猪圆环病毒3型Cap蛋白单克隆抗体的制备及阻断ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.03.029

摘要/Abstract

摘要： 旨在建立检测猪圆环病毒3型（PCV3）抗体的阻断ELISA方法，本研究利用原核表达的PCV3 Cap重组蛋白免疫BALB/c小鼠制备获得了一株分泌阻断效果良好抗体的杂交瘤细胞株2E6。以重组Cap蛋白作为包被抗原，以辣根过氧化物酶（HRP）标记的2E6单克隆抗体作为检测抗体，经条件优化后建立了一种检测PCV3抗体的阻断ELISA方法。用建立的阻断ELISA方法检测50份临床阴性血清，计算阻断率（PI）的临界值，以此来确定该方法的判定标准：当PI≤28.30%时，判定结果为阴性；当PI≥35.05%时，判定结果为阳性；当28.30%<35.05%时，判定为可疑，重复一次试验后如果结果仍为可疑，则判定为阳性。特异性试验表明该方法与猪圆环病毒2型（PCV2）、猪伪狂犬病病毒（PRV）、猪繁殖与呼吸综合征病毒（PRRSV）以及猪瘟病毒（CSFV）的阳性血清均无交叉反应；敏感性试验表明其检测效价可达到1：128；重复性试验表明批内与批间的变异系数均小于10%；符合性检验表明该方法与PCV检测金标准免疫过氧化物酶单层试验（IPMA）比对的Kappa值达0.9，具有高度的一致性。综上所述，本研究建立的阻断ELISA方法具有良好的特异性与较高的符合率，可用于后期进行PCV3抗体的检测，为PCV3的流行病学调查与临床诊断提供技术支持。

关键词: 猪圆环病毒3型, Cap重组蛋白, 单克隆抗体, 阻断ELISA

Abstract: To establish a blocking ELISA method for the detection of porcine circovirus type 3 (PCV3) antibodies, in this study, a hybridoma cell line 2E6, which secretes a good blocking antibody, was obtained by immunization of BALB/c mice with the prokaryotic expression of PCV3 Cap recombinant protein. The recombinant Cap protein was used as the coating antigen, and the horseradish peroxidase (HRP)-labeled 2E6 monoclonal antibody was used as the detection antibody, and the conditions were continuously optimized to finally establish a blocking ELISA method for detecting PCV3 antibody. The established blocking ELISA method was used to test 50 clinical negative sera, and the threshold value of blocking rate (PI) was calculated to determine the cut-off of the method. When PI ≤ 28.30%, the result was determined as negative; when PI ≥ 35.05%, the result was determined as positive; when 28.30%<35.05%, the result was determined as suspicious. The suspicious results should be tested again and the still suspicious results will be judged as positive. Specificity tests showed no cross-reactivity with positive sera for porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine fever virus (CSFV). The sensitivity test showed that the detection potency could reach 1:128. The repetition test showed that the intra- and inter-batch coefficients of variation were less than 10%. The conformity test showed that the method was highly consistent with the gold standard immunoperoxidase monolayer test (IPMA) for PCV detection with a Kappa value of 0.9. In conclusion, the blocking ELISA method established in this study has good specificity and high compliance rate, which can be used for later detection of PCV3 antibodies and provides technical support for epidemiological investigation and clinical diagnosis of PCV3.

Key words: porcine circovirus type 3, Cap recombinant protein, monoclonal antibody, blocking ELISA

中图分类号:

S852.659.2

张宝戈, 黄雅琴, 蔡金双, 朱晨光, 李玉峰. 猪圆环病毒3型Cap蛋白单克隆抗体的制备及阻断ELISA检测方法的建立[J]. 畜牧兽医学报, 2024, 55(3): 1170-1178.

ZHANG Baoge, HUANG Yaqin, CAI Jinshuang, ZHU Chenguang, LI Yufeng. Preparation of Monoclonal Antibodies to Porcine Circovirus Type 3 Cap Protein and the Establishment of a Blocking ELISA Assay[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1170-1178.

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