基于外膜蛋白的鼻气管鸟杆菌抗体间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2024.10.023

Abstract

Abstract: 【Objective】 This study was aimed to develop an ELISA detection method for serum antibodies against Ornithobacterium rhinotracheale (ORT).【Method】 The outer membrane protein OR02 of ORT was cloned and expressed using prokaryotic expression methods.The immunogenicity of the purified expression product was confirmed by Western blotting.The purified product was used as an antigen coated on a solid-phase carrier,and the antigen coating concentration and serum dilution ratio were determined using checkerboard titration method.In addition,the reaction conditions for ELISA detection were optimized,and the cut-off value for the detection method was determined using ORT negative serum.The specificity of the method was evaluated by testing positive sera for other pathogens,and the sensitivity was assessed by testing ORT positive sera at different dilution ratios.Furthermore,a comparison was made with a commercial assay kit to evaluate the concordance of the ELISA method.【Result】 The recombinant protein OR02 was expressed in inclusion bodies with a molecular weight of 58 ku,consistent with the expected size.Western blotting revealed specific binding of the OR02 recombinant protein with positive serum samples for,indicating good immunoreactivity.Using the purified OR02 protein as the coating antigen,the optimal antigen coating concentration was determined to be 2 ng/μL,the dilution of serum sample was 1∶50,the incubation time was 30 min,the dilution of enzyme-linked immunosorbent assay secondary antibody was 1∶5 000 with the incubation time of 30 min,and the optimal substrate color development conditions were incubated for 15 min under dark conditions.The criteria for determining the critical value of positivity and negativity were:D_{450 nm} value of serum≥0.266 was considered positive,while D_{450 nm} value of serum＜0.266 was considered negative.No cross-reactivity was observed when testing positive sera for other pathogens such as Avibacterium paragallinarum (Apg),Newcastle disease virus (NDV),and Infectious bursal disease virus (IBDV).Positive results was still detected when the positive serum with a titer of 1∶64 was diluted at 1∶800.The coincidence rate with commercial kits for the same clinical samples was 91.25%.【Conclusion】 In this study,an indirect ELISA method was developed using recombinant OR02 protein for the detection of serum antibodies against ORT.The method demonstrated good specificity and sensitivity,with a high coincidence rate compared to a commercial kit.It could be considered a reliable tool for the detection of ORT antibodies in chicken serum.

Key words: Ornithobacterium rhinotracheale; outer membrane protein; indirect ELISA; specificity; sensitivity

CLC Number:

S852.61

XU Haojun, LIU Ying, MEI Chen, XU Tong, LI Kai, WANG Hongjun. Development of an Indirect ELISA for Antibody Detection Against Ornithobacterium rhinotracheale Based on Outer Membrane Proteins[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4420-4428.

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Development of an Indirect ELISA for Antibody Detection Against Ornithobacterium rhinotracheale Based on Outer Membrane Proteins

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