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Comparative Study
. 2015;11(2):337-46.
doi: 10.4161/21645515.2014.990851.

A comparative study of two different assay kits for the detection of secreted alkaline phosphatase in HPV antibody neutralization assays

Troy J Kemp  1 , Ken Matsui, Gloriana Shelton, Mahboobeh Safaeian, Ligia A Pinto
Affiliations
Comparative Study

A comparative study of two different assay kits for the detection of secreted alkaline phosphatase in HPV antibody neutralization assays

Troy J Kemp et al. Hum Vaccin Immunother. 2015.

Abstract

To assess immunogenicity and development of antibodies in the context of vaccination, it is critical to quantify titers of neutralizing antibodies. We have been employing the 293TT cell-based neutralization assay system to quantify anti-HPV neutralizing antibodies. In this system, human papillomavirus (HPV) pseudovirion (PsV) particles encapsidating secreted alkaline phosphatase (SEAP) gene are used to measure infection of 293TT cells in 72-hr cell-culture supernatants. SEAP has traditionally been measured by Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (GE). To reduce the cost, and to potentially increase efficiency, we sought a cheaper kit with better detection capability. Performance characteristics of the newer chemiluminescence kit, ZiVa® Ultra SEAP Plus Assay (Ziva) and GE were compared using the 293TT system. Dose titration of HPV PsV 16 or 18 showed that signal-to-noise ratios at 48 and 72 hr post-infection were higher for ZiVa at nearly all doses. ZiVa was superior to GE as it was able to detect SEAP at 48 hr, as well as when lower numbers of 293TT cells were used. The ability of ZiVa to quantitate HPV-16 and -18 neutralizing antibody titers was tested using sera from Cervarix® immunized individuals. Spearman rank correlational analyses showed excellent correlations between the titers obtained with ZiVa and GE for anti-HPV16 (r = 0.9822, p < 0.0001) and anti-HPV18 (r = 0.9832, p < 0.0001) antibodies. We concluded that ZiVa is superior to GE in detecting SEAP, and the antibody titers in sera of vaccinated individuals were similar to those obtained with GE. Thus, Ziva is a suitable alternative to GE.

Keywords: Cervarix®; ELISA, enzyme-linked immunosorbent assay; GE, Great EscAPe™ SEAP Chemiluminescence Kit 2.0; GFP, green fluorescent protein; Gardasil®; HPV, human papillomavirus; L1 and L2, late 1 and late 2 proteins; PsV, pseudovirion; RLU, relative-light-unit; SEAP; VLP, virus-like-particle; ZiVa; ZiVa, ZiVa Ultra SEAP Plus Assay; human papillomavirus; vaccine.

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Figures

Figure 1.
Figure 1.
ZiVa can better detect SEAP than GE. Pseudovirion particles of HPV types 16 and 18 were serially diluted, and used to infect 0.03 × 106 293TT cells. Two-fold serial dilutions were performed starting from 1:1000 to 1:1024000. Supernatants were harvested at 48 and 72 hr post-infection. Each PsV dilution was setup in duplicate wells. Mean ± standard deviation (SD) of 3 independent experiments are shown. (A) Infectivity levels (fold-increase) by PsV16 particles after 48 or 72 hr of incubation are shown. (B) Infectivity levels by PsV18 particles after 48 or 72 hr of incubation are shown. In both (A and B), the same supernatant samples were tested using ZiVa or GE SEAP detection kit. The plots show fold-increase in log10 scale as a function of viral concentration (log10). Some of the error bars are too small to be seen in the graphs.
Figure 2.
Figure 2.
ZiVa can detect SEAP even using limiting numbers of 293TT cells. Different numbers of 293TT cells were plated at the start of the infections by PsV16 or PsV18. The number of cells ranged from 0.002 - 0.3 × 106 cells per well, and 2-fold serial dilutions were performed. Each cell number was tested in triplicates. A fixed dilution factor of 1:100000 for PsV16 (A) and 1:10000 for PsV18 (B) were used to infect 293TT cells. At 48 or 72 hr post-infection, the supernatants were harvested. Cell numbers were plotted in log10 scale. Mean ± SD of 3 independent experiments are shown. Some of the error bars are too small to be seen in the graphs.
Figure 3.
Figure 3.
ZiVa can detect SEAP at lower cell numbers using lower viral dilution concentrations. The experiments were conducted as described in Fig. 2, except higher viral dilution factors were used: 1:600000 for PsV16 and 1:60000 for PsV18. Mean ± SD of 3 independent experiments are shown. Some of the error bars are too small to be seen in the graphs.
Figure 4.
Figure 4.
HPV neutralizing antibody titers measured by ZiVa correlate with those of GE. Cell-culture supernatants from 293TT cell-based neutralization assay from the Uganda Immunogenicity Study were used to quantitate 50% neutralization titers in sera of Cervarix®-immunized individuals using ZiVa or GE. The supernatant samples collected after 72 hr of incubation were used to measure the SEAP activity. Spearman rank-correlational analyses were performed between the titers obtained by ZiVa or GE for both anti-HPV16 and -HPV18 antibodies.
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