Development of a competitive chemiluminescence immunoassay using a monoclonal antibody recognizing 3B of foot-and-mouth disease virus for the rapid detection of antibodies induced by FMDV infection

Abstract

Background: Foot-and-mouth disease (FMD) is a devastating animal disease. Anti-non-structural protein (NSP) antibody detection is very important for confirming suspected cases, evaluating the prevalence of infection, certifying animals for trade and controlling the disease.

Methods: In this study, a competitive chemiluminescence immunoassay (3B-cCLIA) was developed for the rapid detection of antibodies against NSPs in different species of livestock animals using the monoclonal antibody (mAb) 9E2 as a competitive antibody that recognizes NSP 3B.

Results: The cut-off value (50%), diagnostic sensitivity (Dsn) (97.20%, 95.71%, and 96.15%) and diagnostic specificity (Dsp) (99.51%, 99.43%, and 98.36) of the assay were estimated by testing a panel of known-background sera from swine, cattle and sheep, respectively. The accuracy rate of the 3B-cCLIA was further validated and subsequently compared with that of two commercial diagnostic kits. The early diagnostic results showed that antibodies recognizing NSPs developed later (approximately 1-2 days) than antibodies recognizing structural proteins. Furthermore, anti-NSP antibody presence in animals vaccinated multiple times (false positives), especially cattle and sheep, was confirmed, and the false-positive rate increased with the number of vaccinations.

Conclusions: These results indicate that the 3B-cCLIA is suitable for the rapid detection of antibodies against FMDV NSP 3B in a wide range of species.

Keywords: Chemiluminescence immunoassay; Diagnosis; Foot-and-mouth disease virus; Monoclonal antibody; Non-structural protein.

Fig. 1

Receiver operating characteristic (ROC) analysis for the determination of the cut-off value of the 3B-cCLIA. a, c, e Interactive dot diagram of the 3B-cCLIA in testing sera from swine, cattle, and sheep. 0, negative serum samples (n = 410, 175, and 61); 1, positive serum samples (n = 107, 70, and 52). b, d, f Each point on the ROC curve represents a sensitivity–specificity pair in testing the sera from swine, cattle, and sheep

Fig. 2

Early production of antibodies against FMDV SPs and NSPs detected in sera from experimentally challenged swine. a Thirty-two serum samples from four unvaccinated control swine after challenge with FMDV O/Mya98 were collected at 0 dpi and 2–8 dpi and tested using O-LPBE and ID Screen® FMD Type O Competition to detect anti-SP antibodies. The dashed lines _{– – – – –} and …… represent the cut-off values of O-LPBE (reciprocal log 10 ≥ 1.65 was considered positive) and ID Screen® FMD Type O Competition (S/N ≤ 50% was considered positive), respectively. b The above thirty-two serum samples were also tested using the 3B-cCLIA, the 3A + 3B-cCLIA, and two commercial diagnostic kits (3ABC-bELISA and PrioCHECK FMDV NSP ELISA) to detect anti-NSP antibodies. The dashed line (_{– – – – –}) represents the cut-off value of the 3B-cCLIA, 3ABC-bELISA, and PrioCHECK FMDV NSP ELISA (PI ≥ 50 was considered positive), and the dashed line (……) represents the cut-off value of the 3A + 3B-cCLIA (PI ≥ 40 was considered positive). The bar represents the range of values obtained by detecting sera from four swine at the given time

Fig. 3

Intra- and interbatch repeatability performances of the 3B-cCLIA were assessed using four positive and three negative serum samples tested on different days on the same and different batch plates. The dashed line represents the cut-off value of the 3B-cCLIA