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Development of an ELISA based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection

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Abstract

Objectives

To develop a cost-effective ELISA for detection of antibodies against infectious bronchitis virus (IBV) by using a multi-fragment protein as coating antigen.

Results

A multi-fragment antigen, termed BE, which was composed of eight antigenic fragments selected from the three major proteins (S, M, and N) of IBV, was expressed in Escherichia coli. The entire protein had a molecular weight of 61.5 kDa. In addition to it, a smaller truncated protein was also produced; both could react with IBV-positive serum. Next, an indirect ELISA (BE-ELISA) was developed. Coefficients of variation of this assay were lower than 10 %, and no cross-reactivity between the coated antigen BE and antiserum against newcastle disease virus, avian influenza virus, or infectious bursal disease virus was observed. The performance of BE-ELISA was evaluated, and showed 95.4 % coincidence ratio with the whole virus based-ELISA (IDEXX).

Conclusions

The multi-fragment antigen (BE) may represent a promising alternative to the whole virus without safety problems, and this newly established ELISA provides an effective method for anti-IBV antibody detection.

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Acknowledgments

This study was supported by Chinese National Programs for High Technology Research and Development (2011AA10A209), Modern Agro-industry Technology Research System (CARS-41-K09), Applied Basic Research Program of Sichuan Province (2013JY0027), Basic Condition Platform Project of Science and Technology of Sichuan Province (14010136), and Natural Science Foundation of China (31302094).

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. 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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    Authors and Affiliations

    1. 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, Chengdu, 610064, Sichuan Province, China

      Meng-die Ding, Xin Yang, Hong-ning Wang, An-yun Zhang, Zhi-kun Zhang, Wen-qiao Fan & Hai-peng Cao

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    1. Meng-die Ding
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    2. Xin Yang
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    3. Hong-ning Wang
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    Corresponding author

    Correspondence to Hong-ning Wang.

    Additional information

    Meng-die Ding and Xin Yang have contributed equally to this work.

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    Supplementary material 1 (DOCX 2544 kb)

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    Ding, Md., Yang, X., Wang, Hn. et al. Development of an ELISA based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection. Biotechnol Lett 37, 2453–2459 (2015). https://doi.org/10.1007/s10529-015-1935-9

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    • DOI: https://doi.org/10.1007/s10529-015-1935-9

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