An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis
June 19, 2017
An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use SB 239063 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 pathogen (YFV), tick-borne encephalitis pathogen (TBEV), and Saint Louis encephalitis pathogen (SLEV) (16, 31, 38). Flaviviruses are positive-strand RNA infections with genomes of around 11 kb that encode three structural and seven non-structural (NS) protein in the gene purchase C (capsid), M (membrane), E (envelope), NS1, SB 239063 NS2A, NS2B, NS3, NS4A, NS4B, and NS5. WNV is certainly a member from the JEV serocomplex inside the genus C6/36 cells that were contaminated with WNV (NY-99 stress) at a multiplicity of infections of 0.1 (3). At 120 h postinfection, the cells had been scraped through the flask and pelleted by centrifugation at 4,000 rpm for 10 U2AF1 min at 4C. Cell pellets were washed four occasions with borate saline (1.5 M NaCl, 0.5 M H3BO2, 1.0 M NaOH, pH 9.0) SB 239063 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 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 and protocols of Hall et al. (17) as altered 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 occasions. 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 occasions 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 answer 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 SB 239063 samples. RESULTS Determining the diagnostic efficacy of the b-ELISA using serum specimens from Colorado. The 366 human serum specimens from patients presenting with WNV-like symptoms (292 WNV positive and 74 WNV unfavorable) were used to determine overall agreement and sensitivities and specificities of the b-ELISA, based on different diagnostic criteria and PRNT results. To determine the best cutoff values for the b-ELISAs using the WNV-specific MAb 3.1112G and the flavivirus-specific MAb 6B6C-1, three diagnostic criteria were compared. SB 239063 These were the following: (i) the mean result plus 2 SD for the unfavorable control serum samples based on optical densities obtained with the b-ELISA; (ii) the mean results plus 3 SD for the unfavorable control serum specimens based on the optical densities with the b-ELISA; (iii) the 8% and 13% blocking values determined by.