抗人脂蛋白相关磷脂酶A2单抗制备及应用

Development and application of monoclonal antibodies anti-human lipoprotein-associated phospholipase A2

摘要:

本研究旨在制备抗人脂蛋白相关磷脂酶A2 (Lp-PLA2) 单克隆抗体，对单抗进行初步评价，采用免疫层析方法学，建立一种可在社区医疗机构应用的Lp-PLA2快速检测方法。首先从NCBI获得人Lp-PLA2全长基因序列构建表达载体，在CHO-K1细胞中表达Lp-PLA2重组蛋白。以获得的重组蛋白免疫BALB/c小鼠，利用细胞融合技术筛选杂交瘤细胞，小鼠腹水诱生单克隆抗体。通过SDS-PAGE、ELISA等方法评价抗体性能。利用双抗夹心法制备Lp-PLA2量子点荧光免疫层析检测试剂，并使用便携式检测仪器评估试剂性能。制备并筛选得到亲和力达到1×10–8的配对单克隆抗体PLA1与PLA5，抗体亚类为IgG1，能特异性识别血液中Lp-PLA2蛋白。自制Lp-PLA2量子点荧光免疫检测试剂可检测线性范围为20–2 000 ng/mL，与进口试剂的检测相关系数R达到0.99。综上，本研究制备得到一对高亲和力、高特异性的抗人Lp-PLA2单克隆抗体，并建立了Lp-PLA2免疫层析检测方法，该方法检测准确、操作简便，适合Lp-PLA2检测应用于社区医疗机构，为居民心血管健康管理提供了新途径。

Abstract:

The aim of this study is to prepare monoclonal anti-human Lp-PLA2 antibodies, and establish a rapid and accurate immunochromatographic Lp-PLA2 assay used in community medical institution. The gene sequence of human Lp-PLA2 was obtained from NCBI to construct the expression plasmid. Lp-PLA2 protein expressed in CHO-K1 cells was used to immune BALB/c mice. The monoclonal antibodies were produced in mouse ascites after hybridoma cells screening. Antibodies were evaluated by SDS-PAGE, ELISA and other methods. The Lp-PLA2 test strip was prepared based on sandwich method and evaluated with the portable detection instrument. The affinity of the paired antibodies, PLA1 and PLA5, both reached 1×10–8. The antibody subclass was IgG1. Both antibodies recognized the Lp-PLA2 protein in the blood specifically. The Lp-PLA2 test strip was prepared based on sandwich method, with linear range of 20–2000 ng/mL. The Lp-PLA2 test strip correlated well with the diaDexus ELISA test kit. In conclusion, the paired antibodies were successfully prepared with high affinity and specificity. The immunochromatographic test of Lp-PLA2 provided a fast and accurate method to detect the concentration of Lp-PLA2 in blood sample for clinical use in the community medical institution and could contribute to the management of cardiovascular diseases.