Supporting information
Supplementary Table 1—Construction of gene fragments and multi-fragment genes.
Supplementary Table 2—Primers and synthetic nucleotide sequences.
Supplementary Table 3—The A450values of 40 negative chicken sera tested by BE-ELISA.
Supplementary Figure 1—Optimization of BE-ELISA conditions.aThe optimal antigen concentration and serum dilution were determined by checkerboard titration of antigen BE (1.2, 2.4, 3.43, 6, and 8 μg/ml), with 1:100, 1:500, 1:1000, 1:1500, and 1:2000 dilutions of IBV-positive serum and that of chicken negative serum. bBased on these results, the optimal dilution of HRP-conjugated donkey anti-chicken antibody was analyzed at dilutions from 1:2000 to 1:50,000. cUsing the optimized dilutions, the type of blocking buffer was then optimized.
Supporting information
Supplementary Table 1 Construction of gene fragments and multi-fragment genes
Name / Description / Construction and product size / Constructed plasmidsaE4 / 181–210 AA (M gene) / PCR using primers E4-F and E4-R; 114 bp / pE4-19T
E5 / 6–88 AA (N gene) / PCR using primers E5-F and E5-R; 279 bp / pE5-19T
E6 / 118–133 AA (N gene) / Constructed using two synthesized nucleotide strands (E6-F and E6-R) with its sequence optimized for better translation in Escherichia coli
E7 / 218–264 AA (N gene) / PCR using primers E7-F and E7-R; 165 bp / pE7-19T
E8 / 304–385 AA (N gene) / PCR using primers E8-F and E8-R; 273 bp / pE8-19T
E1/2/3 / Splice product of three S fragments (E1, E2, and E3) b / PCR using primers E1-F c and E1/2/3-R; 402 bp / pE1/2/3-19T
E4/5 / Splice product of E4 and E5 / PCR using primers E4-F and E5-R; 381 bp / pE4/5-19T
E6/7/8 / Splice product of E6, E7, and E8 / PCR using primers E6/7/8-F and E8-R; 480 bp / pE6/7/8-19T
BE / Splice product of E1–E8 / PCR using primers E1-F c and E8-R; 1239 bp / pBE-19T; pET32a-BE
NE / Splice product of E5–E8 / PCR using primers NE-F and E8-R; 741 bp / pNE-19T; PGEX-NE
aThe vectors used in this study included pMD19-T, pET32a(+), and PGEX-4T-1
b,c The three S fragments (E1, E2, and E3) and primer E1-F were described in our previous work (Ding et al. 2015)
Supplementary Table 2 Primers and synthetic nucleotide sequences
Name / Sequence (5' to 3') / Restriction siteE4-F / ATAGCCGGCAGTAGTTATCGTATGGTGCAG / NaeI
E4-R / AAAGGCGCCAGTGTCTACTGACTGCTTT / NarI
E5-F / TATGGCGCCTCTAGTGCAACTGGAAAGAC / NarI
E5-R / ATAGGGCCCACTACTTGGGACTGATTTTCT / ApaI
E6-F / CGCGAAAGGTGCGGACACCAAATCTCGTTCTAACCAGGGTA
CCCGTGACGG / ApaI and NarI
E6-R / CGCCGTCACGGGTACCCTGGTTAGAACGAGATTTGGTGTCCG
CACCTTTCGCGGGCC
E7-F / TATGGCGCCTCTTCTAAGGCCGATGAAAT / NarI
E7-R / TGAGCCGGCCTTAATACCTTCCTCATTC / Nae I
E8-F / ATAGCCGGCAGTTCTACTGTGGTCCCAC / NaeI
E8-R / CCCCTCGAGCTAATTGTTCCTCTCCTCAT / Xho I
E1/2/3-R / TAAGCCGGCCGGAACGATGGT / NaeI
E6/7/8-F / AATGGGCCCGCGAAAGGTGC / ApaI
NE-F / ATAGGATCCGCAACTGGAAAGACAGACGC / BamH I
Letters underlined indicate restriction sites, in italic indicate flexible amino acid sequences
Supplementary Table 3The A450values of 40 negative chicken sera tested by BE-ELISA
Seranumber / A450 value
1-4 / 0.17 / 0.13 / 0.139 / 0.069
5-8 / 0.104 / 0.106 / 0.15 / 0.111
9-12 / 0.094 / 0.071 / 0.155 / 0.074
13-16 / 0.094 / 0.068 / 0.073 / 0.124
17-20 / 0.184 / 0.119 / 0.084 / 0.142
21-24 / 0.154 / 0.091 / 0.086 / 0.086
25-28 / 0.11 / 0.139 / 0.079 / 0.09
29-32 / 0.084 / 0.078 / 0.065 / 0.152
33-36 / 0.201 / 0.112 / 0.082 / 0.087
37-40 / 0.168 / 0.176 / 0.105 / 0.092
Forty negative chicken sera diluted at 1:1500 were tested by BE-ELISA. The mean A450 valueand SD were calculated as 0.112 and 0.037, respectively.Thus, the cut-off value using mean ± 3 SD was defined as 0.223
Supplementary Figure 1 Optimization of BE-ELISA conditions. “P” and “N” indicate the A450 value of IBV-positive serum (China Institute of Veterinary Drug Control) andchicken negative serum, respectively. They were tested in triplicate for each condition, and mean values ± SD are shown.“P/N” indicates the ratio between the P and N values. Optimal working conditions were determined to be those that yielded the highest P/N value. aThe optimal antigen concentration and serum dilution were determined by checkerboard titration of antigen BE (1.2, 2.4, 3.43, 6, and 8 μg/ml), with 1:100, 1:500, 1:1000, 1:1500, and 1:2000 dilutions of IBV-positive serum and that of chicken negative serum.The combination that yielded the highest P/N value was an antigen concentration of 3.43 μg/mland a serum dilution of 1:1500.bBased on these results, the optimal dilution of HRP-conjugated donkey anti-chicken antibody was analyzed at a dilution range from 1:2000 to 1:50,000. A dilution of 1:10,000 was determined to be the optimal dilution.cUsing the optimized dilutions, the type of blocking buffer was next optimized. Buffers 1 to 5 represent 1 % (w/v) gelatin in phosphate-buffered saline (PBS), 5 % (w/v) skimmed milk powder in PBS, 10 % (w/v) skimmed milk powder in PBS, 1 % (w/v) BSA in PBS, and 0.5 % (w/v) BSA in PBS, respectively. The buffer with 5 % (w/v) skimmed milk powder in PBS was found to yield the best results
Supplementary Figure 1