China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (10): 4920-4929.doi: 10.16431/j.cnki.1671-7236.2025.10.037

• Preventive Veterinary Medicine • Previous Articles    

Establishment and Application of an Indirect ELISA Antibody Detection Method Based on the Outer Membrane Protein of Chlamydia psittaci in Pigeons

ZHANG Zixuan1, YANG Zhiyuan2, LIU Yuehuan2, YANG Baolin2, GAO Jie3, FENG Qianqian3, LUO Weijue3, LIN Jian2, ZHAO Jicheng2, HUANG Cheng2, HU Ge1   

  1. 1. College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China;
    2. Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China;
    3. Beijing Zhonghai Biotech Co., Ltd., Beijing 100081, China
  • Received:2025-02-17 Published:2025-09-30

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Abstract: 【Objective】 This experiment aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) method applicable to detecting antibodies against Chlamydia psittaci (Cps) in pigeons.【Method】 The codons of the gene encoding the major outer membrane protein (MOMP) of Cps were optimized.After adding trigger factor (TF) at the N-terminus,it was ligated to the pET-30a(+) vector to construct the plasmid pET-30a-M.After induction with IPTG,expression and purification were carried out.Solubility analysis and identification were performed using SDS-PAGE and Western blotting.A checkerboard titration was used to determine the optimal antigen coating concentration and serum dilution,thereby optimizing the indirect ELISA method for detecting pigeon Cps antibodies.The sensitivity of the indirect ELISA method established in this study were compared with that of the indirect hemagglutination assay (IHA).The specificity of this method was evaluated by detecting the positive sera of other common diseases in pigeons.Three positive and three negative sera for Cps were detected respectively to assess its repeatability.The coincidence rate of this indirect ELISA method and the IHA method was evaluated by using 253 pigeon-derived sera (174 clinical serum samples and 79 serum samples after immunization with Cps inactivated vaccine).【Result】 SDS-PAGE and Western blotting results showed that the MOMP protein with a size of approximately 100 ku was successfully expressed in the supernatant after induction and sonication,indicating that this protein was soluble.The optimized conditions of the indirect ELISA method were as follows:Coating with 2 μg/mL antigen at 4 ℃ overnight,blocking with 5% skimmed milk at 37 ℃ for 2 h,serum dilution of 1∶200,serum incubation time of 1 h,HRP-labeled rabbit anti-pigeon IgY incubation for 30 min,and substrate reaction time of 10 min.When the dilution of the positive serum of pigeon Cps was 1∶16,the detection result of the indirect ELISA method was still positive,and the sensitivity was higher than that of the IHA method.There was no cross-reaction between the indirect ELISA method and the positive sera of other common pigeon diseases.Both intra- and inter-assay test results showed that the coefficients of variation of the indirect ELISA method were all less than 10%,indicating that this method had good repeatability.The coincidence rates of the indirect ELISA method established in this study and the IHA method for detecting clinical samples and serum samples after immunization were 80.72% and 85.71% respectively,and the Kappa values were 0.60 and 0.69 respectively.【Conclusion】 The indirect ELISA method established in this experiment exhibited high sensitivity,strong specificity,and excellent repeatability,and was suitable for the detection of serum antibodies against Cps in pigeons.

Key words: pigeons; Chlamydia psittaci; indirect ELISA; indirect hemagglutination assay (IHA); antibodies

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