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

BackgroundRecent, 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.ResultsA 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.ConclusionWell-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 addi...

Specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers

“…Another common diagnostic method is serological testing for the presence of specific antibodies against viral proteins, which is also fast and convenient for epidemiologic investigations. Many tools have been developed for the detection of anti-PEDV antibodies based upon the major structural proteins (such as S, M or N proteins) in serum, colostrum, milk, feces and oral fluid, including indirect immunofluorescence assays (IFA), virus neutralization assays (SN), enzyme-linked immunosorbent assays (ELISA), and fluorescent microsphere immunoassays (FMIA) (Diel et al, 2016;Gerber et al, 2014;Gerber and Opriessnig, 2015;Gimenez-Lirola et al, 2017;Okda et al, 2015). But comparative studies of the above assays using different PEDV structural and nonstructural proteins as antigens are rarely conducted.…”

A convenient colorimetric assay for the quantification of porcine epidemic diarrhea virus and neutralizing antibodies

“…Virus neutralization tests for PEDV often used in the field to determine the level of protective antibodies, as a follow up for vaccination or in herds where animals show clinical signs (Okda et al, 2015) and are also tedious to assess visually, especially for high volume testing. This study is the first to describe methods for a colorimetric V/N tests for PEDV which can significantly reduce the labor effort in performing this test.…”

The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus

“…established an ELISA system using recombinant N protein generated in Escherichia coli 69; Okda et al . established indirect ELISA and FMIA (Fluorescent microsphere immunoassay) based on recombinant N protein, and blocking ELISA based on N protein-specific MAbs45.…”

“…Recently, a one-way cross-reactivity between PEDV strains and TGEV hyperimmune pig antisera was clearly observed, and one or more epitopes on the N-terminal region of N protein were hypothesized to be the root cause of this cross-reactivity45. Thus, the epitopes of N protein used for PEDV diagnosis should be highly PEDV-specific and the reported immunoassays above should be seriously re-examined.…”

“…Presently, the most commonly used diagnostic methods are reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR, both of which have a high efficiency and reliability in detecting viral genetic materials in fecal and intestinal samples. In addition, immunofluorescence assays (IFAs), electron microscopy (EM), immunohistochemistry, in situ hybridization, enzyme-linked immunosorbent assays (ELISAs), immunochromatography assays and fluorescent microsphere immunoassays (FMIAs) have been widely used to diagnose PEDV infection3536373839404142434445. Among these methods, ELISA has the greatest potential for clinical diagnosis on a large scale and the greatest significance for the effective detection and control of PEDV, especially in developing countries, where few pig farms are equipped with qualified laboratories for complicated molecular diagnoses.…”