Calibration and Evaluation of Quantitative Antibody Titers for Varicella-Zoster Virus by Use of the BioPlex 2200

Abstract

Most commercially available enzyme immunoassay-based methods have limited sensitivity to detect antibody responses to varicella-zoster virus (VZV) in vaccinated individuals, who produce lower antibody levels than those with natural infection. However, more sensitive methods are either not commercially available or less amenable to high-throughput testing. The BioPlex 2200 measles, mumps, rubella, and varicella (MMRV) IgG assay (Bio-Rad Laboratories, Hercules, CA) is an automated high-throughput platform based on the microsphere Luminex technology that measures antibodies against measles, mumps, rubella, and varicella viruses simultaneously. Although it has U.S. Food and Drug Administration approval as a qualitative diagnostic test for measles, mumps, rubella, and varicella virus immunity, in this study, we have validated the assay to produce quantitative titers (off label) against the VaccZyme VZV glycoprotein (VZVgp) low-level IgG kit (The Binding Site Ltd., Birmingham, UK) using the World Health Organization international standard. Here, we show that the BioPlex 2200 MMRV IgG assay has sensitivity superior to that of the Zeus enzyme-linked immunosorbent assay (ELISA) VZV IgG assay (Zeus Diagnostics, Branchburg, NJ). Using receiver operating characteristic (ROC) analysis and adjusting the cutoff levels, we improved the sensitivity of the quantitative BioPlex 2200 MMRV IgG assay to 97.4%, while maintaining 100% specificity.

Keywords: BioPlex; antibody titer; immunity; quantitative; seroepidemiology; varicella-zoster virus.

FIG 1

Standard reference curve for the quantitative BioPlex 2200 VZV IgG assay. Relative fluorescence intensity measured for 2-fold serial dilutions of the first WHO VZV IgG international standard. Each point was measured in triplicate, and the average ± standard deviation (SD) values are shown. The curve was fitted using a 4-parameter logic log equation.

FIG 2

Linearity of the quantitative BioPlex 2200 VZV IgG test. Two-fold dilutions of the first VZV international standard were run on the BioPlex 2200, and the RFI were transformed into milli-international units per milliliter using the calibration curve described in Fig. 1. The results show an almost perfect linear correlation coefficient and a slope near 1.00, indicating that the BioPlex method is linear at least up to 25,000 mIU/ml.

FIG 3

Comparison between the BioPlex 2200 and the VaccZyme gpEIA, using a validation set of 148 archival specimens. The vertical blue and red lines identify the VaccZyme gpEIA equivocal zone suggested by the manufacturer, between 100 and 150 mIU/ml, respectively. The horizontal red line at 190 mIU/ml marks the positive cutoff that defines a positive agreement with gpEIA of 97.4%. The horizontal blue line marks the negative cutoff of 152 mIU/ml, which identifies a negative agreement of 100% with the VaccZyme gpEIA (see also Table 1). The dashed lines represent the 95% CI of the regression line.

FIG 4

ROC analysis of the BioPlex 2200 results against the VaccZyme gpEIA results. The area under the curve is 0.999. Results were classified as positive or negative for VZV protective immunity using the manufacturer’s cutoff for the VaccZyme VZV gpEIA of 150 mIU/ml and 100 mIU/ml, respectively. Two samples in the equivocal range were excluded from the ROC analysis.

FIG 5

Correlation between BioPlex 2200 and VaccZyme gpEIA for low-titer samples. Samples that tested below the established cutoff of positivity for the BioPlex 2200 (Fig. 3 and Table 1) were retested by the VaccZyme gpEIA. The results show a significant linear correlation (R² = 0.787, P < 0.0001). The vertical line marks the positive cutoff for the BioPlex 2200 (190 mIU/ml), and the horizontal line marks the positivity cutoff for the VaccZyme VZV gpEIA, at 150 mIU/ml.