夹心ELISA定量检测猪圆环病毒2b型抗原方法的建立

Establishment of a sandwich ELISA for quantitative determination on porcine circovirus type 2b

为建立快速定量检测猪圆环病毒2b型(PCV2b)抗原含量的方法,本研究采用重组抗PCV2b纳米抗体作为捕获抗体,鼠抗PCV2b单克隆抗体为检测抗体,建立了定量检测PCV2b的夹心ELISA方法。该方法不仅能够用于猪圆环疫苗中PCV2b抗原的检测,还可用于大肠杆菌或杆状病毒表达的PCV2b衣壳蛋白(Cap)亚单位疫苗的检测,且与其它猪病毒无交叉反应。该方法检测细胞培养PCV2b的线性范围是104.3TCID50/mL^105.5TCID50/mL,样品回收率为89.3%~105.6%,该方法批内、批间变异系数小于5%,重复性好。该方法与间接免疫荧光方法(IFA)检测相同样品时误差值小于10%,并且ELISA检测结果离散度更小。分别用两种方法定量PCV2b抗原,取相同抗原量免疫猪,其诱导产生的PCV2b抗体效价无显著差异。本研究构建的夹心ELISA方法在定量猪圆环疫苗中PCV2b抗原方面,与传统的IFA方法比较其显示了良好的符合性,并具有更好的重复性和更短的检测周期,是替代IFA方法用于PCV2b定量检测的更优选择。

Abstract： To develop an assay for quantitative determination porcine circovirus type 2b （PCV2b）, a sandwich ELISA was established using a his-tagged nanobody as capture antibody and a mouse monoclonal antibody against PCV2b as detection antibody. This assay was specific for detection of PK-15 cell proliferated PCV2b and E.coli or Baculovirus expressed PCV2b Cap protein but had no cross-reaction with other swine virus. The linear range of this assay was determined to be 104.3 TCID~/mL to 105.5 TCIDsJmL for PCV2b and the recovery ratio of spiked PCV2b was 89.3%-105.6%. The coefficient variation （CV） of intra- and inter-assay were both less than 5%, demonstrating the high repeatability of the assay. Compared with indirect immunof- luorescent assay （IFA）, the virus titers quantified by this ELISA and IFA had no significant difference, and the dispersion was smaller than the latter. The serological response of pigs immunized with PCV2b inactivated vaccine also indicated that the antigen quantified by ELISA was reliable. This assay showed high reproducibility and a short detection time, and it could be used to determine the antigen yields for PCV2b vaccines production.