Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test

Abstract

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

FIG. 1.

(A) Schematic representation of various EAV structural proteins expressed in E. coli. The recombinant full-length proteins derived from EAV ORFs 5, 6, and 7 and their respective truncated versions are depicted. The black bars denote full-length proteins and the white bars denote partial-length proteins. The putative major neutralization sites A (aa 49), B (aa 61), C (aa 67 to 90), and D (aa 99 to 106) located in the N-terminal ectodomain of the GP5 protein are indicated by hatched boxes (1, 4, 6). L, leader sequence; nsp, nonstructural proteins. (B) Western blot analysis of purified bacterial recombinant proteins with EAV protein-specific monoclonal antibodies (MAb) and rabbit (Rb) antisera. Monoclonal or rabbit EAV-specific antibodies used to detect each recombinant protein are indicated at the bottom of the figure. The molecular mass (M.W.) marker is indicated on the right. The molecular masses of recombinant GP5_1-255, GP5_1-116, GP5_75-112, GP5_55-98, N_1-110, N_1-69, M_1-162, and M_88-162 proteins were 32, 17, 7, 8, 15.3, 10.6, 20.6, and 11 kDa, respectively.

FIG. 2.

Comparison of antibody responses to EAV as determined by GP5_55-98 MIA and VNT using sequential serum samples from experimentally infected horses (n = 18). The reciprocal VN titers and MFI values for horses inoculated with different EAV strains, rVBS, 030H, KY84, and CA95G, are plotted against dpi. (A, B, C, and D) VN antibody responses in horses experimentally infected with EAV rVBS (n = 4), EAV 030H (n = 2), EAV KY84 (n = 7), and EAV CA95G (n = 5) as measured by the standard VNT. (E, F, G, and H) VN antibody responses in horses experimentally infected with EAV rVBS (n = 4), EAV 030H (n = 2), EAV KY84 (n = 7), and EAV CA95G (n = 5) as measured by the GP5_55-98 MIA. Designations at the right of each graph represent individual identities of the horses from which samples were obtained.

FIG. 3.

GP5_55-98 and N_1-110 MIA analysis of serially diluted serum samples. Sera from horses with EAV-neutralizing antibodies (+) and negative control equine sera (−) were serially diluted and evaluated in the GP5_55-98 and N_1-110 MIAs. The MFI for each dilution of the standard was determined. Results are reported as MFI per 100 microspheres. Samples are identified by number in the upper right corner. w/, with.

FIG. 4.

ROC curves depicting the sensitivities and specificities of the GP5_55-98 and N_1-110 MIAs compared to the VNT. An ROC curve is a plot of the TPF versus the false-positive fraction (FPF) across all possible cutoff values that can be used to dichotomize the data into positive and negative outcomes. A related parameter of interest is the AUC. Estimated ROC curves (solid lines) with pointwise 95%-credible-interval bands (dashed lines) for the GP5_55-98 MIA and the N_1-110 MIA are indicated.

FIG. 5.

Analysis of the correlation between the GP5_55-98 MIA and the VNT for the detection of anti-EAV antibodies in equine sera. A scatter plot of GP5_55-98 MIA results (MFI values) versus VNT titers determined using 2,500 equine sera is depicted on the log₂ scale. The VNT data were plotted on the x axis, and the MIA data were plotted on the y axis.

FIG. 6.

Aligned deduced amino acid sequences of GP5_55-98 proteins from various laboratory (rVBS and 030H) and field (KY84, CA95G, WA97, and IL93) strains of EAV. Dots indicate the same amino acid as that in the sequence at the top. Letters indicate the amino acid substitution at each site. The predicted N-linked glycosylation sites are underlined. The respective conserved and variable glycosylation sites are indicated by * and ⧫. Neut., neutralization.