抗猪SIgA单克隆抗体的研制

Preparation of Monoclonal Antibodies Against Porcine SIgA

采用饱和硫酸铵盐析、SephadexG-200柱层析和离子交换柱层析技术,从猪初乳中提纯分泌型IgA(SIgA)。经SDS-PAGE电泳鉴定,纯化的SIgA达到电泳纯。以此纯化的SIgA,采用长程免疫法免疫BALB/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG-1500进行细胞融合,第1次融合率为79.7%,第2次融合率为60.3%。建立间接ELISA,用于检测杂交瘤细胞上清中的单克隆抗体,初检阳性率分别为4.1%、16.8%。经2次亚克隆反复筛选获得了2株能稳定分泌抗猪SIgA单克隆抗体的杂交瘤细胞株,命名为CA6、5D4。ELISA和Western-blotting证实,所获得的单抗能特异性针对SIgA。CA6、5D4经反复冻存、复苏及多次传代,仍能分泌高效价抗体,CA6分泌抗体属IgM、κ型,而5D4分泌抗体属IgG1、κ型。

Porcine secretory immunoglobulin A(SIgA) was isolated and purified from colostrums by a procedure of precipitation with saturated ammonium sulfate,chromatography with Sephadex G-200 and ion-exchange chromatography with DEAE52.And it was identified by SDS-PAGE electrophoresis.The BALB/c mice were immunized four times with the purified SIgA,then the spleen cells of the hyperimmunized mouse and SP2/0 cells were fused with PEG-1500.The cell fusion rate of 79.7% was firstly obtained,60.3% secondly obtained.An indirect enzyme-linked immunosorbent assay(ELISA) was developed to detect monoclonal antibodies(McAbs) secreted by hybridoma cell lines.And positive rate of 4.1% were firstly obtained,16.8% secondly obtained.Two hybridoma cell lines secreting monoclonal antibodies,named CA6,5D4 were developed by two subclones.The specificity of McAb against SIgA was identified by means of indirect ELISA and Western-blotting.After reapeated frozen storage,thawed revival and genration,they are still steadily producing high titer of McAbs.The McAbs secreted by CA6 had mouse IgM,κ isotype and the McAbs secreted by 5D4 were found to belong to mouse IgG1,κ isotype.The McAbs we prepared would be very useful in detecting the level of mucosal immune and purifying SIgA.