Strains, serum samples and antibodies
35 ducks at 7 days of age were selected to confirm Salmonella-free ducks by bacterial isolation and identification [35]. After these ducks were raised for 7 days, 5 ducks were randomly selected to collect blood and obtain negative serum control. The remaining ducks were randomly divided into 6 groups (5 per group) according to different pathogenic bacteria, including Salmonella enteritis, Salmonella typhimurium, Salmonella Cottbus, E. coli, Staphylococcus aureus and Streptococcus, above bacteria culture were assigned the 0.2 mL of 6.0 × 106 CFU/mL by three intramuscular injections at 7-day intervals. At week 1 after the last immunization, blood samples from three ducks of each group were used to separate the serum, and then the antibody level of each serum was determined by tube agglutination test [36]. All ducks used for preparing negative serum to Salmonella and positive serum to other bacteria were raised in separate isolators, deprived freely of food and water.
The salmonella positive serum from ducks in present study is from Harbin Animal Husbandry and Veterinary Research Institute, and the serum was identified by agglutination test.
Predicting and screening linear B-cell epitopes
Based on the sequence of the PagN gene, we predicted and screened linear B-cell epitopes using DNA Star software and IEDB website (http://www.iedb.org/). The antigenic index, surface probability and hydrophilicity values are screened.
PagN gene amplification and recombinant plasmid construction
Primers were designed to amplify PagN gene using NCBI reference sequences of Salmonella enteritis (CP050712.1). Upstream primer PagN-F: 5′-CGGGATCCAAAGAAGGGATCTATATCACCGG-3′, downstream primer PagN-R: 5′-CCCAAGCTTAAAGGCGTAAGTAATGCCGAGC-3′, underlined is BamHI and HindIII restriction site, respectively. Using Salmonella enteritis (CVCC3377) genomic DNA as a template, the PagN fragment was amplified by PCR using PagN specific primers, and the gene fragment was purified using a gel extraction kit. After digestion with BamHI and HindIII, the products were purified and ligated to pET-32a(+) by T4 DNA ligase at 16 °C for 8 h，and then transformed into E.coil DH5α competent cells. A single colony on the LB solid medium (containing 100 μg/mL ampicillin) was selected and cultured overnight at 37 °C with shaking at 220 rpm. The positive clones were verified by restriction enzyme digestion and DNA sequencing, yielding pET-32a (+)-PagN.
Expression and purification of the recombinant PagN protein
The recombinant plasmid pET-32a (+)-PagN was transformed into E.coli BL21(DE3) expression strain. Positive clones were picked up and inoculated into 5 mL LB liquid medium with 100 μg/mL of ampicillin overnight at 37 °C. The overnight culture was inoculated into 200 mL LB liquid medium at a ratio of 1:100 and incubated at 37 °C and 220 rpm until the logarithmic growth phase. Expression of recombinant protein was induced by addition of IPTG. The bacterial pellet was collected by centrifugation, the cells were resuspended in phosphate buffered solution (PBS) with 1/10 culture volume and disrupted by sonication. The supernatant and pellet after sonication were collected and subjected to SDS-PAGE to analyze the expression form of the recombinant protein. The recombinant PagN protein was purified using Ni2+-NTA agarose gel affinity chromatography. The bound protein was eluted with elution buffer (20 mmol/L Tris-HCL, 200 mmol/L imidazole). Finally, the target protein was renatured in a gradient of 6 M, 4 M, 2 M urea buffer solution at 4 °C and then the purified protein concentration was measured by BCA protein assay kit (Beyotime, Shanghai, China).
Western blotting assays
The purified recombinant PagN protein was subjected to polyacrylamide gel electrophoresis and then electrotransferred onto a Nitrocellulose (NC) membrane. The NC membrane was blocked at 4 °C in blocking buffer with Tris-buffered saline (TBS) contained 3% BSA and 0.1% Tween 20 (TBST), and then washed 3 times for 5 min in TBST. The blocked membrane was incubated in duck anti-Salmonella serum (dilution 1/500, from Harbin Animal Husbandry and Veterinary Research Institute) at 4 °C for 2 h. The membrane was washed 3 times with TBST, and 1:4000 diluted HRP-goat anti-duck IgG (KPL, Gaithersburg, USA) was added and incubated for 2 h at 4 °C. After washed three times, enhanced chemiluminescent (ECL) developer solution was added to develop the color.
Establishment of the recombinant PagN protein-based iELISA
According to the classical iELISA method [17, 34], the optimal antigen coating concentration and serum dilution of the iELISA test reagents was investigated by checkerboard titration. Briefly, 100 μL of different concentrations (0.25, 0.5, 1.0, and 2.0 μg/mL) of PagN proteins were coated on a 96-well ELISA microplate overnight at 4 °C, 37 °C 1 h or 2 h, respectively. The ELISA plates were then washed three times for 5 min with washing buffer (PBS + 0.5% TWEEN-20). The wells were blocked with 200 μL blocking buffer (1% BSA, 2% BSA, 3% BSA, 3% skimmed milk, 4% skimmed milk and 5% skimmed milk) at 37 °C for 0.5 h, 1 h, 1.5 h, 2.5 h and 3 h, respectively. After three further washes, 100 μL serially diluted the positive and negative serum samples (2-fold dilutions, from 1:25 to 1:400) was added to each well and then incubated at 37 °C for 0.5 h,1 h, 1.5 h or 2 h. After three washes, the samples were incubated with 100 μL of goat anti-duck IgG-horseradish peroxidase conjugate (HRP) (KPL, Gaithersburg, USA) with two-fold serial dilutions (1:2000 to 1:8000) at 37 °C for 0.5 h, 1 h, 1.5 h or 2 h, washed again, and detected with 100 μL 3,3′,5,5′-Tetramethylbenzidine (TMB) color developer at room temperature for 5 min, 10 min,15 min or 20 min and away from light. The reaction was stopped by the addition of 50 μL 2 M H2SO4. All samples were set up in triplicate and measured with a microplate spectrophotometer (model 680, Bio-Rad) at 450 nm. The corresponding positive value (P) was approximately 1.0, the negative value (N) was less than 0.4, and the maximum difference in optical density (P/N) was not less than 2.1, which was considered to be the best reaction [37, 38].
Determination of the cut-off value
Under the best condition, the OD450 values of 38 negative serum of healthy ducks were determined by the iELISA method, and each serum sample was repeated three times. The mean and standard deviation of the OD450 values were calculated. The cut-off value was determined by titration as the mean OD450 (−x) value plus 3 the standard deviation (SD) of the antibody levels of 38 negative serum samples [34]. If the OD450 value of the test sample is higher than the cut-off value, the sample is regards as positive and vice versa.
Sensitivity analysis and specificity of iELISA test
To accurately assess the diagnostic sensitivity of the assay, the Salmonella positive serum was diluted from 1:50 to 1:12800. The highest dilution that produced an OD450 value> the cut-off value was considered as the detection limit of the iELISA assay (three replicates each test serum) [34]. In addition, to evaluate the diagnostic specificity, the iELISA method described above was used to simultaneously detect the OD450 values of positive serum antibodies against duck-derived Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Cottbus, E. coli, Staphylococcus aureus and Streptococcus with three replicates each serum sample. The Salmonella- positive and -negative serum were set as controls.
Repeatability analysis
Under the optimal conditions established, three sero-positive serum samples and one sero- negative serum sample were detected by the iELISA. Four repeats were set for each sample, and the average value, standard deviation and coefficient of variation of each sample were calculated.
Comparison of iELISA with dot-blot
Dot-blot was used to validate the of the developed iELISA. iELISA and Dot-blot were used to detect 100 clinical sera at the same time, and the coincidence rate of them was compared. The steps for Dot-blot are as follows:
1 μg recombinant protein was fully adsorbed on NC membrane at 4 °C. The NC membrane were then washed 4 times for 5 min with TBST buffer. 5% skim milk was added to each well and incubated at 37 °C for 2 h. After 4 further washes, the NC membrane was placed in the positive serum diluted by 5% skimmed milk powder and incubated at 37 °C for 1 h. After 4 washes, the NC membrane was paced in the HRP goat anti-duck IgG by 5% skimmed milk powder and incubated at 37 °C for 1 h. Finally, after washing for 4 times, use enhanced diaminobenzidine (DAB) color developing solution to avoid light and develop 10 min.
Comparing with commercial ELISA test kits for detection of antibodies against Salmonella
To evaluate such iELISA kit using for detection of Salmonella in ducks, 613 clinical serum samples were tested by iELISA established in present study and a commercial ELISA kit (catalog number YJ660391;Shanghai Enzyme-linked Biotechnology Co., Ltd., China), which specifically designed to a double-antigen sandwich ELISA for detecting antibodies to Salmonella in serum by following the manufacturer’s instructions. A purified protein specific for Salmonella was used as a capture antigen and an HRP-conjugate for detecting the antibodies. Agreement between iELISA and the kit results was determined by counting the number of identical results and dividing it by the total number of samples.
Statistical analysis
Statistical analyses were performed using SPSS software (SPSS18.0). All values given in the text are the mean ± SD from the experiment.
