摘要
目的:利用B淋巴细胞杂交瘤技术制备麻痹性贝毒(PSP)的单克隆抗体,以便建立快速、灵敏、有效的毒素检测方法。方法:采用甲醛法将半抗原石房蛤毒素(STX)与血蓝蛋白(KLH)偶联制备成完全抗原STX-KLH,免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,经筛选和克隆,HAT选择培养杂交瘤细胞,利用ELISA方法筛选出分泌抗STX-McAB的杂交瘤细胞株,并通过小鼠体内诱生腹水的方法获得单克隆抗体。结果:共获得4株能稳定分泌麻痹性贝毒抗体的阳性细胞株,建立了分析检测麻痹性贝毒的间接竞争酶联免疫方法。对PSP中STX组分的检出限为20 ng/ml,IC50为220 ng/ml;对GTX2/3的检出限为10 ng/ml,IC50为50 ng/ml。结论:所制备抗体具有高特异性和灵敏性,可用于研制高质量的国产快速检测麻痹性贝毒ELISA试剂盒。
Objective：To develop a rapid, sensitive and effective method for the detection of Paralytic shellfish poisoning （PSP）. Metbods：Monoelonal antibody prepared by B lymphocyte hybridoma technique was used by the formaldehyde method, bapten antigen saxitoxin （STX） and hemoeyanin （KLH） were used to prepare complete antigen STX-KLH, which was used to immune BALB/c mouse. The selected fusion cells were produced by fusing mouse spleen cells with SP2/O myeloma cells, followed by screening and cloning, and cultured with HAT medium. The anti-STX-McAB hybridoma secreting cell lines was obtained by ELISA screening. Monoclonal antibody was produced by mouse in vivo induced ascites method. Results ： We obtained a total of four stable PSP antibody secreting positive cell lines and established a method for detecting PSP by indirect competitive ELISA. Detection limit of ST）（ in PSP was 20 ng/ml, with ICs0 of 220 ng/ml; while detection limit of GTX2/3 was 10 ng/ml, with IC50of 50 ng/ml. Conclusion：The McAB with high specificity and sensitivity can be used to develop domestic high-quality ELISA kit for paralytic shellfish toxins.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2013年第1期69-73,共5页
Chinese Journal of Immunology
基金
国家高技术研究发展计划(863计划)项目(2006AA09Z163)
国家高技术研究发展计划(863计划)项目(2007AA092001)
国家908专项(908-01-ZH3)
国家海洋局海洋赤潮灾害立体监测技术与应用重点实验室基金项目(MATHAB200918)资助
关键词
麻痹性贝毒
石房蛤毒素
单克隆抗体
酶联免疫吸附分析
Paralytic shellfish poisoning （PSP）
Saxitoxin （STX）
Monoclonal antibody （McAB）
Enzyme-linked immu-nosorbent assay（ELISA）
