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

Abstract

TM SEAP Chemiluminescence Kit 2.0; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; L1 and L2, late 1 and late 2 proteins; RLU, relative-light-unitTo 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 TM 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 Ò 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 Ò immunized individuals. Spearman rank correlational analyses showed excellent correlations between the titers obtained with ZiVa and GE for anti-HPV16 (r D 0.9822, p < 0.0001) and anti-HPV18 (r D 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.

Production of Furin‐Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1‐ or L2‐Specific Antibodies

“…Also, the amount of HPV16 and HPV18 PsV particles required to infect 293TT cells to achieve 100-fold induction in signal over the background was at least 10-fold less with Ziva ® . Neutralizing anti-L1 antibody titers we measured by Ziva ® and Great EscAPe™ in sera of Cervarix ® (GlaxoSmithKline) vaccinated individuals were virtually identical, suggesting that Ziva ® can be a suitable alternative to the well-established Great EscAPe (Kemp et al, 2015)…”

“…This L1‐PBNA system measures HPV anti‐L1 antibody titers using PsV particles where little or no L2 proteins on the virion have been cleaved. Using this system, we compared the Clontech's Great EscAPe SEAP Chemiluminescence Kit 2.0 and Jaden Bioscience's Ziva Ultra SEAP Plus Detection Kit (Kemp et al., ). When we compared these 2 kits, we found that Ziva was able to provide higher signals than those of the Clontech's kit.…”

A safe and reliable neutralization assay based on pseudovirus to measure neutralizing antibody titer against poliovirus

“…Many reporter genes can be used to construct the replicon of pseudovirus, such as EGFP, SEAP, β‐gal, luciferase . The report gene can be activated in 30 min and express large amount of exogenous protein in case of virus entry.…”