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[PMC free article] [PubMed] [Google Scholar] 2. where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated. The most medically important flaviviruses include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), and Saint Louis encephalitis virus (SLEV) (16, 31, 38). Flaviviruses are positive-strand RNA viruses with genomes of approximately 11 kb that encode three structural and seven nonstructural (NS) proteins in the gene order C (capsid), M (membrane), E (envelope), NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. WNV is usually a member of the JEV serocomplex within the genus C6/36 cells that had been infected with WNV (NY-99 FN-1501 strain) at a multiplicity of contamination of 0.1 (3). At 120 h postinfection, the cells were scraped from the flask and pelleted by centrifugation at 4,000 rpm for 10 min FN-1501 at 4C. Cell pellets were washed four times with borate saline (1.5 M NaCl, 0.5 M H3BO2, 1.0 M NaOH, pH 9.0) and the final pellet was resuspended in 0.1% sodium dodecyl sulfate and 1% Triton X-100. The cells were sonicated on ice at a 20% output setting for 30 s and centrifuged at 8,000 rpm for 10 min at 4C. Supernatants were aliquoted and stored at ?70C until use. b-ELISA. The b-ELISA for detection of antibodies to WNV FN-1501 was performed using MAb 3.1112G (Chemicon International, Inc., Temecula, CA) and horseradish peroxidase-labeled rabbit anti-mouse IgG (catalog no. 61-6520; Zymed Laboratories). The b-ELISA for detection of antibodies to flaviviruses was performed using horseradish peroxidase-labeled MAb 6B6C-1 (CDC, Fort Collins, CO) using the methods KLRD1 and protocols of Hall et al. (17) as modified by Blitvich et al. (3). MAb 3.1112G is specific for the NS1 glycoprotein of WNV (17). MAb 6B6C-1 is usually specific for the flavivirus E protein (22, 36). Briefly, coating antigen, conjugated antibodies, and monoclonal antibodies were independently titrated against negative and positive control serum samples. The internal 60 wells of an ELISA plate (96-well plate) were coated with WNV antigen produced in C6/36 cells and diluted in coating buffer (carbonate-bicarbonate buffer, pH 9.6) and incubated overnight at 4C. Plates were washed with washing buffer five times. Blocking buffer (phosphate-buffered saline, 0.1% Tween 20, and 5% nonfat dry milk) was added for 40 min at 37C. Plates were washed with washing buffer. Test sera and positive and negative serum controls were diluted 1:10 in blocking buffer and incubated for 2 h at 37C. Wells were then washed five times with washing buffer. MAb diluted in blocking buffer was added and incubated for 1 h at 37C. After washing, for the WNV b-ELISA horseradish peroxidase-labeled rabbit anti-mouse IgG was added for 1 hour at 37C, and for the flavivirus b-ELISA the MAb added was horseradish peroxidase-conjugated 6B6C-1. After washing, 2,2-azinobis(3-ethylbenzthiazolinesulfonic acid) developing solution was added and incubated at 37C. Optical densities (ODs) were measured at 415 nm at different intervals. Optimal dilutions that yielded an value of 0.05. Microsoft Office Excel 2003 was used to calculate the mean, the variance, and 2 and 3 SD values for the unfavorable control samples. RESULTS Determining the diagnostic efficacy of the b-ELISA using serum specimens FN-1501 from Colorado. The 366 human serum specimens from patients presenting with WNV-like symptoms (292 WNV positive and 74 WNV.