[Development and application of monoclonal antibodies anti-human lipoprotein-associated phospholipase A2].
[Development and application of monoclonal antibodies anti-human lipoprotein-associated phospholipase A2].
复制标题
DOI：
10.13345/j.cjb.180320
复制
发表时间：
2019-03-25
期刊：
影响因子：
--
通讯作者：
Cao, Lin
中科院分区：
文献类型：
--
作者：
Xu, Wen;Lu, Zhaoxin;Cao, Lin
本研究的目的是制备抗人Lp-PLA2的单抗，建立一种快速、准确的用于社区医疗机构的Lp-PLA2免疫层析法。从NCBI获得人LP-PLA2基因序列，构建表达载体。用CHO-K1细胞表达的LP-PLA2蛋白免疫BALB/c小鼠。杂交瘤细胞筛选后，在小鼠腹水中产生单抗。用SDS-PAGE、ELISA法等方法检测抗体。采用夹心法制备了LP-PLA2试纸，并用便携式检测仪进行了评价。这对抗体的亲和力均达到1×10⁻⁸。抗体亚类为IgG1。这两种抗体都能识别血液中的LP-PLA2蛋白。采用夹心法制备LP-PLA2试纸，线性范围为20~2000 ng/mL。Lp-PLA2试纸与DiaDexusEL ISA试剂盒具有良好的相关性。综上所述，成功制备了具有较高亲和力和特异性的抗体对。Lp-PLA2免疫层析试验为社区医疗机构临床应用提供了一种快速、准确的检测血样Lp-PLA2浓度的方法，有助于心血管疾病的管理。
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.