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目的 :制备抗胰蛋白酶原激活肽 (TAP)单克隆抗体 (TAPmAb) ,并建立敏感、特异的TAP免疫检测方法。方法 :将人工合成的TAP与血蓝蛋白 (KLH)交联制成免疫原 ,免疫BALB/c小鼠 ,取其脾细胞与Sp2 / 0细胞融合 ,间接ELISA筛选阳性克隆 ,经多次克隆化 ,获得稳定分泌TAPmAb的杂交瘤细胞株 ,并进行鉴定 ,进而建立检测TAP的ELSIA竞争法。结果 :获得10株能稳定分泌TAPmAb的杂交瘤细胞株 ,除 1株为IgG1外 ,余 9株均为IgM类 ;效价达 1∶10 5～ 1∶10 6;与BSA、人白蛋白、胰蛋白酶原、淀粉酶均无交叉反应 ,中和抑制试验表明培养上清、腹水中的抗体能明显被TAP中和。建立了检测TAP的ELISA竞争法 ,灵敏度为 0 .6 9μg/L ,批内变异系数为 9.10 % ,批间变异系数为 10 .33%。用该法测定急性胰腺炎组患者及对照组患者TAP含量分别为 5 7.35 μg/L及 9.30 μg/L (P <0 .0 1)。结论 :成功制备抗TAPmAb ,并建立了敏感的ELISA竞争法。
Objective: To prepare TAP monoclonal antibody (TAP mAb) and to establish a sensitive and specific TAP immunoassay. Methods: Synthesized TAP and KLH were used as immunogen to immunize BALB / c mice. The spleen cells were fused with Sp2 / 0 cells and the positive clones were screened by indirect ELISA. After several clones , To obtain a stable secretion of TAPmAb hybridoma cell lines and identification, and then establish the ELSIA competition TAP detection. Results: Ten hybridoma cell lines secreting TAP mAb were obtained, except for one strain of IgG1, all of the nine strains were IgM; the titer was 1:10 5 ~ 1:10 6; Trypsinogen, amylase no cross-reaction, neutralization inhibition test showed that the culture supernatant, ascites antibodies can be significantly TAP neutralization. The competitive ELISA method for the detection of TAP was established. The sensitivity was 0.69 μg / L, the intra-assay CV was 9.10% and the inter-assay CV was 10.33%. The TAP levels of patients with acute pancreatitis and control group measured by this method were respectively 5 7.35 μg / L and 9.30 μg / L (P <0.01). Conclusion: The anti-TAP mAb was successfully prepared and a sensitive ELISA competition was established.