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Clinical and Vaccine Immunology, February 2007, p. 134-138, Vol. 14, No. 2
1071-412X/07/$08.00+0     doi:10.1128/CVI.00322-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Monoclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying West Nile Virus-Neutralizing Antibodies in Horse Sera

National Veterinary Research and Quarantine Service, 480 Anyang-6 dong, Anyang, Gyeonggi 430-824, Republic of Korea,¹ College of Veterinary Medicine, Chungbuk National University, 48 Gaeshin-dong, Heungduk-Ku, Cheongju, Chungbuk 361-763, Republic of Korea,² Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011³

Received 7 September 2006/ Returned for modification 16 October 2006/ Accepted 16 November 2006

A rapid immunoassay for detecting and quantifying West Nile virus (WNV)-neutralizing antibodies in sera was developed as an alternative to the plaque reduction neutralization test (PRNT), the gold standard test for WNV. The assay is a competitive, enzyme-linked immunosorbent assay using neutralizing monoclonal antibody 5E8 (NT-ELISA). A cutoff percent inhibition (PI) value of 35% (mean PI plus 3 standard deviations), with a specificity of 99%, was established based on analysis of 246 serum samples from horses free of WNV. The NT-ELISA detected neutralizing antibodies in all sera collected 7 or 14 days postinoculation from mice (n = 11) infected with lineage I (strain NY385-99) or II (strain B956) WNV. When sera from WNV-vaccinated horses (n = 212) were tested by NT-ELISA and PRNT, the NT-ELISA gave a positive result for 96.1% (173/180) of the PRNT-positive sera and 3.1% (1/32) of the PRNT-negative sera. Discrepancies between the two tests were observed mainly with sera with low PRNT₉₀ titers (expressed as the reciprocal of the highest dilution yielding 90% reduction in the number of plaques) for WNV or low PIs by NT-ELISA. The overall agreement (k value) between the two tests was 0.86. A good correlation (r² = 0.77) was also observed between the tests for endpoint titration of sera (n = 116). In conclusion, the newly developed NT-ELISA may be a good alternative serologic assay for detecting WNV that can be used for large-scale testing of WNV-neutralizing antibodies in multiple species.

Published ahead of print on 29 November 2006.