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 specificity (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 field 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 verified the false-positive phenomenon with animals vaccinated multiple times (Table S1).
Serum samples collected in the field: 173 field 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 purification 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 identified epitopes are “92EYIEKA97”, which is located in 3A, and “23EGPYAGPLE31”, 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 purified by affinity 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 purified 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, purified 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 five 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 Scientific, 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:
PI% = (1-CLs/CLn)×100%,
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 file 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 field. 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.