Tao Sirui, Duan Yu, Zha Yinhe, Tong Xiaxia, He Yulong, Feng Huapeng, Shu Jianhong
College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Zhejiang Hom-Sun Biosciences Co., Ltd., Shaoxing 312000, China.
Vet Sci. 2025 Jan 9;12(1):40. doi: 10.3390/vetsci12010040.
(PCV2) is the main and primary causative agent of Postweaning Multisystemic Wasting Syndrome (PMWS). To date, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescent assay (IFA), and enzyme linked immunosorbent assay (ELISA) are the most commonly diagnostic methods for detecting PCV2 antigens. However, these methods require specialized equipment and technical expertise and are suitable for laboratory use only. This study aims to develop an immunochromatographic strip and a magnetic chemiluminescence immunoassay for the detection of PCV2 antigens. The recombinant protein was constructed using a prokaryotic expression system, and the polyclonal antibody was obtained by animal experiments. Polystyrene microspheres are used as solid phase carriers to covalently bind to the amino groups of proteins to form immunoprobes. Monodisperse beads are covalently bound to antigens or antibodies as solid phases to bind antibodies or antigens in the liquid phase in a superior manner, thereby capturing and separating antigens and antibodies in the liquid phase. The immunochromatographic strip is qualitative detection method, this method can detect PCV2a strain, PCV2b strain, and PCV2d strain. The immunochromatographic strip had minimum detection limits of 10TCID/0.1 mL, 10TCID/0.1 mL, and 10TCID/0.1 mL for PCV2a/LG, PCV2b/SH, and PCV2d/JH. The results of testing PEDV (CV777 strain), PRV (HB2000 strain), CSFV (WH-09 strain), PRRS (JXA1-R strain), PPV (WH-1 strain), and ASFV (SD strain) were negative. The agreement between the immunochromatographic strip and the ELISA kit was 93.33% (140/150) and the Kappa was 0.866 (Kappa > 0.81). On the premise of ensuring sensitivity, the most important feature of the immunochromatographic strip is that this method can save time when testing; results can be obtained within 5 to 10 min. Magnetic chemiluminescence immunoassay is quantitative detection method; this method can detect PCV2 Cap proteins in swine serum, the linear range of this method was 0.25 ng/mL to 32 ng/mL and R of the standard curve was 0.9993. The limit of detection (LOD) is 0.051 ng/mL and the limit of quantitation (LOQ) is 0.068 ng/mL. The agreement between the magnetic chemiluminescence immunoassay and the ELISA kit (test PCV2 Cap proteins) was 97.14% (68/70). This method took less than 30 min to achieve results, which is less than the ELISA kit. The results of this study show that immunochromatographic strip and magnetic chemiluminescence immunoassay for PCV2 antigens had great sensitivity and specificity, which lays the foundation for PCV2 clinical detection.
猪圆环病毒2型（PCV2）是断奶后多系统消耗综合征（PMWS）的主要致病原。迄今为止，免疫过氧化物酶单层试验（IPMA）、间接免疫荧光试验（IFA）和酶联免疫吸附试验（ELISA）是检测PCV2抗原最常用的诊断方法。然而，这些方法需要专门的设备和技术专长，仅适用于实验室使用。本研究旨在开发用于检测PCV2抗原的免疫层析试纸条和磁化学发光免疫分析法。使用原核表达系统构建重组蛋白，并通过动物实验获得多克隆抗体。聚苯乙烯微球用作固相载体，与蛋白质的氨基共价结合以形成免疫探针。单分散微球与抗原或抗体共价结合作为固相，以优异的方式结合液相中的抗体或抗原，从而捕获和分离液相中的抗原和抗体。免疫层析试纸条是定性检测方法，该方法可检测PCV2a毒株、PCV2b毒株和PCV2d毒株。免疫层析试纸条对PCV2a/LG、PCV2b/SH和PCV2d/JH的最低检测限分别为10TCID/0.1 mL、10TCID/0.1 mL和10TCID/0.1 mL。检测猪流行性腹泻病毒（CV777毒株）、伪狂犬病病毒（HB2000毒株）、猪瘟病毒（WH - 09毒株）、猪繁殖与呼吸综合征病毒（JXA1 - R毒株）、猪细小病毒（WH - 1毒株）和非洲猪瘟病毒（SD毒株）的结果均为阴性。免疫层析试纸条与ELISA试剂盒的一致性为93.33%（140/150），Kappa值为0.866（Kappa＞0.81）。在确保灵敏度的前提下，免疫层析试纸条最重要的特点是该方法在检测时可节省时间；5至10分钟内即可获得结果。磁化学发光免疫分析法是定量检测方法；该方法可检测猪血清中的PCV2 Cap蛋白，此方法的线性范围为0.25 ng/mL至32 ng/mL，标准曲线的R为0.9993。检测限（LOD）为0.051 ng/mL，定量限（LOQ）为0.068 ng/mL。磁化学发光免疫分析法与ELISA试剂盒（检测PCV2 Cap蛋白）的一致性为97.14%（68/70）。该方法不到30分钟即可获得结果，比ELISA试剂盒所需时间少。本研究结果表明，用于检测PCV2抗原的免疫层析试纸条和磁化学发光免疫分析法具有很高的灵敏度和特异性，为PCV2的临床检测奠定了基础。
