  number       = {13},
  pages        = {957--967},
  publisher    = {Mary Ann Liebert, Inc.},
  series       = {AIDS Research and Human Retroviruses},
  title        = {A new cell line-based neutralization assay for primary HIV type 1 isolates},
  url          = {http://dx.doi.org/10.1089/088922202760265623},
  volume       = {18},
  year         = {2002},
}