title: Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.
abstract: Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.
authors: Department of Veterinary Science, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku, Tokyo 162-8640, Japan.
authors: Yoshihiro
authors: Y
authors: Kaku
authors: Akira
authors: A
authors: Noguchi
authors: Glenn A
authors: GA
authors: Marsh
authors: Jennifer A
authors: JA
authors: Barr
authors: Akiko
authors: A
authors: Okutani
authors: Kozue
authors: K
authors: Hotta
authors: Boldbaatar
authors: B
authors: Bazartseren
authors: Shuetsu
authors: S
authors: Fukushi
authors: Christopher C
authors: CC
authors: Broder
authors: Akio
authors: A
authors: Yamada
authors: Satoshi
authors: S
authors: Inoue
authors: Lin-Fa
authors: LF
authors: Wang
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis* / Q000235:genetics; D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009500:Neutralization Tests / Q000379:methods*; D045405:Nipah Virus / Q000276:immunology*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D018116:Vesiculovirus / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D014773:Virology / Q000379:methods*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase