1 Supporting information
2 Supplementary Table 1—Construction of gene fragments and multi-fragment genes.
3 Supplementary Table 2—Primers and synthetic nucleotide sequences.
4 Supplementary Table 3—The A
450 values of 40 negative chicken sera tested by BE-ELISA.
5 Supplementary Figure 1—Optimization of BE-ELISA conditions. a The optimal antigen concentration and
6 serum dilution were determined by checkerboard titration of antigen BE (1.2, 2.4, 3.43, 6, and 8 μg/ml),
7 with 1:100, 1:500, 1:1000, 1:1500, and 1:2000 dilutions of IBV-positive serum and that of chicken
8 negative serum. b Based on these results, the optimal dilution of HRP-conjugated donkey anti-chicken
9 antibody was analyzed at dilutions from 1:2000 to 1:50,000. c Using the optimized dilutions, the type of
10 blocking buffer was then optimized.
11
12

13 Supporting information
14 Supplementary Table 1 Construction of gene fragments and multi-fragment genes e
Nam Description Construction and product size Constructed plasmids a
E4 181–210 AA (M gene)
E5 6–88 AA (N gene)
PCR using primers E4-F and E4-R; 114 bp
PCR using primers E5-F and E5-R; 279 bp pE4-19T pE5-19T
E6
E7
E8
118–133 AA (N gene)
218–264 AA (N gene)
304–385 AA (N gene)
Constructed using two synthesized nucleotide strands (E6-F and E6-
R) with its sequence optimized for better translation in Escherichia
coli
PCR using primers E7-F and E7-R; 165 bp
PCR using primers E8-F and E8-R; 273 bp
E
1/2/3
Splice product of three S fragments (E1, E2, and E3) b
PCR using primers E1-F bp c and E
1/2/3
-R; 402
E
4/5
Splice product of E4 and E5 PCR using primers E4-F and E5-R; 381 bp pE7-19T pE8-19T pE
1/2/3
-19T pE
4/5
-19T
E
6/7/8
Splice product of E6, E7, and PCR using primers E
6/7/8
-F and E8-R; 480 bp
E8
BE Splice product of E1–E8 pE
6/7/8
-19T
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
15 a The vectors used in this study included pMD19-T, pET32a(+), and PGEX-4T-1

16 b,c The three S fragments (E1, E2, and E3) and primer E1-F were described in our previous work (Ding et
17 al. 2015 )
18

19 Supplementary Table 2 Primers and synthetic nucleotide sequences
Name Sequence (5' to 3')
E4-F ATAGCCGGCAGTAGTTATCGTATGGTGCAG
E4-R AAAGGCGCCAGTGTCTACTGACTGCTTT
E5-F TATGGCGCCTCTAGTGCAACTGGAAAGAC
E5-R ATAGGGCCCACTACTTGGGACTGATTTTCT
E6-F CGCGAAAGGTGCGGACACCAAATCTCGTTCTAACCAGGGTA
CCCGTGACGG
E6-R CGCCGTCACGGGTACCCTGGTTAGAACGAGATTTGGTGTCCG
CACCTTTCGCGGGCC
E7-F TATGGCGCCTCTTCTAAGGCCGATGAAAT
E7-R TGAGCCGGCCTTAATACCTTCCTCATTC
E8-F ATAGCCGGCAGTTCTACTGTGGTCCCAC
E8-R CCCCTCGAGCTA ATTGTTCCTCTCCTCAT
E
1/2/3
-R TAAGCCGGCCGGAACGATGGT
E
6/7/8
-F AATGGGCCCGCGAAAGGTGC
NE-F ATAGGATCCGCAACTGGAAAGACAGACGC
20 Letters underlined indicate restriction sites, in italic indicate flexible amino acid sequences
21
Restriction site
Nae I
NarI
NarI
Apa I
Apa I and NarI
NarI
Nae I
Nae I
Xho I
Nae I
Apa I
BamH I

22 Supplementary Table 3 The A
450 values of 40 negative chicken sera tested by BE-ELISA
Sera A
450
value number
1-4
5-8
9-12
0.17
0.104
0.094
0.13
0.106
0.071
0.139
0.15
0.155
0.069
0.111
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
23 Forty negative chicken sera diluted at 1:1500 were tested by BE-ELISA. The mean A
450
value and SD were
24 calculated as 0.112 and 0.037, respectively. Thus, the cut-off value using mean ± 3 SD was defined as
25 0.223
26

27 Supplementary Figure 1 Optimization of BE-ELISA conditions. “P” and “N” indicate the A
450
value of IBV-
28 positive serum (China Institute of Veterinary Drug Control) and chicken negative serum, respectively.
29 They were tested in triplicate for each condition, and mean values ± SD are shown. “P/N” indicates the
30 ratio between the P and N values. Optimal working conditions were determined to be those that yielded
31 the highest P/N value. a The optimal antigen concentration and serum dilution were determined by
32 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,
33 and 1:2000 dilutions of IBV-positive serum and that of chicken negative serum. The combination that
34 yielded the highest P/N value was an antigen concentration of 3.43 μg/ml and a serum dilution of
35 1:1500. b Based on these results, the optimal dilution of HRP-conjugated donkey anti-chicken antibody
36 was analyzed at a dilution range from 1:2000 to 1:50,000. A dilution of 1:10,000 was determined to be
37 the optimal dilution. c Using the optimized dilutions, the type of blocking buffer was next optimized.
38 Buffers 1 to 5 represent 1 % (w/v) gelatin in phosphate-buffered saline (PBS), 5 % (w/v) skimmed milk
39 powder in PBS, 10 % (w/v) skimmed milk powder in PBS, 1 % (w/v) BSA in PBS, and 0.5 % (w/v) BSA in
40 PBS, respectively. The buffer with 5 % (w/v) skimmed milk powder in PBS was found to yield the best
41 results
42

43 Supplementary Figure 1
44
45