2.1 Sera
Cattles were experimentally infected with different capripoxvirus isolates, kindly provided by the Lanzhou veterinary research Institute, and sera were collected at various time intervals following vaccination. Cattle were infected with LSDV with Gansu in China isolates. Orf virus-positive sera were collected from sheep experimentally infected with the orf virus (18). The animal experiment was conducted under the approval of the China Science Centre for Human and Animal Health Animal Care Committee using the China Council on Animal Care guidelines for research animals. Cattle sera from native animals or animals infected with LSDV, either experimentally or in field outbreaks, were obtained from Lanzhou.
2.2 Construction of A33R expression vector
A primer set (A33R-F/A33R-R) targeting A33R gene sequence (201–591), encoding for a truncated length A33R without the trans-membrane region as well as C-terminal region (aa) was designed based on a reference gene sequence available in GenBank (Acc. # DQ184476), forward primer A33R-F 5'- ccgGAATTCATGTTAGCATTTTTTAATAATAC-3', and reverse primer A33R-R 5'- ccgAAGCTTTTAAAAAAAAGATCTTACACAG-3'. These oligonucleotides had added restriction enzyme sites (underlined) for EcoRI and Hind III, respectively, at the 5′ ends and primer tags (small letters).
The DNA extracted from LSDV-2019 /Xinjiang using a viral DNA extraction kit (TaKaRa) as the manufacturer's protocol was used as a template in a PCR reaction mixture. A partial A33R gene was amplified using gene-specific primers (A33-F and A33-R). The PCR reaction mixture (50 μl ) was comprised of primers (10 pmol of each) along with 5×Taq PCR Master mix (MBI Fermentas, USA) and a final volume made up by nuclease-free-water. The cycling condition was: initial denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 94°C for the 40 s, annealing at 55°C for 40 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The amplified PCR product and pET30a vector were digested with EcoRI and Hind III enzymes and gel purified. In a standard reaction, the insert was ligated into the pET30a vector, and the recombinant plasmid (pET30a-A33R) was transferred into E. coli JM109 strain initially and subsequently into expression host cells E. coli BL21 (DE3) cells. The recombinant strain with pET30a-A33R plasmid named RpET30a-A33R and sequenced by Shanghai Biological Co., Ltd.
2.3 Expression, purification of the recombinant A33 fusion protein
RpET30a-A33R was cultured at 37°C for 3 h with 200 rpm agitation after the OD600 value was reached 0.6, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and kept at 37℃ for 6 h. The sediments were collected and washed three times using PBS. The protein of LSDV A33 was purified by Ni-bind@ Resin (GE Healthcare bio-science, Sweden). The purified protein was dialyzed against phosphate-buffered saline (PBS), diluted 4:1 in glycerol after SDS-PAGE analysis, and stored at -70°C before use in ELISA and immunization.
For confirmation, the proteins were resolved by electrophoresis on 12% Bis-Tris polyacrylamide gels (Shanghai Sangon Biotech, China) and transferred to polyvinylidene fluoride membranes (Immobilon-P Transfer membranes, Millipore). Membranes were blocked for 12 h at 4°C in 5% (wt/vol) Tris-buffered saline supplemented with 0.1% Tween 20 (TBST)-diluted milk (BSA, Amresco) buffer. Membranes were incubated with primary antibody diluted in 5% (wt/vol) BSA and 1 × TBST at room temperature. The detection was carried out with 1:400 diluted LSDV positive serum as primary antibody and 1:15000 diluted rabbit anti-bovine IgG horseradish peroxidase (HRP) conjugate (Stan cru, USA) as a secondary antibody. The blots were detected by the enhanced chemiluminescence detection kit (#1705062, Bio-Rad) after incubation with an appropriate secondary antibody conjugated to horseradish peroxidase. All the membranes were imaged with the ChemiDoc XRS + system (Bio-Rad).
2.4 The Polyclonal antibody preparation
Three experimental rabbits of three-month-olds were immunized subcutaneously with A33R protein antigen diluted in PBS and emulsified with an equal volume of complete Freud's adjuvant (Sigma, USA). The rabbits were injected two times at an interval of 14 d with the same concentration using incomplete Freud's adjuvant as a boost. Test bleeding was done after 14 d of final immunization, and an indirect ELISA confirmed the titer of the specific antibody.
2.5 Development of the competitive ELISA
The competitive ELISA conditions were optimized by checkerboard titration using serial dilutions of A33 protein tested against serial dilutions of known positive and negative bovine sera.
A checkerboard titration was performed to determine the optimal working dilution of the coating antigen, serum, competitive antibody, and horseradish peroxidase-labeled mouse anti-rabbit IgG (HRP-IgG) (Santa Cruz, sc-2357 ) using a 96-well ELISA plate. Antigen coating concentrations were 16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml, 1 μg/ml and 0.5 μg/ml per well, and serum dilutions were 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64. The dilutions that gave the smallest difference between positive and negative serum (P/N) by absorbance at 450 nm were selected. Test sera included positive, negative, and blank sample controls, controls without serum.
After optimization, competitive ELISA was performed using the following procedure. Ninety-six well ELISA plates (Costar) were diluted in carbonate buffer with 1000 folds (protein concentration 2 mg/ml) coated with 100 μl and incubated overnight at 4°C. Then plates were sealed with 5 g/LCasein (Sigma) and incubated for 2 h at 37°C. After three washes with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST), serum samples were diluted 1:2 in dilution buffer (PBS), in a 50 μl volume well, the competitive antibody in a 50 μl volume well mixing and incubated for 30 min at 37°C, internal controls including positive, negative controls serum and 100% control wells without serum. After four washes, horseradish peroxidase-conjugated mouse anti-rabbit serum was added in the same dilution buffer at an appropriate working concentration, 100 μl per well, and incubated at 37°C for 30 min. After three washes, color was developed with 3,3’,5,5’-tetramethylbenzidine (TMB, Sigma), and the reaction was stopped after 15 min with 2 M H2SO4. The optical density (OD) of the samples were read at 450 nm with a microplate reader (Model 680. Bio-Rad).
The Percent inhibition (PI) value = (1- mean OD value at 450 nm of sample/ mean OD of 100 % control well without serum at 450 nm)×100. Those normally applied in studies conducted in our laboratory were needed for considering a run valid: mean OD of 100 % control wells =1.5±0.5,the PI of positive control ≥60 %.
2.6 Panel sera employed for the validation of the method
A panel sample was employed, including 605 negative sera and 110 LSDV positive sera, divided into detailed categories.
350 field samples without LSDV exposure, collection of serum samples was performed in 3 Gansu province farms during 2017 (no LSD in china).
100 field samples of Yak without LSDV exposure,collection of serum samples was performed in Gansu province farms during 2018(there has no LSD in china).
155 field samples without LSDV exposure, collection of serum samples was performed in 2 Gansu province farms during 2020, and these were confirmed based on Capripox Double Antigen multi-species ELISA (ID.Vet).
40 positive sera came from the experimental infection (Lanzhou Veterinary Research Institute P3) and conformed based on PCR detection and Capripox Double Antigen multi-species ELISA kit (ID.Vet ).
37 positive sera came from the immune bovine with inactivated Goatpox vaccine (Lanzhou veterinary research institute) and conformed based on Virus Neutralization Test.
33 positive sera with LSDV exposure were collected in Guangdong province, Xinjiang, and Hebei provinces and confirmed based on Capripox Double Antigen multi-species ELISA (ID.Vet).
1 positive serum of FMDV, 1 positive serum of BVDV, 1 positive serum of Brucella, 1 positive serum of bluetongue, and 1 Orf virus-positive sera were stocked in Lanzhou Veterinary Research Institute.
2.7 Cut-off estimation, statistical validation, and determination of the C-ELISA
A receiver operating characteristic (ROC) curve is a graphical representation of the relative effects of the false negative and false positive rates for every possible cut-off, and it is an effective method of evaluating the quality or performance of diagnostic tests. Therefore, the discriminating power of the competitive ELISA for anti-A33R antibody detection was evaluated by a ROC curves analysis (19) using Medcalc statistical software version 20.008 (https://www.medcalc.org/), trial version. The accuracy of a diagnostic test is displayed in an interactive dot diagram,where data of the negative and positive groups are displayed as dots on two vertical axes.
2.8 Validation, repeatability, and comparison of the c-ELISA
The coefficient of variation (CV) was calculated between plates (inter-assay variation) and within the same plate (intra-assay variation) for 30 sera samples (20 positive, 10 negative) to validate the test and evaluate repeatability. For inter-assay CV, each sample was tested on three different plates on different occasions, and for intra-assay CV, four replicates within each plate were assayed. A total of 30 experimentally generated evaluated by the ELISA and MNT.