title: A comparative study of two different assay kits for the detection of secreted alkaline phosphatase in HPV antibody neutralization assays.
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.
authors.affiliation: a Human Papillomavirus (HPV) Immunology Laboratory; Leidos Biomedical Research, Inc. ; Frederick National Laboratory for Cancer Research ; Frederick , MD USA.
authors.forename: Troy J
authors.initials: TJ
authors.lastname: Kemp
authors.forename: Ken
authors.initials: K
authors.lastname: Matsui
authors.forename: Gloriana
authors.initials: G
authors.lastname: Shelton
authors.forename: Mahboobeh
authors.initials: M
authors.lastname: Safaeian
authors.forename: Ligia A
authors.initials: LA
authors.lastname: Pinto
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis*; D057134:Antibodies, Neutralizing / Q000097:blood*; D002648:Child; D005260:Female; D052162:Human papillomavirus 16 / Q000276:immunology*; D052161:Human papillomavirus 18 / Q000276:immunology*; D006801:Humans; D009500:Neutralization Tests / Q000379:methods*; D053918:Papillomavirus Vaccines / Q000008:administration & dosage / Q000276:immunology*; D022701:Virosomes / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D053918:Papillomavirus Vaccines; D022701:Virosomes; D000469:Alkaline Phosphatase
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