摘要： 通过细胞融合建立分泌抗植酸酶单克隆抗体(mAb)的杂交瘤细胞株。采用体内诱生腹水法制备单克隆抗体,并对其免疫学特性进行鉴定。结果表明,筛选出2株杂交瘤细胞,其中,5C3细胞株产生单抗亚型为IgG1型,效价为1∶(1.02×106),IC50=100.07 ng/mL,亲和常数为6.70×109L/mol,与市售常规植酸酶、包被植酸酶及浓缩植酸酶的IC50分别达到130.43,126.16,125.33 ng/mL,交叉反应率分别为76.72%,79.32%和79.84%。与高温失活(100℃水煮5 min)的麸皮植酸酶、普通植酸酶、包被植酸酶及浓缩植酸酶IC50分别为4 205.45,4 015.86,3 593.19,3 610.40ng/mL,交叉反应率分别为2.38%,2.49%,2.78%,2.77%。说明成功筛选了杂交瘤细胞株,该细胞株能分泌抗植酸酶mAb,该植酸酶mAb对植酸酶亲和力高、特异性强、灵敏度好,为植酸酶ELISA检测试剂盒及试纸条研制奠定了基础。
关键词:
植酸酶,
杂交瘤,
单克隆抗体,
ELISA
Abstract: The hybridoma lines that secrete phytase monoclonal antibody(mAb) were established through cell fusion.Phytase mAb was prepared by inducing ascites in mice.The immunological characteristics of the mAb were identified.The result indicated that two hybridomas were selected.The subtype of mAb generated by 5C3 hybridoma was IgG1,the titer was 1 ∶ 1.02×106,IC50 was 100.07 ng/mL and Kaff was 6.70×109 L/mol.Phytase mAb IC50 to normal,coated and concentrated phytase was 130.43,126.16,125.33 ng/mL,and cross-reactivity was 76.72%,79.32% and 79.84%,respectively.Phytase mAb IC50 to inactivated(boiled for 5 min) wheat bran phytase,normal,coated and concentrated phytase was 4 205.45,4 015.86,3 593.19,3 610.40 ng/mL,and the cross-reactivity was 2.38%,2.49%,2.78% and 2.77%,respectively.In conclusion,hybridoma lines that secrete phytase mAb were established,phytase mAb with high affinity and specificity and sensitive was prepared.The phytase mAb may lay a foundation for the subsequent ELISA kit and strip development.
Key words:
Phytase,
Hybridoma mAb,
ELISA
中图分类号:
胡骁飞, 魏凤仙, 李青梅, 职爱民, 王方雨, 孙亚宁, 柴书军, 付晓鹤. 植酸酶单克隆抗体的制备及鉴定[J]. 华北农学报, 2012, 27(5): 44-48. doi: 10.3969/j.issn.1000-7091.2012.05.009.
HU Xiao-fei, WEI Feng-xian, LI Qing-mei, ZHI Ai-min, WANG Fang-yu, SUN Ya-ning, CHAI Shu-jun, FU Xiao-he. Preparation and Characteristic of Monoclonal Antibody against Phytase[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(5): 44-48. doi: 10.3969/j.issn.1000-7091.2012.05.009.