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Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus a

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Meng-die Ding , Hong-ning Wang , Hai-peng Cao , Wen-qiao Fan , Bing-cun Ma , Peng-wei a

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Xu , An-yun Zhang & Xin Yang

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School of Life Science, Sichuan University, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, “985 Project” Science Innovative Platform for Resource and Environment Protection of Southwestern China, Chengdu, China Published online: 02 Apr 2015.

Click for updates To cite this article: Meng-die Ding, Hong-ning Wang, Hai-peng Cao, Wen-qiao Fan, Bing-cun Ma, Peng-wei Xu, An-yun Zhang & Xin Yang (2015): Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1025692 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1025692

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Bioscience, Biotechnology, and Biochemistry, 2015

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus Meng-die Ding, Hong-ning Wang*, Hai-peng Cao, Wen-qiao Fan, Bing-cun Ma, Peng-wei Xu, An-yun Zhang and Xin Yang School of Life Science, Sichuan University, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, “985 Project” Science Innovative Platform for Resource and Environment Protection of Southwestern China, Chengdu, China Received January 7, 2015; accepted February 23, 2015

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http://dx.doi.org/10.1080/09168451.2015.1025692

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166–247 aa, S1 gene; 501–515 aa, S1 gene; 8–30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV. Key words:

infectious bronchitis virus (IBV); multi-epitope antigen; antibodies; ELISA

Infectious bronchitis (IB) is a serious and highly contagious disease of chickens caused by infectious bronchitis virus (IBV), a member of the family Coronaviridae (order Nidovirales) and genus Coronavirus. Control of such a highly contiguous disease mainly relies on vaccination. Hence, a determination of the immune status of chicken is important for controlling IB outbreaks in large flocks. Enzyme-linked immunosorbent assay (ELISA) is a rapid, simple, and sensitive method and has been widely used in the serological profiling of viruses.1) Currently, the commercial ELISA kit (IDEXX Laboratories, Westbrook, USA) for detecting antibodies against IBV is available and is coated with the whole IBV (M41 strain). However, it is expensive and

the production, purification, and inactivation of viral suspension are time-consuming and labor-intensive. In contrast, the recombinant protein-based ELISA is relatively inexpensive, sensitive, and reproducible. Moreover, the recombinant protein has an advantage over whole virus preparation as it forms immunodominant epitopes and is devoid of any non-specific moieties in whole cell preparation.2) IBV genome encodes four structural proteins, spike glycoprotein (S), membrane glycoprotein (M), phosphorylated nucleocapsid protein (N), and the small membrane glycoprotein (E). S glycoprotein on the outside of the virus is cleaved post-translationally by cellular proteases into amino-terminal S1 (535 amino acids; 90 kDa) and carboxyl-terminal S2 (627 amino acids; 84 kDa) subunits.3,4) When developing a protein-based ELISA for detecting antibodies against IBV, S and N proteins were the preferred choices as they were both a strong immunogen. But as a result of that, N protein, which is produced abundantly during infection, is highly conserved and immunogenic, thus the N protein-based ELISA methods were always developed for diagnosis of IBV infection in chickens.5–9) It is well known that S protein is responsible for virus-neutralizing antibody and protection,10,11) hence using S protein as antigen to develop an ELISA method is more suitable for evaluating the IB vaccine efficiency. As the heterologous expression of the full-length S protein is difficult as it is a large protein (180 kDa) with intensive hydrophobicity and is highly glycosylated,12) an ELISA assay (S-fg ELISA) using partial S protein for serum antibody detection against IBV was developed in the previous study.13) It was found to be a convenient, economical, and efficient method, which demonstrated that it was feasible to utilize S protein fragment to replace the whole S protein for sero-surveillance. While it also showed clearly that only two antigenic sites (S1-F and S2-G) contained in the S-fg protein (381–555 aa) made contribution to the antigenicity and the cross-reactivity

*Corresponding author. Emails: [email protected]; [email protected] Abbreviations: IBV, infectious bronchitis virus; E. coli, Escherichia coli; PBS, phosphate buffer; CBS, carbonate buffer; P/N, the ratio of OD450 values between the positive and the negative serum. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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of the S-fg ELISA, which were located with the region of S1 subunit C terminus and S2 subunit N terminus. Otherwise, additional antigenic regions of S protein were identified in subsequent researches,12,14) and these findings enable the designation of the S protein fragment with improved versions and with enhanced detection potential. In recent years, approaches based on multi-epitope antigen, which are devoid of redundant sequences, and which permit a high epitope density (sensitivity) and careful choice of unique specific epitopes (specificity), had been used in the detection of antibodies against viruses, such as Dengue virus, Hepatitis C virus, and Hepatitis G virus C, and had achieved both a good degree of sensitivity and a greater specificity in results.15−17) In this study, we constructed a multiepitope antigen of S protein (SE) to establish an IBVspecific antibody ELISA, which would be expected to be useful in evaluating IBV vaccine efficiency. The SE protein consisted of three S fragments, E1 (166–247 aa, S1 gene), E2 (501–515 aa, S1 gene), and E3 (8–30 aa, S2 gene). It was designed by arranging the three fragments in tandem in the order of E1–E2–E3 and was expressed in Escherichia coli BL21(DE3). It harbored several antigenic sites in the conserved regions, including Sp1 (194–209 aa), Sp2 (209–228 aa), and Sp3 (245–260 aa, QYNTGNFSDGLYPFTN),14,18) as well as two conformation-independent epitopes Sp5 (518–532 aa) and Sp7 (566–584 aa).14) Therefore, the aim of this study was to establish an indirect ELISA based on the multi-epitope antigen SE and to evaluate its potential in the detection of IBV antibodies.

Materials and methods Strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Prediction and screening of linear B-cell epitopes. Based on the IBV sequence reported previously (GenBank Accession Number: DQ834384.1), linear B-cell epitopes of S protein were predicted with bioinformatics tools ABCpred server (Saha and Raghava; Table 1.

Strains E. coli DH5α E. coli BL21 (DE3) Plasmids pET32a(+)

pSE-19T pET32a-SE

RT-PCR amplification. Fragment E1 was amplified by PCR using the primer pair E1-F/E1-R (Table 2). The total RNA was extracted from IBV M41 strain using TRIzol reagent (TaKaRa) and cDNA was synthesized using PrimeScrip® RT reagent Kit Perfect Real Time (TaKaRa). Briefly, in a total volume of 50 μL, 3 μL viral cDNA, 1 μL each primer, 4 μL dNTPs, 0.5 μL (2.5 U) rTaq DNA polymerase (TaKaRa), and 5 μL 10× buffer supplied by the manufacturer were combined. The rest was supplemented by ddH2O. The used conditions were: reverse transcription (37 °C, 15 min; 85 °C, 5 s), initial denaturation 94 °C (5 min), followed by 30 thermal cycles of denaturation (95 °C, 40 s), annealing (54 °C, 40 s), and extension (72 °C, 30 s), followed by a final extension (72 °C, 10 min). The resulting product was resolved on a 2% agarose gel and purified using gel extraction kit (Omega). Then it was inserted into vector pMD19-T to construct recombinant vector pE119T, and the vector was subsequently characterized by PCR and DNA sequencing analysis. The nucleotide sequences of E2 and E3 had been optimized for better translation in E. coli by a computer software (http://genomes.urv.es/OPTIMIZER/),21,22) and were designed to include restriction sites as shown in Table 2. In brief, 5 μL complementary oligonucleotides E2-F/E2-R or E3-F/E3-R synthesized by Sangon (Shanghai, China) and 30 μL ddH2O were mixed in a tube, heated at 95 °C for 20 min followed by cooling to room temperature. Phosphorylation of E2 and E3 was performed using a mixed solution system consisting of 5 μL 10 mmol/L ATP, 2 μL T4 Polynucleotide Kinase, 5 μL 10× T4 DNA Polynucleotide Kinase Buffer (TaKaRa), and 8 μL ddH2O, reacting at 37 °C for 30 min. The resulting products were resolved on a 2% agarose gel and were purified using gel extraction kit (Omega). Gene splicing and construction of recombinant plasmids. We designed the multi-epitope antigen gene

Bacterial strains and plasmids used in this study.

Bacteria strain or plasmid

pMD19-T pE1–19T

http://www.imtech.res.in/raghava/abcpred/ ABC_submission.html)19) and BepiPred 1.0 server (Larsen et al.; http://www.cbs.dtu.dk/services/BepiPred/).20)

Properties

Source or reference Purchased from Takara Purchased from Takara

T7 promoter; His-tag; lac I; MCS; Apr; 5.9 kb For gene cloning; Apr; 2.69 kb Derivative of pMD19-T which contained the E1 gene in this work; Apr, 2.95 kb Derivative of pMD19-T which contained SE gene in this work; Apr; 3.09 kb Derivative of pET32a(+) in which 0.399 kb of the SE gene was inserted into the BamHI/XhoI sites; Apr; 6.29 kb

Note: Apr, ampicillin resistance.

Kindly provided by professor Linhan Bai of School of Life Science, Sichuan University Purchased from Takara This work This work This work

An ELISA method for antibodies detection against IBV Table 2.

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Primers and synthetic sequences.

Fragment

Sequence (5′–3′)

E1-F E1-R SE-F SE-R E2-F E2-R E3-F E3-R

AGC GGATCC TCCGTATATTTAAATGGTG TAT GGGCCC TGGACC ATTAATAAAAGGAT AGC GGATCC TCCGTATATTTAAATGGTG AGC CTCGAG CTA CGGAACGATGGT CTCTGGTGGTAAACTGGTTGGTATCCTGACCTCTCGTAACGAAACC GGCGCG GG CGCCCGCGCCGGTTTCGTTACGAGAGGTCAGGATACCAACCAGTTTACCACCAGA GGGCC CGCCAACTGCCCGTACGTTTCTTACGGTAAATTCTGCATCAAACCGGACGGTTCTATCGCGACCATCGTTCCG GCC GGCCGGAACGATGGTCGCGATAGAACCGTCCGGTTTGATGCAGAATTTACCGTAAGAAACGTACGGGCAGTTGG

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Note: Letters in italic means restriction sites, underlined means flexible amino acid sequences.

SE by arranging the three S gene fragments in tandem in the order of E1–E2–E3, which is flanked by BamHI and XhoI restriction enzyme sequences, as shown in Fig. 1. PCR product of fragment E1 was digested with ApaI and was linked with fragments E2, E3 by T4 DNA ligase at 16 °C overnight. Then the splice product was used as template to amplify the SE gene using the primer pair SE-F/SE-R (Table 2) and the PCR product was inserted into vector pMD19-T. The recombinant plasmid pSE-19T confirmed by sequencing was doubledigested with BamHI and XhoI. The resulting product SE gene with a stop codon (UAG) was ligated into the multiple-cloning site region downstream of the TrxTag/His-Tag/S-Tag of pET32a(+) vector, followed by transformation into E. coli DH5α-competent cells. Recombinant plasmid, namely pET32a-SE, was extracted and initially checked by PCR and digestions and followed by automated DNA sequence analysis. Protein expression and purification. For expression, the plasmid pET32a-SE confirmed by sequencing was transformed into E. coli BL21(DE3) cells by thermal stimulation and expressed in lysogeny broth (LB) medium supplemented with 60 μg/mL ampicillin at 37 °C to an OD600 of 0.4–0.6, followed by induction with 1 mmol/L isopropyl β-D-thiogalactoside (IPTG) for a further 4 h with agitation. The E. coli BL21(DE3) cells with empty pET-32a(+) vector were cultured as a contrast. The cells were harvested by centrifugation at 12,000 rpm for 10 min at 4 °C, and then resuspended in phosphate buffer (PBS, pH 7.4). The cells were lysed by enzymolysis and sonication. It was found that the recombinant protein containing the Trx-Tag/His-Tag/S-Tag was expressed in a form

Fig. 1. Schematic representation of the generation of the multi-epitope antigen (SE). Notes: The black blocks represent the gene fragments. The numbers indicate amino acid positions. The blank blocks represent the flexible amino acids (GPGP or GAGA).

of inclusion body. So the inclusion bodies were collected and purified at denaturing conditions using a Ni-NTA Sefinose column (BBI, Shanghai, China), according to the manufacturer’s instructions. In brief, the inclusion bodies were dissolved with Ni-DenatureGuHCl buffer (100 mM NaH2PO4, 300 mM NaCl, 6 M GuHCl, pH 8.0) about 5 mL per mL of pellet, and were added into a Ni-NTA Sefinose column, which was equilibrated previously with Ni-Denatureurea buffer (100 mM NaH2PO4, 300 mM NaCl, 8 M urea, pH 8.0). The same buffer was passed through the column to wash and Ni-Denature-250 buffer (100 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 8 M urea, pH 8.0) was used to elute the SE fusion protein. In order to remove imidazole, which was necessary for subsequent application in ELISA, dialysis was performed for the purified SE protein with PBS (pH 7.4). Purity was verified by SDSPAGE, and the protein content was determined by modified Bradford protein assay kit (BBI). Samples were stored at −20 °C until use. While the tag protein expressed by the empty pET-32a(+) vector (Trx-Tag/ His-Tag/S-Tag/His-Tag) was expressed as a soluble protein, and was purified under natural conditions according to the manufacturer’s instructions.

Western blot analysis. To evaluate the antigenicity, the SE protein was subjected to 12% SDS-PAGE followed by transferring to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk powder in TBST (containing 0.05% Tween 20) for 2 h at 37 °C. It was then washed three times with 1× TBST and incubated with IBV positive serum (1:100 dilution with PBS, China Institute of Veterinary Drug Control, Beijing, China) or negative serum for 1 h at 37 °C. The membrane was washed again, as above, and incubated with Rabbit antichicken IgG (H+L), HRP conjugated (1:2000 dilution, biosynthesis biotechnology Company, Beijing, China) for a further 1 h at 37 °C. The blot was washed and developed by incubation in DAB substrate solution (HRP-DAB chromogenic substrate Kit, BBI) for 5 min at RT in dark. The reaction was stopped with distilled water.

Optimization of the SE-ELISA. The coating buffers, including 0.01 M PBS (pH 7.4), 0.01 M NaOH

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(pH 12), 0.05 M carbonate buffer (CBS, pH 9.6), and blocking buffers, including 1% gelatin in PBS, 5% skimmed milk powder in PBS, 10% skimmed milk powder in PBS, 1% BSA in PBS, and 0.5% BSA in PBS, were optimized for the SE-ELISA. The optimal antigen concentration and the serum dilution for the SE-ELISA were determined using a checkerboard titration. Different coating antigen dilutions (1:100, 1:150, 1:250, 1:350, and 1:900) and chicken serum diluted at 1:100, 1:250, 1:500, 1:1000, 1:1500, and 1:2000 were tested with secondary HRP-conjugated AffiniPure Donkey Anti-Chicken IgY(IgG) (H+L) (BBI) diluted at 1:2000, 1:5000, 1:10,000, 1:20,000, and 1:50,000. All combinations were repeated twice and the optimal results were determined to be those which provided the greatest ratio of OD450 values between the positive and the negative serum (P/N). The SE-ELISA was performed as follows: SE protein (100 μL/well) diluted in coating buffer according to the optimal concentration was coated onto 96-well microwell plate (Corning) and incubated overnight at 4 °C. Plates were washed three times with PBST (PBS with 0.05% (v/v) Tween-20) and then blocked with blocking buffer (200 μL/well) by incubation at 37 °C for 2 h. After washing three times with PBST, serum samples (100 μL/well) diluted with PBS (containing 0.01% BSA) were added for 1 h and incubated at 37 °C. Plates were washed three times with PBST again before addition of the secondary antibody (100 μL/well). After final three washes, tetramethylbenzidine (TMB, 100 μL/well) was added to each well. The plates were incubated for 5–10 min in the dark at RT and the reaction was terminated using 2 M H2SO4 (Solution D, 50 μL/well). Plates were read at 450 nm using Bio-Rad Model 680 microplate ELISA reader.

Results

Cut-off value, specificity, and reproducibility of the SE-ELISA. The cut-off value for the optimized SEELISA was determined using OD450 values obtained from 40 negative serum samples (collected from unvaccinated chickens). The specificity of the SEELISA was examined with positive serum against Newcastle disease virus, Infectious bursal disease virus, and avian influenza. The reproducibility of the SE-ELISA was evaluated by testing 10 selected chicken serum samples. The coefficient of variation (CV) was used to evaluate the inter-assay variation (between plates) and the intra-assay variation (within a plate). Each sample was tested in four different plates on different occasions to determine the inter-assay CV and four replicates within each plate were used to calculate the intra-assay CV.

Optimization of the SE-ELISA The optimal coating buffer for the SE-ELISA was 0.05 M CBS (pH 9.6) as it can obtain the maximal P/N at 11.125 (1.157/0.104), while the P/N values of the buffers 0.01 M PBS (pH 7.4) and 0.01 M NaOH (pH 12) were 10.088 (1.150/0.114) and 10.629 (1.116/ 0.105), respectively. Five percent skimmed milk powder in PBS was identified to be the optimal blocking buffer for the SE-ELISA (Table 4). As shown in Fig. 3, when the antigen was diluted at 1:350 (3.689 μg/mL) and the primary antiserum (chicken serum samples) were diluted at 1:1000, the OD450 value difference of the positive antiserum and negative antiserum was the maximal. Based on these results, the optimal secondary antibody dilution was identified as 1:10,000, as given in Table 5.

Comparison of the SE-ELISA and IDEXX ELISA. The sensitivity and specificity of the SE-ELISA were evaluated in comparison to a commercial ELISA kit (IDEXX) by testing 164 chicken serum samples from a chicken field.

Cut-off value, specificity, and reproducibility of the SE-ELISA The mean OD450 and SD of all 40 negative serum by SE-ELISA were 0.141 and 0.0635, as given in Table 6. Thus the cut-off value using the mean ± 3 S.D was defined at 0.332. Heterologous serums to the viruses of

Prediction and screening of B-cell epitopes for IBV S protein Based on two predictors, the potential B-cell epitopes on S protein of IBV were predicted and their amino acids were: 38–67, 106–121, 192–207, 216–226, 276–291, 316–348, 293–302, 406–421, 518–532, 531– 545, 566–584, 683–698, 704–716, 1020–1032, and 1138–1162. On the basis of the prediction results and the antigenic sites defined previously, three fragments with good antigenicity were chosen, designated E1, E2, and E3. Their amino acid sequences and locations are given in Table 3. E1 (166–247 aa) and E2 (501–515 aa) were located on S1 protein, E3 (8–30 aa) was located on S2 protein.

Construction of recombinant plasmids Fragment E1 was amplified by PCR and 264 bp in length. E2 and E3 were synthesized and their sequences had been optimized for better translation in E. coli (data not shown). The multi-epitope gene SE constructed by gene splicing (E1, E2, and E3 in tandem) through enzyme digestions and PCR amplification was 399 bp in length. It was successfully inserted into vector pET32a(+) to obtain the recombinant expression plasmid pET32a-SE.

Expression and purification The multiple antigen, namely SE, was expressed in a form of inclusion body after induced by IPTG (Fig. 2(B)). It was purified and identified to be approximately 32 kDa by SDS-PAGE analysis (Fig. 2(B)). Western blot result showed it can react with IBV positive serum (Fig. 2(C)).

166–247 aa (S1 gene)

501–515 aa (S1 gene) 8–30 aa (S2 gene)

E1

E2

E3

Position

NCPYVSYGKFCIKPDGSIATIVP

SGGKLVGILTSRNET

SVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKALAYFVNGTAQDVILCDGSPRGLLACQYNTGNFSDGFYPFIN

Amino acids sequence

Antigenic gene fragments selected in this study.

Names

Table 3.

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Broadly cross-reactive and immunodominant region located on S2 subunit N terminus11,34) A conformation-independent Sp7 (566–584 aa), largely conserved in all IBV strains14) Prediction results of ABCpred

Immunogenic region 240–255 aa and antigenic regions Sp1 (194–209 aa), Sp2 (209–228 aa)14,18) Prediction results of ABCpred and Bepipred Conformation-independent epitope Sp5 (518–532 aa)14)

References for selection

An ELISA method for antibodies detection against IBV 5

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Fig. 2. Analysis of SE protein. Notes: (A) SDS-PAGE analysis of recombinant SE protein. M: protein molecular weight marker, low range (Takara). Lanes 1 and 4, the proteins expressed by IPTG-induced and uninduced bacterial cells containing the empty pET32a(+) vector. The tag protein (Trx-Tag/His-Tag/S-Tag/His-Tag) was about 21 kDa. Lanes 2 and 3, the proteins expressed by IPTG-induced and uninduced bacterial cells containing the recombinant plasmid pET32a-SE, note a fusion protein about 32 kDa with the tag protein (Trx-Tag/His-Tag/S-Tag) weight in 18.3 kDa present in induced bacterial cells. (B) SDS-PAGE analysis of purified SE protein and the tag protein. Lanes 1 and 2, supernatant and pellet from the sonicated IPTG-induced bacterial cells containing the empty pET32a(+) vector, the tag protein was expressed in a form of soluble protein. Lanes 3 and 4, supernatant and pellet from the sonicated IPTG-induced bacterial cells containing the recombinant plasmid pET32a-SE, the SE protein was expressed in a form of inclusion body. Lane 5, purified His-tag protein. Lane 6, purified SE protein. (C) Western blot analysis. Lanes 1 and 2, the results of the SE protein and the His-tag protein with IBV positive serum. Lanes 3 and 4, the results of the SE protein and the His-tag protein with negative serum. M: protein molecular weight marker stained with amido black 10B, low range (Takara). M1: pre-stained protein marker (Blue Plus Protein marker, TransGen). Only the SE protein can react with IBV positive serum. Table 4.

Optimation of blocking buffers for the SE-ELISA.

Blocking buffers P N P/N

1% gelatin

5% skimmed milk powder

10% skimmed milk powder

1% BSA

0.5% BSA

1.169 0.112 10.438

0.948 0.09 10.533

0.869 0.099 8.778

1.113 0.135 8.244

0.964 0.104 9.269

Note: “P” means the OD450 value of IBV positive serum; “N” means the OD450 value of negative serum.

ND, IBD, and AI were shown negative by SE-ELISA (OD450 0.164–0.178). By testing 10 selected serum samples in quadruplicate, the inter-assay CV was observed to range from 2.75 to 5.49%, with a median value of 4.23%, and the intra-assay CV was observed to range from 1.66 to 5.32%, with a median value of 3.17%.

Fig. 3. Evaluation of the multi-epitope protein SE with positive and negative serums to IBV. Note: Microplates were coated with different concentrations of the SE protein and reacted with different dilutions of IBV positive and negative chicken serum.

Comparison of the SE-ELISA and IDEXX ELISA As shown in Table 7, the relative sensitivity of SE-ELISA was 92.38%, the specificity was 89.83%, and the accuracy was 91.46%. Out of the 105 IDEXX ELISA-positive serum samples, 97 of the samples were determined to be positive by SE-ELISA. In addition, out of 59 IDEXX ELISA-negative serum samples, 53 of the samples were determined to be negative.

An ELISA method for antibodies detection against IBV Table 5.

7

Optimal secondary antibody dilution for the SE-ELISA.

Dilutions

1:2000

1:5000

1:10,000

1:20,000

1:30,000

1:40,000

1:50,000

P N P/N

1.948 0.417 4.67

1.531 0.252 6.08

1.045 0.16 6.53

0.626 0.101 6.20

0.462 0.092 5.02

0.367 0.08 4.59

0.344 0.115 2.99

Notes: “P” means the OD450 value of IBV positive serum; “N” means the OD450 value of negative serum.

Table 6. SE-ELISA. 0.139 0.091 0.114 0.156 0.076

OD450 value of 40 negative serum samples of the

0.128 0.104 0.097 0.1 0.174

0.159 0.15 0.099 0.1 0.104

0.152 0.164 0.095 0.053 0.255

0.11 0.157 0.127 0.191 0.116

0.075 0.13 0.079 0.125 0.136

0.105 0.097 0.115 0.166 0.121

0.224 0.138 0.284 0.383 0.245

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Notes: Mean OD450 and SD of all 40 negative serum by SE-ELISA were 0.141 and 0.0635. Thus the cut-off value using the mean ± 3 SD was defined at 0.332.

Table 7. Results

Comparison of the SE-ELISA and IDEXX ELISA. IDEXX ELISA

SE-ELISA

Common serum samples

105 59 164

103 61 164

97 53 150

+ − Total

Note: Relative sensitivity, (97/105) × 100 = 92.38%; Relative specificity, (53/59) × 100 = 89.83%; Relative accuracy, (150/164) × 100 = 91.46%.

Discussion ELISA is convenient for evaluating the vaccination response, especially for viruses which have different serotypes like IBV, since it can catch different antibodies because of the plentiful coated antigens. In general, the coating antigen is inactivated virus or recombinant protein. In recent years, ELISA methods based on multiple antigen or synthetic peptides that have been developed have shown improved sensitivity and specificity.15−17,23−28) One of the purposes we expressed IBV S protein in the form of the multi-epitope antigen is to explore whether it is an alternative for the fulllength S protein which is difficult to obtain. The three gene fragments of SE protein we constructed were mainly located in the conserved regions due to two considerations. One is that there are several serotypes of IBV vaccines employed in poultry farms due to the high variability of S1 gene sequence, including the most common vaccines like M41, H120, H52, as well as others like 4/91, 28/86. On the other hand, the mutations of S1 gene sequence are not always associated with the new antigenic sites, IB viruses of different serotypes and genotypes not only have different epitopes, but also share common epitopes that are of importance in cross-immunity,4,29) thus we can screen epitopes from the conserved regions which could have the cross-reactivity. Homology analysis of amino acid sequences for the fragments E1 (166–247 aa) and E2 (501–515 aa) showed an identity of 85.4–100% and 86.7–100% among some published IBV strains (M41, H120, H52, 28/86, 4/91, and W93), respectively. The epitope E3 (Sp7, 566–584 aa) was also largely conserved in all IBV strains.14)

Additionally, the epitopes of the SE protein harbored were mainly linear but not conformational as the epitopes of S protein identified in the literature were mostly short peptides to be conformation independent, and a number of software tools developed and applied in recent years were mainly focused on linear B-cell epitope prediction.19,20,30−33) We designed the SE by taking into account both the identified antigenic sites and the predicted epitopes to make it more reliable and perfect as the defined epitopes of S1 and S2 protein are not enough. Moreover, it is difficult to obtain the original conformation of conformational epitopes in heterogeneous expression systems. In order to keep each fragment independent, the restriction enzyme sequences (ApaI, NarI) and additional added nucleotide sequences between E1 and E2, E2 and E3 can translate into flexible amino acids (GPGP or GAGA) to separate them from each other. The SE protein was expressed in E. coli which is not glycosylated as the antigen sites it harbored were linear epitopes. In the early work, we analyzed the nucleotide sequences of fragments E2 and E3 using E. coli Codon Usage Analyzer 2.1 (http://www.faculty.ucr.edu/~mma duro/codonusage/usage.htm) as their complementary oligonucleotides were obtained by synthesis. It was found that they both had codons, whose usage frequency was lower than 25% in E. coli and the ratios were 40 and 52%, respectively. Then the nucleotide sequences were optimized for better translation in E. coli (data not shown), and this may be helpful for the high-level expression of SE protein in some degree (Fig. 2(A)). The SE protein was expressed in the form of inclusion body at 37 °C, we attempted to reduce the culture temperature to 30 °C to obtain the soluble protein, but without success. Furthermore, we found that the expression quantity at the two temperatures had little difference. According to the western blot result (Fig. 2(C)), we can believe that the SE protein has a good antigenicity and it is feasible using the SE protein to replace the full-length S protein as coated antigen to establish an ELISA method. Many reacting conditions were considered and optimized in the construction of the SE-ELISA. First, using a checkerboard titration, the optimal coated antigen concentration and dilutions of the primary antibody and the second antibody, as well as the coating buffer, blocking buffer were determined. The tested serum samples were a 1:1000 dilution for the SE-ELISA developed in this study, while at a 1:500 dilution was recommended by the commercial ELISA kit (IDEXX), indicating that the multi-epitope antigen SE had achieved a good sensitivity which may be attributed to its high epitope density (Fig. 1). Second, considering that the cut-off value selection is one of the most

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important factors in developing ELISA, a panel of serum (n = 40) collected from unvaccinated chickens were used to confirm the cut-off value of the SE-ELISA, which could make the criteria valid for application in the chicken farms. Furthermore, the reproducibility and specificity of the SE-ELISA were examined using this cut-off value (0.332) and the results showed that the method was repeatable with a low variation and had no reactivity with several other avian virus serums. At last, agreement between the SE-ELISA and IDEXX ELISA was studied in evaluating 164 serum samples. Although 14 serum gave discordant results, six of which were positive only in SE-ELISA, while eight of which were positive only in IDEXX ELISA, the two methods have a high agreement ratio (91.46%). In conclusion, a multi-epitope antigen of S protein was developed and can be successfully used in the SE-ELISA method to detect IBV-specific antibodies in chicken serum samples with a good sensitivity and specificity. For further application at a large scale in evaluating the IB vaccine efficaciously, additional studies are still needed to establish the deadline for different poultry farms.

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Disclosure statement [15]

No potential conflict of interest was reported by the authors.

Funding This work was supported by National 863 Plan [grant number 2011AA10A209]; Modern Agro-industry Technology Research System [grant number CARS-41-K09]; Applied Basic Research Program of Sichuan Province [grant number 2013JY0027].

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