Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus

Abstract

Background: Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies.

Results: A recombinant North American nucleoprotein (NP) based iELISA was developed and validated along with a bELISA using newly developed PEDV-NP specific biotinylated monoclonal antibodies (mAbs) and an FMIA using magnetic beads coupled with expressed NA PEDV-NP. Receiver operating characteristic (ROC) analysis was performed using swine serum samples (iELISA n = 1486, bELISA n = 1186, FMIA n = 1420). The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. The iELISA and bELISA showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. Inter-rater (kappa) agreement was calculated to be 0.941 between iELISA and IFA, 0.945 between bELISA and IFA and 0.932 between FMIA and IFA. Similar comparative kappa values were observed between the iELISA, bELISA and FMIA, which demonstrated a significant level of testing agreement among the three assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted with these assays. All three assays detected seroconversion of naïve animals within 6-9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples.

Conclusion: Well-validated iELISA, bELISA and FMIA assays for the detection of PEDV antibodies were developed and showed good correlation with IFA and each other. Each assay format has advantages that dictate how they will be used in the field. Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies. In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. Measurement of neutralizing antibody responses using the FFN assay may provide a valuable tool for assessment of vaccine candidates or protective immunity.

Fig. 1

Purification of antibody capture antigen. SDS-PAGE/Coomassie blue staining of E. coli expressed and purified NA PEDV-NP antigen used to coat ELISA microtiter plates and FMIA microspheres. Molecular weight ladder (MW) PEDV-NP (51 kDa)

Fig. 2

Antigen/antibody specificity. Western blot analysis showing detection of recombinant expressed NA PEDV-NP protein and specificity of the monoclonal antibody used in the bELISA. L- Molecular weight ladder. A- anti-PEDV-NP mAb 6–29. B- anti-polyhistidine mAb. C- anti PEDV-NP convalescent swine serum

Fig. 3

Serum dilution optimization for both ELISA assays and FMIA. Reference serum standard was titrated 2-fold in antigen coated wells at a fixed concentration in order to gauge a maximum signal-to-noise ratio for each assay a iELISA, b bELISA, c FMIA. Arrows show the optimum dilution of swine serum from which the highest signal to noise ratio was achieved

Fig. 4

Receiver operator characteristic (ROC) validation and determination of diagnostic sensitivity and specificity of the PEDV-NP iELISA, bELISA and FMIA assays. Diagnostic sensitivity and specificity were calculated using serum samples from a known PED-uninfected and PED-infected population. ROC analysis was performed using MedCalc version 11.1.1.0 (MedCalc software, Mariakerke, Belgium). In each panel, the dot plot on the left represents the negative testing population, and the dot plot on the right represents the positive population. The horizontal line bisecting the dot plots represents the cutoff value that gives the optimal diagnostic sensitivity and specificity. a Serum iELISA, b Serum bELISA, c Serum FMIA

Fig. 5

Kinetic time course antibody evaluation. Antibody time course kinetics were calculated for each of the ELISAs and FMIA using serum samples from experimentally infected pigs collected at weekly intervals. The horizontal line indicates the diagnostic cutoff for each test. All three tests demonstrate similar kinetic curve responses via their calculated S/P values. a Antibody kinetic time course via iELISA, b Antibody kinetic time course via bELISA, c Antibody kinetic time course via FMIA

Fig. 6

FFN antibody and FMIA isotype time course evaluation. Using serum collected over time from experimentally infected pigs, the FMIA demonstrates the kinetic nucleoprotein-directed, isotype-specific response of IgG and IgM in serum. In addition, the data show a concomitant appearance of neutralizing antibodies as soon as 14 DPI

Fig. 7

Assessment of neutralizing antibody titers in different sample matrices following PEDV exposure. FFN titers were detected in various sample matrices including colostrum (n = 25), milk (n = 23) and serum (n = 27) collected at the time of farrowing and weekly for two weeks post-farrowing. Error bars indicate a 95 % confident interval for mean titers indicated by horizontal lines