大豆Kunitz胰蛋白酶抑制因子单克隆抗体的制备与鉴定

为制备免疫学特性良好的大豆Kunitz胰蛋白酶抑制因子(Kunitz trypsin inhibitor,KTI)单克隆抗体,以50μg/只的免疫剂量将KTI免疫BALB/c小鼠,利用间接ELISA和间接竞争ELISA鉴定多抗血清效价和敏感性,选择血清效价和敏感性较优的小鼠进行细胞融合,多次筛选得到稳定分泌KTI单克隆抗体(Monoclonal antibody,mAb)的杂交瘤细胞株,采用体内诱生腹水法制备mAb并对其免疫学特性进行鉴定。结果表明,1号小鼠经免疫后效价最高且敏感性最好,半数抑制浓度(IC50)为157.33ng/mL,经细胞融合筛选得到4E6-E9阳性杂交瘤细胞株,所制备的抗体效价达到1:1 638 400,亚型为IgG1型,亲和常数为1.45×108 L/mol,IC50为28.39ng/mL,且特异性强。说明试验所制备的mAb免疫学特性良好,能够为建立灵敏、特异的KTI免疫学检测方法提供良好的抗体保障。

Preparation and identification of the monoclonal antibody of Kunitz trypsin inhibitor from soybean

To prepare monoclonal antibodies against soybean Kunitz trypsin inhibitor(KTI)with good immunological characteristics.BALB/c mice were used to immunize with 50μg of KTI,the antiserum titer and sensitivity were identified via indirect ELISA and indirect competitive ELISA.The mice which serum has better titer and sensitivity were selected for cell fusing,then he hybridoma cell strain which stabilization secreted KTI monoclonal antibody(mAb)were screened.Thereafter,mAb was prepared by inducing ascites in vivo and identified the immunological characteristics.The result indicated that the NO.1 mouse was confirmed to produce the antibody with the highest titre,IC50 was 157.33 ng/mL.After cell fusing,4 E6-E9 positive hybrid tumor cell lines were obtained,and the 4 E6-E9 mAb titer was above 1:1 638 400,of which subtype was IgG1,with affinity constant 1.45×108 L/mol,IC5028.39 ng/mL,and high specificity.Our results demonstrated the mAb had good immunological characteristics,and could provide a good antibody basis for the sensitivity and specificity immunological detection of KTI.