纳米SiO<sub>2</sub>富集水样重金属镉-hicELISA检测方法的建立

周叶; 杨树宇; 高祎琳; 陈俊杰; 刘长忠; 王自良; 姜金庆

doi:10.13386/j.issn1002-0306.2020100133

纳米SiO₂富集水样重金属镉-hicELISA检测方法的建立

doi: 10.13386/j.issn1002-0306.2020100133

河南科技学院动物科技学院，河南新乡 453003

基金项目: 中原千人计划-中原科技创新领军人才项目（204200510010）；河南省高校科技创新团队（20IRTSTHN025）；河南省重点科技攻关项目（202102110090）

详细信息

作者简介:
周叶（1995−）, 女, 硕士研究生, 研究方向: 动物源食品安全免疫学快速检测技术, E-mail: yezhou9502@163.com

通讯作者:
姜金庆（1972−）, 男, 博士, 教授, 研究方向: 动物疫病防控与残留物免疫检测, E-mail: jjq5678@126.com

中图分类号: TS252.1
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- 被引次数: 0
出版历程
- 收稿日期: 2020-10-20
- 网络出版日期: 2021-06-08
- 刊出日期: 2021-07-07

Determination of Cadmium in Water Samples by Nano-SiO₂ Preconcentration with hicELISA

College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China

摘要

摘要: 目的：探究纳米SiO₂富集分离水样中Cd²⁺的条件，制备抗Cd²⁺单克隆抗体，建立水样中痕量Cd²⁺异源性间接竞争ELISA（heterologous indirect competitive ELISA，hicELISA）快速检测方法。方法：以纳米SiO₂富集、EDTA-2Na竞争洗脱Cd²⁺，ICP法测定处理过程中的Cd²⁺含量。人工合成Cd-ITCBE-BSA免疫Balb/C小鼠，制备Cd²⁺单克隆抗体。人工合成异源性包被抗原，建立镉离子hicELISA检测方法。结果：纳米SiO₂对Cd²⁺的吸附容量为13.3 mg/g，富集倍数可达20倍，EDTA-2Na可有效洗脱被富集的Cd²⁺。以Cd-ITCBE-BSA为免疫原，小鼠血清效价达到1:1.28×10⁴；腹水效价为1:2.0×10⁵，亲和力Ka为8.1×10⁸L/moL。以Hg-ITCBE-OVA包板建立的hicELISA检测限为2.9 μg/L，与Hg²⁺、Cu²⁺、Zn²⁺、Pb²⁺、Cr³⁺、Mo⁶⁺、Fe³⁺、Co²⁺无明显交叉反应。在湖水、自来水、超纯水样品中镉的加标回收率为92.47%~102.86%。结论：建立的纳米SiO₂富集重金属镉-hicELISA检测方法灵敏、特异、准确，能用于水样中镉离子的检测。
- 纳米SiO₂ /
- 重金属 /
- 镉离子 /
- hicELISA /
- 异源性检测
Abstract: Objective: Explore the conditions of preconcentration and separation of Cd²⁺ in water samples by nano-SiO₂, prepare monoclonal antibody against Cd²⁺ and establish a rapid detection method of trace Cd²⁺ in water samples by heterologous indirect competitive ELISA (hicELISA). Methods: Cd²⁺ was preconcentrated by nano-SiO₂ and eluted by EDTA-2Na. The content of Cd²⁺ was determined by ICP. Balb/C mice were immunized with Cd-ITCBE-BSA to prepare Cd²⁺ monoclonal antibody. The heterologous coating antigen was synthesized to establish the detection method of cadmium ion hicELISA. Results: The adsorption capacity of Nano-SiO₂for Cd²⁺ was 13.3 mg/g, and the enrichment multiple was 20 times. EDTA could be used as the effective eluent for the pretreatment. Using Cd-ITCBE-BSA as immunogen, the titer of serum was 1:1.28×10⁴, The titer of mAb in ascites was 1:2.0×10⁵ and the affinity constantwas 8.1×10⁸ L/mol. The detection limit of hicELISA was 2.9 μg/L. There was no significant cross reaction with Hg²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Cr³⁺, Mo⁶⁺, Fe³⁺, Co²⁺. The recoveries of cadmium in lake water, tap water and ultrapure water were 92.47%~102.86%. Conclusion: The hicELISA method was sensitive, specific and accurate, and could be used for the detection of cadmium in water samples.
- nano-SiO₂ /
- heavy metal /
- cadmium ion /
- hicELISA /
- heterologous detection

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图 1 人工抗原紫外扫描图

Figure 1. UV spectra of artificial antigen

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图 2 小鼠多抗血清效价测定

Figure 2. Titer determination of mouse polyclonal antibody

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图 3 小鼠多抗血清对Cd-EDTA的抑制曲线

Figure 3. Inhibition curve of Cd-EDTA by mouse polyclonal antibody serum

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图 4 细胞传代培养上清中抗体效价变化

Figure 4. Detection of antibody titer in the supernatant of cell subculture

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图 5 Cd-EDTA mAb的K_a 测定曲线

Figure 5. Association constant curve of Cd-EDTA mAb

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图 6 hicELISA抑制曲线图

Figure 6. Inhibition curve of hicELISA

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表 1 纳米SiO₂对Cd²⁺的富集

Table 1. Enrichment of Cd²⁺ and by nano-SiO₂

镉标准溶液（mg/L）	SiO₂用量（mg）	A液Cd²⁺浓度（mg/L）	B液Cd²⁺浓度（mg/L）	吸附容量Q_e（mg/g）
5	30	0.08±0.03	98.30±0.05	3.28
10	30	0.19±0.02	197.20±0.06	6.54
20	30	0.07±0.02	406.14±0.08	13.28

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表 2 包被原与抗体的最佳浓度测定

Table 2. Determination of optimum concentration of coating antigen and antibody

包被原	阳性吸收值	阴性吸收值	包被浓度（μg/mL）	抗体稀释倍数
Cd-ITCBE-OVA	1.45	0.18	1	51200
Hg-ITCBE-OVA	1.35	0.15	2	51200
Cd-DTPA-OVA	0.57	0.12	4	2000
Cd-EDTA-OVA	0.65	0.16	8	4000

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表 3 hicELISA检测方法的特异性

Table 3. Specificity of hicELISA

化合物	IC₅₀（μg/L）	交叉反应率
Cd²⁺-EDTA	44.8	100
Hg²⁺-EDTA	574.8	7.8
Cu²⁺-EDTA	792.6	5.6
Zn²⁺-EDTA	>6.4×10³	＜0.5
Pb²⁺-EDTA	>6.4×10³	＜0.5
Cr³⁺-EDTA	>6.4×10³	＜0.5
Mo⁶⁺-EDTA	>6.4×10³	＜0.5
Fe³⁺-EDTA	>6.4×10³	＜0.5
Co²⁺-EDTA	>6.4×10³	＜0.5

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表 4 不同样品中Cd²⁺的添加回收试验

Table 4. Recovery test of Cd²⁺ in different samples

样品	加标浓度（μg/L）	回收浓度（μg/L）	回收率（%）	相对标准差RSD （%）
湖水	10.00	9.67±0.32	96.70±3.20	2.93
	30.00	29.89±2.03	99.63±6.80	2.91
	50.00	51.43±5.15	102.86±10.30	3.41
自来水	10.00	9.64±0.36	96.40±3.60	2.51
	30.00	27.74±1.58	92.47±5.30	3.25
	50.00	47.67±3.90	95.34±7.80	2.08
纯水	10.00	9.98±0.51	99.80±5.10	0.94
	30.00	29.54±1.52	98.47±5.10	0.91
	50.00	50.03±3.28	100.06±6.50	1.21

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