Development of a Competitive Chemiluminescence Immunoassay Using a Monoclonal Antibody Recognizing 3B of Foot-and-mouth Disease Virus for Differentiating Infected From Vaccinated Animals

Background: Foot-and-mouth disease (FMD) is a devastating animal disease. Differentiation of infected from vaccinated animals (DIVA) is very important for conrming 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) for DIVA was developed for the rapid detection of antibodies against non-structural proteins (NSPs) in different species of livestock animals using monoclonal antibody 9E2 as a competitive antibody that recognizes NSP 3B. Results: The cut-off (50%), diagnostic sensitivity (97.20%, 95.71%, and 96.15%) and diagnostic specicity (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. The accuracy rate of 3B-cCLIA was further validated followed by its comparison with two commercial diagnostic kits. The early diagnostic performance showed that antibodies to NSPs occurred later (about 1–2 days) than antibodies to structural proteins. Furthermore, NSP antibodies present in animals vaccinated multiple times (false-positive), especially in cattle and sheep, were conrmed, and the false-positive rate increased with the number of vaccinations. Conclusions: These results indicated that 3B-cCLIA is suitable to rapidly detect antibodies against FMDV NSP 3B in a wide range of species for DIVA. times, we attempted to use two mAbs recognizing NSP 3A and 3B to develop 3A + 3B-cCLIA. In addition, the early diagnostic performance, repeatability, and stability of 3B-cCLIA were also evaluated. These results indicated that 3B-cCLIA can be a promising tool for large-scale serological surveys in vaccinated areas for DIVA.

To date, slaughtering infected and contacted animals together with prohibiting the import of animal and animal products from FMD endemic countries have been adopted to prevent the disease in FMD-free nations. Considering the economic costs, a vaccination policy was adopted for control and eradication of the disease in endemic countries [6,7]. However, vaccination with inactivated vaccines raises other issues, such as the differentiation of FMDV-infected from vaccinated animals (DIVA) and the production of carrier animals to shed the virus [6,7]. Therefore, detecting antibodies against NSPs has been one of the widely preferred and applied diagnostic methods for DIVA and helps in identifying the subclinical infections, evaluate the prevalence of infection and control the disease [8] because a series of purifying techniques remove the majority of NSPs from the inactivated vaccine during production [3,9].
In previous study, the mAb 9E2 recognizing NSP 3B that can be used to develop the diagnostic method for DIVA has been preliminary veri ed.
However, mAb 2G5 recognizing NSP 3A is not suitable for use alone in establishing a diagnostic method because of the mutation and deletion of the epitope identi ed by mAb 2G5 [3]. In the study, we used mAb 9E2 as the detection antibody to develop a competitive chemiluminescence immunoassay (termed 3B-cCLIA) for the rapid detection of NSP antibodies. To decrease the false positives in cattle and sheep vaccinated multiple times, we attempted to use two mAbs recognizing NSP 3A and 3B to develop 3A + 3B-cCLIA.

Serum samples
Serum samples from naïve animals: Serum samples from clinically healthy and unvaccinated animals, including 310, 175, and 61 samples from swine, cattle, and sheep, respectively, were collected and tested using liquid-phase blocking ELISA of FMDV O (O-LPBE) and A-LPBE (presented negative results; titer, < 1:4). The diagnostic speci city (Dsp) and cut-off value were calculated using these serum samples (Table  S1).
Serum samples from infected animals: Collection of 107 serum samples was carried out from swine infected with FMDV A/GDMM/2013 or O/Mya98 at 7-25 days post infection (dpi) in the Animal Biological Safety Level 3 (ABSL-3) Laboratory at Lanzhou Veterinary Research Institute (Lanzhou, China); 70 serum samples were collected from cattle infected with FMDV (A/GDMM/2013 or O/Mya98) at 8-20 dpi; 52 serum samples were collected from sheep with clinical symptoms in the eld and tested as NSP-positive using two commercial diagnostic kits (3ABC-bELISA and PrioCHECK FMDV NSP ELISA). The diagnostic sensitivity (Dsn) and cut-off value were calculated using these samples. In addition, a total of 32 sera collected from four unvaccinated control swine experimentally challenged with FMDV O/Mya98 at 0 dpi and 2-8 dpi were used for comparing the early diagnostic performance of 3B-cCLIA and two commercial diagnostic kits and identify seroconversion to FMDV SP and NSP (Table S1).
Serum samples from vaccinated animals: One hundred serum samples were collected at 21 days post vaccination (dpv) from swine vaccinated with FMDV O univalent multiple-epitope recombinant vaccine developed by our research group. The Dsp and cut-off value were calculated using these samples. In addition, a collection of 120 serum samples was carried out from sows vaccinated 3-15 times with commercial O/A divalent inactivated vaccines. Similarly, a collection of 129 serum samples was carried out from dairy cows vaccinated with commercial O/A divalent inactivated vaccines every four months for a total of 2, 5 or 10 vaccinations. Similarly, collection of seventy-seven serum samples was carried out from 15 sheep vaccinated 1-3 times with laboratory-made FMDV A/AF72, O/Mya98/BY/2010, or Asia 1/JS05 univalent inactivated vaccine. These samples were employed for evaluating the diagnostic performances of 3B-cCLIA and two commercial diagnostic kits and veri ed the false-positive phenomenon with animals vaccinated multiple times (Table S1).
Serum samples collected in the eld: 173 eld sera collected from swine suspected of FMDV infection during 2010-2018. These sera were used for comparing the coincidence rates between 3B-cCLIA and two commercial diagnostic kits (Table S1).
Serum samples from other virus-infected swine: In this study, one serum sample from classical swine fever virus (CSFV)-infected swine, one from senecavirus A (SVA)-infected swine, one from porcine parvovirus (PPV)-infected swine, one from porcine reproductive and respiratory syndrome virus (PRRSV)-infected swine, and two from porcine circovirus type 2 (PCV2)-infected swine were investigated (Table S1).
Control sera: The serum sample derived from swine infected with FMDV O/Mya98 at 25 dpi served as the standard positive serum (P51). The percentage inhibition (PI) of the serum in the 3ABC-bELISA and PrioCHECK FMDV NSP ELISA was 92% and 93%, respectively. The standard negative serum sample (P734) was taken from clinically healthy swine who had not been immunized against FMD. The serum was tested with O-LPBE and A-LPBE (produced negative result; titer, < 1:4), 3ABC-bELISA (produced negative result; PI = 1%), and PrioCHECK FMDV NSP ELISA (produced negative result; PI = -7%).

Antigen and antibodies
The 3ABC coding region of FMDV A/GDMM/2013 mutated at positions 46 aa and 163 aa was cloned into the pProEXHTB plasmid. The expression and puri cation of the recombinant 3ABC protein has been described [13].
The mAbs against the 3ABC protein, designated 2G5 and 9E2, were obtained at our laboratory, and their minimally identi ed epitopes are " 92 EYIEKA 97 ", which is located in 3A, and " 23 EGPYAGPLE 31 ", which is located in 3B [3]. MAbs 2G5 and 9E2 were largely produced by injecting hybridomas into the peritoneal cavities of BALB/c mice and puri ed by a nity protein G column chromatography. Then, the mAbs 2G5 and 9E2 were conjugated with horseradish peroxidase (HRP). The polyclonal antibody was obtained by inoculating rabbits with the puri ed 3ABC protein.

Development of competitive CLIA using mAb against NSP 3B
Checkerboard titration was used to optimize the conditions of 3B-cCLIA. The coating of mAb 2G5 onto 96-well white plates (Costar) was carried out at 1, 0.5, 0.25, and 0.125 μg/mL concentrations in a 100-μL volume followed by overnight incubation at 4 °C. After washing, puri ed 3ABC protein was diluted to 0.5, 0.25, and 0.125 μg/mL in PBST (PBS containing 0.05% Tween-20) and added to each well, and the plate was incubated for 1 h at 37 °C. After three PBST washes, each well received 200 μL of blocking buffer, followed by incubation at 37 °C for 2 h. Then, the serial dilution of standard positive serum (P51) and standard negative serum (P734) was carried out with dilution buffer (10% equine serum, 1% casein in PBST) at 1:2.5-1:20 dilutions, and 50 μL of the serum was transferred to each well. Simultaneously, each well was added 50 μL of HRP-conjugated mAb 9E2 (9E2-HRP) at concentrations of 0.08, 0.04, 0.02, 0.01, 0.005, and 0.0025 μg/mL followed by incubation of the plate at room temperature. After being washed ve times, chemiluminescence (CL) substrate (KEY-BIO, Beijing, China) including 50 μL of solution A (luminol and luminous enhancer) and 50 μL of solution B (peroxide solution) were added. The signals of CL were measured with Varioskan lux (Thermo Scienti c, USA) after 5 min. The determination of the optimum mAb 2G5 concentration, 3ABC protein concentration, serum dilution, 9E2-HRP concentration, and incubation time was carried out based on the ratios of CL values of standard negative serum to standard positive serum (N/P).
3B-cCLIA was carried out in optimal conditions. The following formula was used to calculate the PI: Where mean CL values of the standard negative serum control are represented by CLn while the CL values of the test samples are represented by CLs.
A mean CLn ≥ 6,000,000 and mean PI of the standard positive control > 80% indicate assay validity.
Similarly, 3A+3B-cCLIA was developed, and detailed information is presented in the Additional le 1.

Cut-off value, Dsn, and Dsp
The cut-off value of 3B-cCLIA was determined by testing 875 serum samples from different origins. NSP-negative sera from swine (n = 410), cattle (n = 175), and sheep (n = 61) were used to estimate the Dsp of each species, and NSP-positive sera from swine (n = 107), cattle (n = 70), and sheep (n = 52) were used to estimate the Dsn of each species using MedCalc software.

Comparison of accuracy rates and diagnostic performances
The accuracy rates of two commercial diagnostic kits were also evaluated using the abovementioned 875 NSP-negative and positive serum samples. The diagnostic performances of 3B-cCLIA, 3A+3B-cCLIA and two commercial diagnostic kits were compared by testing 430 serum samples, including 120, 60, and 77 samples from vaccinated swine, cattle, and sheep, respectively, and 173 samples from the eld. In addition, serum samples from four swine challenged with FMDV O/Mya98 collected at different times were utilized to evaluate and compare the early diagnostic performance of the four assays.
Estimation of the repeatability and stability of 3B-cCLIA To calculate the intra-and inter-batch repeatability performances, three replicates of seven serum samples with varying PI levels were evaluated on various days using plates coated in the same and different batches. To determine the shelf life of the coated plates, they were vacuum-packed and stored at 37 °C for 15 days or at 4 °C for 1 year after blocking.

Comparison of early diagnostic performance
Seroconversion to NSP and SP was studied in four unvaccinated control swine post-challenge using O-LPBE and ID Screen® FMD Type O Competition to detect the SP antibody and using 3B-cCLIA, 3A + 3B-cCLIA, 3ABC-bELISA, and PrioCHECK FMDV NSP ELISA to detect the NSP antibodies. Antibodies against SPs were rst detected at 5 dpi, and all the sera examined were positive at 7 dpi (Fig. 2a). Antibodies against NSPs were detected early at 7 dpi and identi ed as positive in all swine at 8 dpi except for the PrioCHECK FMDV NSP ELISA (Fig. 2b). Therefore, seroconversion to NSP occurred approximately 1-2 days later than that to SP.
Repeatability and stability of 3B-cCLIA The assay was highly repeatable, as determined by four positive and three negative serum samples tested on different days on same and different batch plates (Fig. 3).
The standard negative serum (P734) and standard positive serum (P51) were examined while using 3B-cCLIA plates stored at 37°C for 15 days or at 4°C for 1 year. The CL values of P734 were > 6,000,000, and the PI value of P51 was > 80%. The ndings showed that 3B-cCLIA plates may be preserved for up to 15 days at 37°C and up to 1 year at 4°C.

Discussion
DIVA tests are important for serological surveillance by providing evidence of vaccinated herds infected with or freedom from FMD [1,3,17].
To avoid omitting animals infected with different serotype isolates, identifying conserved mAbs is important in developing a diagnostic method for DIVA. The epitope " 23 EGPYAGPLE 31 " identi ed by mAb 9E2 is located in 3B and is well conserved among different serotypes of FMDV, which has been veri ed in a previous study [3]. Therefore, in this study, a competitive CLIA (3B-cCLIA) using mAb 2G5 as the capture antibody to capture puri ed 3ABC protein and 9E2-HRP as the detection antibody was developed for DIVA. The cut-off PI value of 3B-cCLIA was determined to be 50% by testing panels of serum samples from different origins. Based on the cut-off PI of 50%, Dsn (97.20%, 95.71%, and 96.15%) and Dsp (99.51%, 99.43%, and 98.36) were determined in naïve and infected swine, cattle and sheep, respectively (Fig. 1). The PrioCHECK FMDV NSP ELISA had good Dsp (accuracy rate 100%) in testing naïve sera but had low Dsn in testing infected sera when compared with 3B-cCLIA and 3ABC-bELISA.
Although most NSPs are removed from the inactivated vaccine, residual NSPs are still present in the inactivated vaccine [3] and can induce the corresponding antibodies after repeated vaccination, especially in cattle [11,12,[21][22][23], which will interfere with the DIVA test based on detecting NSP antibodies and affect the serological surveillance and evaluation of the infection status [19]. This phenomenon was also con rmed in this study, in which dairy cows vaccinated multiple times with the traditional inactivated vaccine produced a high false-positive rate using 3B-cCLIA and two commercial diagnostic kits (Tables 1 and 2). In addition, the false-positive rate in sheep vaccinated three times also increased (Tables 1 and 2). However, the false positives in sows vaccinated multiple times were almost equal to those in negative sera (Tables 1 and 2), as found in previous reports [12,18]; this may be due to different immune systems in different species. False positives increased with the number of vaccinations. To address this problem, we attempted to use two mAbs recognizing NSP 3A and 3B to develop 3A + 3B-cCLIA. In theory, only when the two antibodies were simultaneously present in serum, the serum was considered to be positive, thereby using the assay to try to decrease the false-positive rate in vaccinated animals. However, the results showed that 3A + 3B-cCLIA could not solve this false-positive problem (Tables 1 and 2).
Sera from animals vaccinated multiple times are usually weak positive. Therefore, it is a strategy to increase the cut-off (compromise true sensitivity) to decrease the false-positive rates in testing vaccinated animals. Another approach is to establish the correlation between residual NSPs in the inactivated vaccines with different rounds of puri cation and the NSP antibody in different species that were inoculated different times with the corresponding inactivated vaccine, thereby conforming to a threshold value of residual NSPs in the inactivated vaccine to guide vaccine manufacturers to improve vaccine purity. In addition, the development and use of new FMD vaccines that do not contain NSP components, such as marker vaccines [24,25], virus-like particle VLP vaccines [26], multiple-epitope recombinant vaccines [27], and synthetic peptide vaccines, could eliminate this barrier.
In this study, seroconversion to NSP occurred later than that to SP, which is similar to previous reports [11,21]. The seroconversion time of the PrioCHECK FMDV NSP ELISA was delayed compared with that of the other three methods, which explains the lower Dsn of the PrioCHECK FMDV NSP ELISA in detecting sera from infected animals.

Conclusion
A competitive CLIA that could rapidly detect antibodies against FMDV NSP 3B was developed in this study. 3ABC-bELISA and PrioCHECK FMDV NSP ELISA use the blocking ELISA format and need to be washed twice in process, but 3B-cCLIA uses the competitive format, only needs to be washed once and requires 15 min to obtain results. In addition, the early diagnostic performance, repeatability, and stability of 3B-cCLIA were also evaluated. These results indicated that 3B-cCLIA can be a promising tool for large-scale serological surveys in vaccinated areas for DIVA.  Early production of antibodies against FMDV SPs and NSPs 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, ID Screen® FMD Type O Competition to detect SP antibodies. The dashed lines -----and …… represent the cut-off values of O-LPBE and ID Screen® FMD Type O Competition, respectively. (b) Thirty-two serum samples were tested using 3B-cCLIA, 3A+3B-cCLIA, and two commercial diagnostic kits (3ABC-bELISA and PrioCHECK FMDV NSP ELISA) to detect NSP antibodies. The dashed line (-----) represents the cut-off value of 3B-cCLIA, 3ABC-bELISA, and PrioCHECK FMDV NSP ELISA, and the dashed line (……) represents the cut-off value of 3A+3B-cCLIA. The bar represents the range of values obtained by detecting sera from four swine at the given time.

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

Supplementary Files
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