Animal serum samples
Standardization
Standardization of recombinant proteins was performed using 16 goat serum samples, ten positive samples from animals experimentally infected with C. pseudotuberculosis as confirmed by isolating bacteria from caseous lesions; and six negative serum samples from animals from CL nonendemic areas.
Validation
In the first validation step, recombinant SodC protein was selected to be tested with positive serum control samples from 50 goats and 45 sheep in which C. pseudotuberculosis infection was confirmed by isolating bacteria from caseous lesions. Samples from 50 goats and 45 sheep from CL nonendemic areas in southern Brazil, where there are strict controls for introducing new animals, were used as a negative control (Barral et al. 2019).
The second stage included 756 field samples of small ruminants from the semiarid region of the state of Bahia, Brazil, of which 400 were goat samples and 356 were sheep samples. All samples used had prior confirmation of the presence or absence of humoral response to C. pseudotuberculosis by indirect ELISA using bacterium secreted antigens (Carminati et al. 2003).
All procedures involving animals were performed according to the recommendations of the Animal Ethics Committee (AEC) of the Institute of Health Sciences, Federal University of Bahia, under protocol No. 4958051018.
Antigenic evaluation of recombinant proteins
Recombinant protein generation
The sequences of the target proteins were retrieved from NCBI GenBank: NanH [gb| ADK28179.1]; SodC [gb| ADK28404.1]; PknG [gb| ADK29622.1]; SpaC [gb| ADK29663.1]; DTxR [gb| WP_048653406.1]; Trx [gb| ANQ80418.1]; TrxR [gb| ANQ80417.1]; LexA [gb| ANQ79549.1].
The rDTxR, rTrx, rTrxR, and rLexA recombinant proteins used in this study were kindly provided by the Multiuser Center for Biomolecular Innovation, IBILCE/UNESP, São José do Rio Preto, SP, Brazil. These proteins were produced and synthesized as described by Kabsch (2010).
The genes coding for C. pseudotuberculosis SodC, SpaC, NanH, and PknG proteins were synthesized and cloned individually in the commercial vector pD444-NH (DNA 2.0 Inc., USA) (https://www.atum.bio), with the original codons replaced by Escherichia coli-optimized codons. The recombinant plasmids were transformed into E. coli BL21 (DE3) Star and purified using affinity chromatography, as previously described (Simionatto et al. 2010). Fractions containing recombinant proteins were identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and quantified using the Lowry protein assay (Bio-Rad Laboratories, CA, USA) and following the manufacturer’s instructions.
Indirect ELISA standardization using recombinant proteins
A checkerboard procedure with different antigen concentrations, serum sample dilutions, and anti-IgG goat and sheep antigens tested in combination was used to screen recombinant antigens and standardize ELISA. 96-well polystyrene plates (GREINER Bio-One, São Paulo, Brazil) were sensitized with 100 µL of each of the recombinant proteins (at concentrations of 0.1, 0.5, 1.0, and 2.0 μg/mL−1), diluted in 0.05 M carbonate/bicarbonate buffer (pH 9.6), and incubated at 4ºC for 16 h. The plates were washed with 0.01 M PBS, 0.05% Tween 20 (PBS-T) and blocked with 200 µL of casein (5%) per well for two h. After washing with PBS-T, 50 µL/well of control sera (positive and negative) were added at 1:50, 1:100, and 1:200 dilutions in PBS-T containing 1% casein, and incubated at 37ºC for one h. After being washed five more times, 50 µL/well of the anti-IgG goat or sheep antibody conjugated with peroxidase (Bethyl, Montgomery, USA) was added at dilutions of 1:5,000, 1:10,000, 1:20,000, and 1:30,000 in PBS-T containing 1% casein, and incubated at 37ºC for 45 min. Color development was performed with the addition of 50 µL/well of 1:2-O-phenylenediamine substrate (OPD) (Sigma-Aldrich, St. Louis, USA) at 22ºC, away from light, for 20 min. The reaction was arrested by adding 25 μL/well of 4N H2SO4. The mean optical density (OD) at 492 nm was determined using a microtiter plate reader (THERMO PLACA, Miami, USA).
The interplate OD was corrected for each standardized ELISA mode by multiplying the correction factor (FtC) between plates by the OD reading (Zwirner 1996). The corrected OD (cD) was calculated using the following formula: FtC = mean OD of the standard positive of the reference plate /Mean OD of the standard positive of each plate. Therefore, cD = OD × FtC.
ELISA validation with recombinant SodC protein
After identifying the optimal antigen concentration (0.1 μg/mL−1), serum dilutions (1:100) and goat and sheep secondary antibodies (1:20,000), recombinant SodC protein was selected to validate the ELISA using goat and sheep sera. The ELISA was used with antigens secreted from C. pseudotuberculosis, as standardized by Carminatti et al. (2003) for comparison. Serum samples were considered positive when the reaction exhibited an OD > mean plus two OD standard deviations obtained for negative controls (Patarroyo et al. 2002).
Statistical analysis
To evaluate the specificity, sensitivity, and cut-off point of the ELISA immunoassay, the data obtained were analyzed using the Receiver Operating Characteristic (ROC). SPSS software v.23 for Windows was used for statistical analysis. The graphics were generated through the GrapfPad Prism 8 and Microsoft office 2013 package.