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. 2014 Jul:409:131-46.
doi: 10.1016/j.jim.2013.11.022. Epub 2013 Dec 1.

Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1

Marcella Sarzotti-Kelsoe  1 , Robert T Bailer  2 , Ellen Turk  2 , Chen-li Lin  2 , Miroslawa Bilska  3 , Kelli M Greene  3 , Hongmei Gao  3 , Christopher A Todd  3 , Daniel A Ozaki  3 , Michael S Seaman  4 , John R Mascola  2 , David C Montefiori  3
Affiliations

Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1

Marcella Sarzotti-Kelsoe et al. J Immunol Methods. 2014 Jul.

Abstract

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of pre-clinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format.

Keywords: Assay validation; GCLP; HIV; Neutralizing antibody; TZM-bl cells.

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Figures

Figure 1
Figure 1. Linear range of input TZM-bl cell numbers that support HIV-1 infection
DEAE-dextran was present at 30 μg/ml. Luminescence was measured in solid black plates after 48 hrs.
Figure 2
Figure 2. Linear range of infection in TZM-bl cells
DEAE-dextran was present at 30 μg/ml after the addition of cells. Luc activity was quantified by luminescence 48 hrs later.
Figure 3
Figure 3. Neutralization curves in TZM-bl cells at different input virus doses
Average RLU in cell control and virus control wells, respectively: 1:250 dilution (926 vs. 11,813; range = 10,887); 1:50 dilution (997 vs. 55,285; range = 54,287); 1:10 dilution (1,059 vs. 242,013; range = 240,954); 1:2 dilution (1,194 vs. 551,651; range = 550,457).
Figure 4
Figure 4. Nonspecific activity of normal human serum samples
Six serum samples assayed at a 1:20 dilution against 17 Env-pseudotyped viruses.
Figure 5
Figure 5. Neutralization curves with s.d. bars
HIV-1-positive serum DUMC-1 was assayed against molecularly cloned Env-pseudotyped viruses 6101.10, QH0515.01 and Du151.02 in triplicate wells for each dilution of serum sample.
Figure 6
Figure 6. Inter-assay and inter-operator variability
TriMab was assayed multiple times against Env-pseudotyped viruses 3988.25, QH0692.42, SS1196.01 and BG1168.01. ID50 values of each curve are shown next to the operator who performed the assay.
Figure 7
Figure 7. Reproducibility of the TZM-bl assay in domestic and international laboratories
Proficiency testing results (ID50 titers) from participating laboratories testing two representative serologic reagents (A, B: reagent 1) (C, D: reagent 2), against six Env-pseudotyped viruses, over a four-year period. Panels A (n=14) and C (n=14) show the reproducibility data obtained from repeat testing by the Duke Laboratory and NVITAL, panels B (n=66) and D (n=66) show data from repeat testing by all participating labs analyzed. Bars indicate the upper and lower limits of the acceptance ranges.
Figure 8
Figure 8. Comparison of the TZM-bl assay to the U87.CD4.CCR5 assay (Monogram) using 2G12 and 2F5 as standards
Values on the y-axis are μg/ml. TZM-bl assay (white); U87.CD4.CCR5 assay (black).
Figure 9
Figure 9. Effect of altering the length of time of infection prior to measuring luminescence
RLU in cell control (background) and SS1196.01 Env-pseudotyped virus control wells were, respectively: 48 hrs = 1,068 vs 65,158; 72 hrs = 1,224 vs 70,756; 96 hrs = 1,604 vs 60,621.
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