Development and Evaluation of an Immunochromatographic Strip and a Magnetic Chemiluminescence Immunoassay for Detection of Porcine Circovirus Type 2 Antigen
用于检测猪圆环病毒2型抗原的免疫层析试纸条与磁化学发光免疫分析法的开发与评价
📄 英文摘要 English Abstract
Porcine circovirus type 2 (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 102.89TCID50/0.1 mL, 103.19TCID50/0.1 mL, and 103.49TCID50/0.1 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 R2 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.
📄 中文摘要 Chinese Abstract
📋 英文结构化总结 English Structured Summary
全文整理
Header:
Background
Porcine circovirus type 2 (PCV2) is considered to be the smallest single stranded circular DNA virus known to directly infect mammals. The genome consists of 1766 nucleotides and 11 specific open reading frames (ORF). The main specific ORF is ORF1 and ORF2. ORF1 encodes two replication-related proteins (Rep and Rep’) and the capsid protein (Cap) encoded by ORF2 is the only structural protein of PCV2. At present, the Cap protein is considered to be the most immunogenic main protein of PCV2, with a molecular weight of about 27.8 kDa, consisting of 233–234 amino acids, and is closely related to PCV2 infection. PCV2 has caused great harm to the pig industry. With the deepening of research, it was found that PCV2 not only caused Postweaning Multisystemic Wasting Syndrome (PMWS), but also caused porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory syndrome (PRDC), reproductive failure (RF), and congenital tremors (CT). To date, polymerase chain reaction (PCR), indirect fluorescence assay (IFA), immunoperoxidase monolayer assay (IPMA), in situ hybridization (ISH), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) had been widely used to detect PCV2 antibodies and antigens. 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.
Header:
Methods
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. Magnetic chemiluminescence immunoassay is quantitative detection method. The recombinant Cap protein of PCV2 was expressed in Escherichia coli and obtained a high-purity protein by affinity chromatography purification. The protein was used to immunize rabbits, and polyclonal antibodies were successfully obtained and purified.
Header:
Results
The immunochromatographic strip can detect PCV2a strain, PCV2b strain, and PCV2d strain. The immunochromatographic strip had minimum detection limits of 10^2.89 TCID50/0.1 mL, 10^3.19 TCID50/0.1 mL, and 10^3.49 TCID50/0.1 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 can detect PCV2 Cap proteins in swine serum; the linear range of this method was 0.25 ng/mL to 32 ng/mL and R² of the standard curve was 0.9993. 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.
Header:
Data Summary
The immunochromatographic strip had minimum detection limits of 10^2.89 TCID50/0.1 mL, 10^3.19 TCID50/0.1 mL, and 10^3.49 TCID50/0.1 mL for PCV2a/LG, PCV2b/SH, and PCV2d/JH. The agreement between the immunochromatographic strip and the ELISA kit was 93.33% (140/150) and the Kappa was 0.866. The magnetic chemiluminescence immunoassay linear range was 0.25 ng/mL to 32 ng/mL with R² of 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 was 97.14% (68/70).
Header:
Conclusions
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.
Header:
Practical Significance
The immunochromatographic strip is a qualitative method that can save time when testing; results can be obtained within 5 to 10 min. The magnetic chemiluminescence immunoassay is a quantitative method that took less than 30 min to achieve results, which is less than the ELISA kit. Both methods can be used for PCV2 clinical detection in swine serum and field settings.
📋 中文结构化总结 Chinese Structured Summary
背景:
猪圆环病毒2型（PCV2）被认为是已知直接感染哺乳动物的最小单链环状DNA病毒。其基因组由1766个核苷酸和11个特异性开放阅读框（ORF）组成。主要的特异性ORF为ORF1和ORF2。ORF1编码两种复制相关蛋白（Rep和Rep'），ORF2编码的衣壳蛋白（Cap）是PCV2唯一的结构蛋白。目前，Cap蛋白被认为是PCV2最具免疫原性的主要蛋白，分子量约为27.8 kDa，由233–234个氨基酸组成，与PCV2感染密切相关。PCV2对养猪业造成了巨大危害。随着研究的深入，发现PCV2不仅引起断奶后多系统衰竭综合征（PMWS），还可引起猪皮炎与肾病综合征（PDNS）、猪呼吸道综合征（PRDC）、繁殖障碍（RF）和先天性震颤（CT）。迄今为止，聚合酶链式反应（PCR）、间接荧光试验（IFA）、免疫过氧化物酶单层试验（IPMA）、原位杂交（ISH）、免疫组化（IHC）和酶联免疫吸附试验（ELISA）已被广泛用于检测PCV2抗体和抗原。这些方法需要专业设备和技术专长，仅适用于实验室使用。本研究旨在开发一种免疫层析试纸条和磁化学发光免疫分析法用于PCV2抗原的检测。
方法:
采用原核表达系统构建重组蛋白，通过动物实验获得多克隆抗体。以聚苯乙烯微球作为固相载体，与蛋白质的氨基共价结合形成免疫探针。单分散微球与抗原或抗体共价结合作为固相，以更佳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。免疫层析试纸条为定性检测方法。磁化学发光免疫分析法为定量检测方法。PCV2重组Cap蛋白在大肠杆菌中表达，经亲和层析纯化获得高纯度蛋白。用该蛋白免疫家兔，成功获得并纯化了多克隆抗体。
结果:
免疫层析试纸条可检测PCV2a株、PCV2b株和PCV2d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10^2.89 TCID50/0.1 mL、10^3.19 TCID50/0.1 mL和10^3.49 TCID50/0.1 mL。检测PEDV（CV777株）、PRV（HB2000株）、CSFV（WH-09株）、PRRS（JXA1-R株）、PPV（WH-1株）和ASFV（SD株）的结果均为阴性。免疫层析试纸条与ELISA试剂盒的一致率为93.33%（140/150），Kappa值为0.866（Kappa > 0.81）。在保证灵敏度的前提下，免疫层析试纸条最重要的特点是该方法可节省检测时间，5至10分钟内即可获得结果。磁化学发光免疫分析法可检测猪血清中的PCV2 Cap蛋白；该方法的线性范围为0.25 ng/mL至32 ng/mL，标准曲线R²为0.9993。磁化学发光免疫分析法与ELISA试剂盒（检测PCV2 Cap蛋白）的一致率为97.14%（68/70）。该方法在不到30分钟内即可获得结果。
数据汇总:
免疫层析试纸条对PCV2a/LG、PCV2b/SH和PCV2d/JH的最低检测限分别为10^2.89 TCID50/0.1 mL、10^3.19 TCID50/0.1 mL和10^3.49 TCID50/0.1 mL。免疫层析试纸条与ELISA试剂盒的一致率为93.33%（140/150），Kappa值为0.866。磁化学发光免疫分析法的线性范围为0.25 ng/mL至32 ng/mL，R²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试剂盒的一致率为97.14%（68/70）。
结论:
本研究结果表明，免疫层析试纸条和磁化学发光免疫分析法检测PCV2抗原具有较高的灵敏度和特异性，为PCV2的临床检测奠定了基础。
实际意义:
免疫层析试纸条是一种定性方法，可节省检测时间，5至10分钟内即可获得结果。磁化学发光免疫分析法是一种定量方法，在不到30分钟内即可获得结果，耗时少于ELISA试剂盒。两种方法均可用于猪血清中PCV2的临床检测和现场检测。