Optimization and proficiency testing of a pseudovirus-based assay for detection of HIV-1 neutralizing antibody in China.
Among the neutralizing antibody evaluation assays, the single-cycle pseudovirus infection assay is high-throughput and can provide rapid, sensitive and reproducible measurements after a single cycle of infection. Cell counts, pseudovirus inoculation levels, amount of diethylaminoethyl-dextran (DEAE-dextran), and the nonspecific effects of serum and plasma were tested to identify the optimal conditions for a neutralizing antibody assay based on pseudoviruses. Optimal conditions for cell counts, pseudovirus inoculation, and amount of DEAE-dextran were 1 × 10(4)cells/well, 200TCID(50)/well, and 15 μg/ml, respectively. Compared with serum samples, high-concentration anticoagulants reduced the relative light unit (RLU) value. The RLU value increased sharply initially but then decreased slowly with dilution of the plasma sample. Test kits containing 10 HIV-1 CRF07/08_BC pseudovirus strains and 10 plasma samples from individuals infected with HIV-1 CRF07/08_BC were assembled into two packages and distributed to nine laboratories with a standard operating procedure included. For the 10 laboratories that evaluated the test, 17 of 44 (37%) laboratory pairs were considered equivalent. A statistical qualification rule was developed based on the testing results from 5 experienced laboratories, where a laboratory qualified if at least 83% of values lied within the acceptable range.
Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Department of Cell Biology, National Institutes for Food and Drug Control (NIFDC), Beijing, China.
Jianhui
J
Nie
Wenbo
W
Wang
Zhiheng
Z
Wen
Aijing
A
Song
Kunxue
K
Hong
Shan
S
Lu
Ping
P
Zhong
Jianqing
J
Xu
Wei
W
Kong
Jingyun
J
Li
Hong
H
Shang
Hong
H
Ling
Li
L
Ruan
Youchun
Y
Wang
D057134:Antibodies, Neutralizing / Q000097:blood* / Q000276:immunology; D002452:Cell Count; D002460:Cell Line; D002681:China; D003588:Cytopathogenic Effect, Viral; D003637:DEAE-Dextran / Q000737:chemistry; D015483:HIV Antibodies / Q000097:blood* / Q000276:immunology; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D020128:Inhibitory Concentration 50; D059021:Laboratory Proficiency Testing / Q000379:methods / Q000592:standards*; D009500:Neutralization Tests / Q000379:methods* / Q000592:standards; D011933:Reagent Kits, Diagnostic / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D057134:Antibodies, Neutralizing; D015483:HIV Antibodies; D011933:Reagent Kits, Diagnostic; D003637:DEAE-Dextran