Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

Abstract

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

FIG. 1.

Comparison of Ag capture ELISA, TaqMan, and traditional RT-PCR used to test pools of naturally infected mosquitoes. TaqMan Ct values represent the threshold cycle number at which fluorescence increases above a fixed threshold value. Symbols: ▴, pools positive by TaqMan, Ag capture ELISA, and RT-PCR; ▪, pools positive by TaqMan and Ag capture ELISA but equivocal or negative by RT-PCR; ♦, pool positive by TaqMan and RT-PCR, but negative by Ag capture ELISA; •, pools positive by TaqMan only. ELISA plates were read at 450 nm; all symbols below the dotted line represent negative results by Ag capture ELISA.

FIG. 2.

Sensitivity and specificity of the Ag capture ELISA used to test organ homogenates of naturally infected American crows. (A) Analysis of samples positive (•) and negative (▪) for virus isolation by Ag capture ELISA and TaqMan. A ▪ with a superimposed white × represents a sample which could not be confirmed as positive by the Ag capture inhibition test. A total of 35 WN virus-positive samples from 73 homogenates tested are shown. Symbols below the horizontal dotted line indicate samples negative by Ag-Cap ELISA; symbols to the right of the arrow (TaqMan Ct = 37) indicate samples below the threshold Ct for TaqMan (Ct is the threshold cycle number at which fluorescence increases above a fixed threshold value). The insert shows the linear relationship between virus titer (log₁₀ PFU/0.005 ml) and Ct value for a set of RNA standards from a previously titrated WN virus seed included in the TaqMan assay. (B) Cross-reactivity of avian organ homogenates tested in Ag capture assays for both WN and SLE viral Ags. The absorbance ratio is calculated as A₄₅₀ of test sample/A₄₅₀ of normal control sample; ratios of ≥2.0 are considered significant. The dotted and dashed lines show the 2.0 absorbance ratio thresholds for the SLE and WN virus Ag capture ELISAs, respectively.