A new cell line-based neutralization assay for primary HIV type 1 isolates
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/329936
- author
- Shi, Y ; Albert, J ; Francis, G ; Holmes, H and Fenyö, Eva Maria LU
- organization
- publishing date
- 2002
- 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
- 2016-04-01 12:24:13
- date last changed
- 2022-01-27 03:16:23
@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}},
doi = {{10.1089/088922202760265623}},
volume = {{18}},
year = {{2002}},
}