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

doi:10.19586/j.2095-2341.2026.0042

生物技术进展 ›› 2026, Vol. 16 ›› Issue (4): 822-832.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

司朝朝¹(), 朱早兵², 刘冬冬¹, 汪永平¹, 杨立桃¹()

^1.上海交通大学生命科学技术学院，代谢与发育科学国际合作联合实验室，上海 200240
^2.江苏省农业科学院植物保护研究所，南京 210014

收稿日期:2026-02-27 接受日期:2026-05-06 出版日期:2026-07-25 发布日期:2026-09-11
通信作者: 杨立桃
作者简介:司朝朝 E-mail: woshisizhao@126.com；
基金资助:
上海市科技创新行动计划项目(24390713700);上海市科技创新行动计划项目(24DZ2201300)

Preparation of a Monoclonal Antibody Against SpCas9 Protein and Application of a Sandwich ELISA for Screening Gene-edited Rice

Zhaozhao SI¹(), Zaobing ZHU², Dongdong LIU¹, Yongping WANG¹, Litao YANG¹()

^1.Joint International Research Laboratory of Metabolic and Developmental Sciences，School of Life Sciences and Biotechnology，Shanghai Jiao Tong University，Shanghai 200240，China
^2.Institute of Plant Protection，Jiangsu Academy of Agricultural Sciences，Nanjing 210014，China

Received:2026-02-27 Accepted:2026-05-06 Online:2026-07-25 Published:2026-09-11
Contact: Litao YANG

摘要/Abstract

摘要：

在基因编辑作物的研发中，转化植株中SpCas9蛋白的快速定量检测对于阳性植株早期筛选及转基因成分消除验证至关重要，但目前适用于植物基质的可靠检测方法尚不完善。以大肠杆菌原核表达的重组SpCas9蛋白为免疫原，系统制备了鼠源单克隆抗体（mAb）和兔源多克隆抗体（pAb），通过12株mAb与pAb的系统配对筛选，建立了以#10 mAb为捕获抗体、pAb为检测抗体的夹心酶联免疫吸附（enzyme-linked immunosorbent assay, ELISA）定量检测方法，并依据方法学验证标准对其分析性能进行了系统评价。结果表明，所制备的mAb和pAb效价均超过1∶729 000。建立的夹心ELISA标准曲线在1.50~48.00 ng·mL^-1范围内线性良好（R2=0.998 4），检测限为0.56 ng·mL^-1；批内和批间变异系数分别为0.98%~3.90%和0.70%~7.34%；加标回收率为97.0%~109.1%；稀释线性回收率为96.8%~114.1%；与SaCas9、FnCas12a、BrCas12b、LwaCas13a蛋白均无交叉反应。该方法经Western blotting验证具有良好的特异性，并成功应用于T₂代基因编辑水稻叶片的检测：16株阳性株系的SpCas9蛋白含量为10.30±0.13~150.26±0.25 ng·g^-1鲜重，株系间差异达14.6倍，野生型水稻检测结果均为阴性（低于检测限）。研究建立的夹心ELISA方法灵敏、特异、重复性好，适用于植物复杂基质，可作为基因编辑作物研发中阳性植株筛选、Cas9表达水平评估及无外源成分纯合材料快速鉴定的高效、低成本技术工具。

关键词: SpCas9蛋白, 单克隆抗体, 夹心ELISA, 基因编辑水稻, 快速检测

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

中图分类号:

Q-33

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.

Zhaozhao SI, Zaobing ZHU, Dongdong LIU, Yongping WANG, Litao YANG. Preparation of a Monoclonal Antibody Against SpCas9 Protein and Application of a Sandwich ELISA for Screening Gene-edited Rice[J]. Current Biotechnology, 2026, 16(4): 822-832.

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链接本文: https://swjsjz.magtechjournal.com/CN/10.19586/j.2095-2341.2026.0042

https://swjsjz.magtechjournal.com/CN/Y2026/V16/I4/822

图/表 13

图1 SpCas9重组蛋白表达及纯化过程A：表达重组蛋白，M—蛋白标准分子量，1—包涵体，2—上清；B：重组蛋白的纯化过程，1~5泳道分别为50、100、200、250、500 mmol·L-1咪唑洗脱液，6—流出液；C：纯化后的重组蛋白。黑色箭头标记位置是Cas9重组蛋白

Fig. 1 Expression and purification of recombinant SpCas9 protein

图2 多克隆抗体与单克隆抗体效价测定结果A：多克隆抗体效价；B：单克隆抗体效价；1~12代表不同杂交瘤细胞分泌的单克隆抗体

Fig. 2 The titers of pAb and mAbs

表1 单克隆抗体亚型及轻链鉴定结果

Table 1 Isotype and light chain characterization of monoclonal antibodies

抗体编号	亚类	抗体编号	亚类
#1	IgG2a，κ	#7	IgG1，κ
#2	IgG1，κ	#8	IgG1，κ
#3	IgG1，κ	#9	IgG2b，κ
#4	IgG1，κ	#10	IgG2a，κ
#5	IgG1，κ	#11	IgG1，κ
#6	IgG1，κ	#12	IgG1，κ

表2 单克隆抗体的配对筛选

Table 2 Selection of paired antibodies

抗体编号	标准品浓度/（ng·mL^-1）
抗体编号	1 000	100	10	0
#1	—	3.341 5	1.025 1	0.328 4
#2	3.565 3	2.070 2	0.477 2	0.164 4
#3	3.663 0	2.971 3	0.821 7	0.210 4
#4	3.623 2	3.403 2	1.093 3	0.174 0
#5	3.598 2	1.756 4	0.416 5	0.223 0
#6	3.642 4	3.214 5	0.918 2	0.169 1
#7	3.505 5	2.397 0	0.658 0	0.135 7
#8	3.325 2	2.727 9	0.685 3	0.111 2
#9	3.933 2	2.708 9	0.645 0	0.189 9
#10	3.526 3	3.451 0	1.305 3	0.167 4
#11	2.383 5	0.807 7	0.196 5	0.123 9
#12	3.614 3	2.799 6	0.653 1	0.170 5

图3 Western blot验证抗SpCas9抗体的特异性A：10#单克隆抗体实验结果；B：多克隆抗体实验结果。M—蛋白标准分子量，1—SpCas9蛋白标准品（10 ng），2—SpCas9蛋白标准品（2 ng），3—#6 SpCas9基因编辑水稻，4—#4 SpCas9基因编辑水稻，5—野生型水稻，黑色箭头标记位置是Cas9蛋白，#4、#6对应结果2.6的基因编辑水稻样本

Fig. 3 Western blot analysis of the specificity of anti-SpCas9 antibodies

图4 夹心ELISA检测Cas9重组蛋白的标准曲线

Fig. 4 The standard curve of Cas9 protein detected by sandwich ELISA

表3 批内和批间变异系数

Table 3 Intra-assay and Inter-assay coefficients of variation

		标准品浓度/（ng·mL^-1）
		48.0	24.0	12.0	6.0	3.0	1.5
批内	平均值±标准差	48.0±2.3	24.3±0.6	11.6±0.1	6.4±0.3	2.9±0.1	1.4±0.1
批内	变异系数/%	3.0	2.6	1.2	3.9	1.0	1.3
批间	平均值±标准差	47.8±0.5	24.0±0.2	12.0±0.3	6.1±0.2	3.0±0.1	1.5±0.1
批间	变异系数/%	1.0	0.7	2.4	4.0	3.3	7.3

表4 水稻叶片提取液基质效应评价

Table 4 Evaluation of the matrix effect in rice leaf extracts

基质稀释倍数	原液	1∶2	1∶4	1∶8	1∶16	1∶32
回收率±标准差/%	110.5±4.6	109.7±3.1	100.4±1.7	97.2±1.3	96.0±1.5	99.3±1.7
变异系数/%	4.2	2.8	1.7	1.3	1.5	1.7

表5 水稻叶片提取液中加标SpCas9蛋白的回收率

Table 5 Recovery of SpCas9 protein spiked rice leaf extracts

标准品浓度/（ng·mL^-1）	48.0	24.0	12.0	6.0	3.0	1.5
回收率±标准差/%	97.0±2.8	97.7±0.7	102.±2.6	106.6±3.1	109.1±2.0	108.4±1.5
变异系数/%	2.93	0.68	2.52	2.87	1.86	1.36

表6 SpCas9基因编辑水稻叶片提取液稀释线性评价

Table 6 Dilution linearity of extracts from SpCas9-edited rice leaves

样品	稀释倍数	检测浓度/（ng·mL^-1）	稀释回收率/%
#4	原液	14.8±0.4	—
	1∶2	15.2±0.3	102.9
	1∶4	14.4±0.7	97.5
	1∶8	14.3±0.8	96.8
#6	原液	25.9±0.3	—
	1∶2	28.2±0.2	109.0
	1∶4	28.9±0.6	111.8
	1∶8	29.5±0.1	114.1

图5 SpCas9 ELISA与4种Cas蛋白的交叉反应分析

Fig. 5 Cross-reactivity analysis of the SpCas9 ELISA with four Cas proteins

图6 水稻叶片SpCas9基因PCR扩增结果注：1~16为SpCas9基因编辑水稻；17~18为野生型水稻。

Fig. 6 PCR amplification results of SpCas9 gene in rice leaves

表7 基因编辑水稻叶片中Cas9蛋白含量检测结果

Table 7 Detection results of Cas9 protein content in gene-edited rice leaves

样品编号	Cas9蛋白含量/（ng·g^-1鲜重）	样品编号	Cas9蛋白含量/(ng·g^-1鲜重)
#1	39.18±1.57	#10	21.61±0.16
#2	27.64±0.34	#11	20.85±0.41
#3	25.52±1.12	#12	16.31±0.19
#4	112.62±0.55	#13	10.30±0.13
#5	27.92±0.25	#14	34.82±1.79
#6	150.26±0.25	#15	10.92±0.68
#7	57.03±0.65	#16	15.44±0.43
#8	47.71±0.25	#17	未检出(<LOD)
#9	24.01±0.05	#18	未检出(<LOD)

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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

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