Abstract:

Rapid and accurate detection of SpCas9 protein in transformed plants is essential for positive plant selection and transgene elimination during gene-edited crop development， yet reliable detection methods validated for plant matrices remain limited. In this study， mouse monoclonal antibodies （mAbs） and rabbit polyclonal antibodies （pAbs） were generated against prokaryotically 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 sandwich 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. Intra-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 detected in wild-type rice samples （below the detection limit）. The established sandwich ELISA exhibited high sensitivity， specificity， 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.

Key words: SpCas9 protein, monoclonal antibody, sandwich ELISA, gene-edited rice, rapid detection