─株特异抗人血浆Lp（a）单克隆抗体（A5g3）的制备及特征分析

PRODUCTION AND CHARACTERIZATION OF A MOUSE MONOCLONAL ANTIBODY (A5g3)SPECIFIC FOR HUMAN Lp(a)

用序列超速离心和柱层析分离纯化的人血浆脂蛋白（ａ）（Ｌｐ（ａ））免疫Ｂａｌｂ／ｃ小鼠，得到具有对Ｌｐ（ａ）高抗体活性的小鼠脾细胞，与小鼠ＮＳ１骨髓瘤细胞经聚乙二醇（ＰＥＧ）杂文融合，建立分泌特异抗人血浆Ｌｐ（ａ）的单克隆杂交瘤细胞系，对其中的─株单克隆细胞株──Ａ５ｇ３进行了初步分析。Ａ５ｇ３分泌的单克隆抗体类型为ＩｇＧ（２ａ），经间接ＥＬＩＳＡ法分析与Ｌｐ（ａ）有高度反应性，与低密度脂蛋白（ＬＤＬ）及血浆纤溶酶原（Ｐｇ）无反应，免疫印迹结果也表明，Ａ５ｇ３可与纯化的或血浆中的Ｌｐ（ａ）结合，与血浆中的其它成分和纯化的ＬＤＬ及Ｐｇ均不结合，因此Ａ５ｇ３是特异抗人血浆Ｌｐ（ａ）的单克隆抗体。Ａ５ｇ３与还原后的Ｌｐ（ａ）也不结合，说明其与Ｌｐ（ａ）的结合依赖于抗原的完整结构，具有此种特性的Ｌｐ（ａ）特异抗体国内少见报道。

Extensive evidence has suggested that Lp(a)may be an independent risk factor for atherosclerosis.The isolation of a mouse monoclonal antibody,A5g3,monospecific for Lp(a).Eight-week-old Balb/c mice were immunized by injection of purified Lp (a)(purified by sequencial ultracentrifugation following chromatography on Sepharose 6B).The highest immunoreactivity spleen cells were mixed with NS1 myeloma cells.The fusion was conducted by 50% PEG(MW ,1 500).Supernatants were screened for the presence of antibodies to Lp(a)by ELISA. The cells corresponding to positive wells were subcloned at limiting dilution.The McAb secreted by A5g3 was well characterized and the Ig class and subclass were determined as IgG and IgG(2a).In ELISA,the McAb A5g3 had a high degree of reactivity with Lp(a)and reacted little to LDL or plasminogen. Immunoblotting results showed that the McAb only recognized Lp(a)band and did not reacted to other composition of plasma and reduced Lp(a).Therefore,it was concluded that the McAb A5g3 identifies an antigen unique to Lp(a)and the antigen reactive on Lp(a)was dependent on its integrity.