生物技术进展2026，Vol.16Issue(4)：822-832,11.DOI:10.19586/j.2095-2341.2026.0042
SpCas9蛋白单克隆抗体的制备及夹心ELISA检测方法在基因编辑水稻筛选中的应用
Preparation of a Monoclonal Antibody Against SpCas9 Protein and Application of a Sandwich ELISA for Screening Gene-edited Rice
摘要
Abstract
Rapid and accurate detection of SpCas9 protein in transformed plants is essential for positive plant selection and trans-gene elimination during gene-edited crop development,yet reliable detection methods validated for plant matrices remain limit-ed.In this study,mouse monoclonal antibodies(mAbs)and rabbit polyclonal antibodies(pAbs)were generated against prokary-otically expressed recombinant SpCas9 protein.A sandwich enzyme-linked immunosorbent assay(ELISA)was established by systematic pairing of 12 mAbs with pAbs,using mAb#10 as the capture antibody and pAbs as the detection antibody,followed by comprehensive analytical performance validation.The results showed that all antibody titers exceeded 1∶729 000.The sand-wich ELISA showed excellent linearity over 1.50～48.00 ng·mL-1(R²=0.998 4)with a limit of detection(LOD)of 0.56 ng·mL-1.In-tra-assay and inter-assay coefficients of variation were 0.98%～3.90%and 0.70%～7.34%,respectively.Spike recoveries ranged from 97.0%to 109.1%,and dilution linearity recoveries from 96.8%to 114.1%.No cross-reactivity was observed with SaCas9,FnCas12a,BrCas12b or LwaCas13a.The specificity of the sandwich ELISA was validated by Western blotting,and the assay was applied to T2-generation gene-edited rice leaves.Among 16 SpCas9-positive rice lines,SpCas9 protein contents ranged from 10.30±0.13 to 150.26±0.25 ng·g-1 fresh weight,with a 14.6-fold variation among the lines.No detectable SpCas9 protein was de-tected in wild-type rice samples(below the detection limit).The established sandwich ELISA exhibited high sensitivity,specific-ity,and reproducibility,and was suitable for analysis in complex plant matrices.This method provides a rapid and cost-effective tool for screening positive gene-edited plants,evaluating Cas9 expression levels,and identifying transgene-free homozygous lines during gene-edited crop development.关键词
SpCas9蛋白/单克隆抗体/夹心ELISA/基因编辑水稻/快速检测Key words
SpCas9 protein/monoclonal antibody/sandwich ELISA/gene-edited rice/rapid detection分类
生物科学引用本文复制引用
司朝朝,朱早兵,刘冬冬,汪永平,杨立桃..SpCas9蛋白单克隆抗体的制备及夹心ELISA检测方法在基因编辑水稻筛选中的应用[J].生物技术进展,2026,16(4):822-832,11.基金项目
上海市科技创新行动计划项目(24390713700 （）
24DZ2201300). （）
