抗人精浆磷脂酶A2单克隆抗体细胞株的建立与鉴定

目的 :制备并鉴定人精浆磷脂酶A2 (PLA2 )的单克隆抗体 (McAb)。　方法 :通过PEG沉淀、SephacrylS 30 0柱层析、DEAE SephadexA 2 5柱层析及羟基磷灰石 (HA)柱层析分离纯化人精浆中的PLA2 ,并以SDS PAGE鉴定其分子量及蛋白浓度 ;免疫BALB/C小鼠制备抗PLA2McAb ,通过ELISA技术、Western Blot方法鉴定其敏感性及特异性。　结果 :从精浆中提纯的PLA2的相对分子质量 (Mr)约为 3490 0 ,纯化倍数约为 2 4 5倍。制备的McAb经鼠Ig亚类分析为IgMKappa型 ,敏感性可达 1∶5 6～ 1∶5 8,Western Blot证明其特异结合的抗原即为PLA2蛋白酶。结论 :Mr为 3490 0的PLA2可能为一种新型PLA2。该PLA2的McAb的制备将为研究人精浆中PLA2与男性生殖能力的关系提供广阔的前景

Construction and Identification for Cell Strain of Anti-human Seminal Plasma Phospholipase A2 Monoclonal Antibody

OBJECTIVES: To establish and evaluate the anti-human seminal plasma phospholipase A2 (PLA2) monoclonal antibody (McAb). METHODS: After having been separated and purified from human seminal plasma by PEG precipitation, Sephacryl S-300 column chromatography, DEAE-Sephadex A-25 column chromatography and HA column chromatography, PLA2 was regarded as an antigen to immune BALB/C mouse to produce anti-human seminal plasma PLA2 McAb. The PLA2 McAb sensitivity and specificity were performed by ELISA technique and Western-blot analysis, respectively. RESULTS: The molecular weight of PLA2 depurated with 245 fold purification from human seminal plasma was about 34,900, while the sensitivity and typing of its McAb were 1:5(6)-1:5(8) and IgM (kappa) with a satisfied Western-Blot results. CONCLUSIONS: The PLA2, which had not been reported in international and domestic papers, may be a new type of PLA2. The establish of its McAb will provide significant tools for the research of the relationship between PLA2 in human seminal plasma and male fertility.