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 A450 values of 40 negative chicken sera tested by BE-ELISA.

Supplementary Figure 1—Optimization of BE-ELISA conditions. a The 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. b Based on these results, the optimal dilution of HRP-conjugated donkey anti-chicken

antibody was analyzed at dilutions from 1:2000 to 1:50,000. c Using the optimized dilutions, the type of

blocking buffer was then optimized.

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Supporting information

Supplementary Table 1 Construction of gene fragments and multi-fragment genes

Nam

e

Description Construction and product size Constructed

plasmids a

E4 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

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a The 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)

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Supplementary Table 2 Primers and synthetic nucleotide sequences

Name Sequence (5' to 3') Restriction site

E4-F ATAGCCGGCAGTAGTTATCGTATGGTGCAG Nae I

E4-R AAAGGCGCCAGTGTCTACTGACTGCTTT NarI

E5-F TATGGCGCCTCTAGTGCAACTGGAAAGAC NarI

E5-R ATAGGGCCCACTACTTGGGACTGATTTTCT Apa I

E6-F CGCGAAAGGTGCGGACACCAAATCTCGTTCTAACCAGGGTA

CCCGTGACGG Apa I and NarI

E6-R CGCCGTCACGGGTACCCTGGTTAGAACGAGATTTGGTGTCCG

CACCTTTCGCGGGCC

E7-F TATGGCGCCTCTTCTAAGGCCGATGAAAT NarI

E7-R TGAGCCGGCCTTAATACCTTCCTCATTC Nae I

E8-F ATAGCCGGCAGTTCTACTGTGGTCCCAC Nae I

E8-R CCCCTCGAGCTA ATTGTTCCTCTCCTCAT Xho I

E1/2/3-R TAAGCCGGCCGGAACGATGGT Nae I

E6/7/8-F AATGGGCCCGCGAAAGGTGC Apa I

NE-F ATAGGATCCGCAACTGGAAAGACAGACGC BamH I

Letters underlined indicate restriction sites, in italic indicate flexible amino acid sequences

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Supplementary Table 3 The A450 values of 40 negative chicken sera tested by BE-ELISA

Sera

number

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 value and SD were

calculated as 0.112 and 0.037, respectively. Thus, the cut-off value using mean ± 3 SD was defined as

0.223

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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) and chicken 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. a The 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/ml and a serum dilution of

1:1500. b Based 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. c Using 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

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Supplementary Figure 138

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