摘要
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。
AIM: To prepare monoclonal antibody against trypsinogen activation peptide(TAP), and develop an immunoassay of TAP for the early diagnosis of acute pancreatitis. METHODS: Chemically synthesized TAP was conjugated to KLH as immunogen to immunize BALB/c mice . The spleen cells were fused with Sp2/0 cells. Clones secreting specific monoclonal antibody were screened by indirect ELISA. After cloning, several hybridoma cell clones stably producing anti-TAP monoclonal antibody were obtained. The mAbs were identified and the detection of TAP concentration by competitive ELISA was established. RESULTS: Ten hybridoma cell clones which could produce monoclonal antibodies to TAP were obtained. The isotypes of the ten mAbs were all IgM, except one (IgG1). Ascites titers were between 1∶105 to 1∶106. Specificity analysis proved that all these mAbs reacted only with TAP and had no cross-reaction to Bovine serum (BSA), human albumin, trypsinase, amylase. Neutralisation test showed that these mAbs could be evidently neutralized by TAP. A competitive ELISA for the measurement of TAP was developed, with a sensitivity of 0.69 μg/L. The average intra- and inter-assay coefficient of variation(CV) were 9.10% and 10.33% respectively. The median unrinary TAP concentration was 57.35 μg/L for acute pancreatitis patients and 9.30 μg/L for controls (P<0.01). CONCLUSON: The mAbs against TAP were prepared successfully and a competitive ELISA method for detecting TAP concentration was established.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第4期425-428,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
广州市教育局重点课题基金资助 (No .0 1 2 9)
