An improved microtiter assay for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.
BACKGROUND
The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay.


METHODS
In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor(s), the tested sera or plasma was removed by a washout procedure after initial 3-6 h culture in the assay.


RESULTS
This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method.


CONCLUSIONS
The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.
Zhang
Chiyu
C
Laboratory for Molecular and Cell Immunology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China. zhangcy1999@hotmail.com
Chen
Yunyun
Y
Ben
Kunlong
K
D015492:AIDS Serodiagnosis / Q000379:methods; D002455:Cell Division; D015483:HIV Antibodies / Q000097:blood*; D016655:HIV Core Protein p24 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D009500:Neutralization Tests / Q000379:methods*; D013778:Tetrazolium Salts; D013844:Thiazoles
D015483:HIV Antibodies; D016655:HIV Core Protein p24; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue