Development and evaluation of chemiluminescence enzyme-linked immunoassay for residue detection of three organophosphorus pesticides
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摘要: 利用能同时识别对硫磷、甲基对硫磷和杀螟硫磷的宽谱特异性单克隆抗体，建立了同时测定这3种有机磷农药残留的化学发光酶联免疫分析方法（chemiluminescence enzyme immunoassay，CLEIA），比较了间接竞争（ic-CLEIA）和直接竞争（dc-CLEIA）2种反应模式，优化了相关理化参数，确立了最适反应条件。结果表明：间接竞争CLEIA法检测对硫磷、甲基对硫磷和杀螟硫磷的抑制中浓度（IC50）分别为5.57、2.30和2.62 μg/kg，检测线性范围分别为0.39~100、0.39~25和0.39~25 μg/kg；直接竞争CLEIA法检测对硫磷、甲基对硫磷和杀螟硫磷的抑制中浓度（IC50）分别为5.43、1.34和1.24 μg/kg，检测线性范围分别为0.39~100、0.10~25和0.10~25 μg/kg。所建立的CLEIA方法基本能满足对硫磷、甲基对硫磷和杀螟硫磷在谷物和果蔬中最大残留限量的检测要求，为研制有机磷农药多残留检测试剂盒提供了技术依据。
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关键词:
- 化学发光酶联免疫分析 /
- 有机磷农药 /
- 残留 /
- 单克隆抗体 /
- 宽谱特异性
Abstract: In this study, chemiluminescence enzyme-linked immunoassay (CLEIA) for the detection of the three organophosphorus pesticides was established and optimized. A monoclonal antibody with broad-specificity to parathion, parathion-methyl, and fenitrothion was employed. The direct and indirect competitive CLEIAs (dc-CLEIA and ic-CLEIA) were compared, and the physical and chemical parameters in those two reaction models were optimized. The half-maximum inhibition concentration (IC50) and the linear range from ic-CLEIA was respectively 5.57 μg/kg and 0.39-100 μg/kg for parathion, 2.30 μg/kg and 0.39-25 μg/kg for parathion-methyl, and 2.62 μg/kg and 0.39 to 25 μg/kg for fenitrothion. The half-maximum inhibition concentration (IC50) and the linear range for dc-CLEIA was respectively 5.43 μg/kg and 0.39-100 μg/kg for parathion, 1.34 μg/kg and 0.10-25 μg/kg for parathion-methyl, and 1.24 μg/kg and 0.10-25 μg/kg for fenitrothion. The novel methods for the detection of the above mentioned organophosphorus pesticides can meet the requirements of their maximum residue limits in fruits and vegetables, which paves the way to produce rapid-test kits for the detection of organophosphorus pesticides. -
图式 1 两种半抗原、对硫磷及类似物的化学结构
图式 1. Chemical structures of the two haptens used in the immunoassay, parathion and the similar analytes
表 1 宽谱特异性抗体7B2对3种农药的交叉反应率
Table 1. Cross reactivity of the broad-specific antibody 7B2 toward the three pesticides
农药Pesticide 7B2 (PA-QA1-7B2) IC20
/(μg/kg)IC50
/(μg/kg)交叉反应率CR/% 对硫磷parathion 4.42 26.20 100.00 甲基对硫磷parathion-methyl 1.73 11.90 220.20 杀螟硫磷fenitrothion 0.72 7.30 358.90 
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表 2 反应缓冲液中离子强度对CLEIA的影响
Table 2. Effects of ionic strength in reaction buffers on CLEIA performances
反应缓冲液Reaction buffer pH 离子强度IS/(mol/L) IC50/(μg/kg) IC20/(μg/kg) 最大发光值RLUmax 最大发光值/IC50RLUmax/IC50 ic-CLEIA 8.5 0.01 18.74 4.68 1.93 × 106 1.03 × 105 0.05 11.91 2.67 1.75 × 106 1.47 × 105 0.10 4.52 1.15 7.60 × 105 1.68 × 105 dc-CLEIA 8.5 0.01 23.62 4.75 5.81 × 105 2.45 × 104 0.05 18.09 3.83 5.98 × 105 3.31 × 104 0.10 4.54 0.49 6.86 × 105 1.51 × 105
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表 3 ic-CLEIA和dc-CLEIA方法理化参数比较
Table 3. Physical and chemical parameters of ic-CLEIA and dc-CLEIA
理化参数Physicochemical parameters ic-CLEIA dc-CLEIA 抗原用量Antigen 2 μg/mL 0.078 μg/mL 抗体用量Antibody 0.312 5 μg/mL 2 μg/mL 底物液体积Substrate volume 150 mL 120 mL 有机溶剂Organic solvent 10%甲醇10% Methanol 10%甲醇10% Methanol 反应缓冲液种类及pHThe type and pH of reaction buffer PB (8.5) PB (8.5) 反应缓冲液离子强度Ionic strength of reaction buffer 0.10 mol/L 0.10 mol/L 实验时间Reaction time 约2 hAbout 2 h 约1 hAbout 1 h 线性范围及决定系数Linear range and coefficient of determination 对硫磷parathion 0.39~100 μg/kg (R2 > 0.98) 0.39~100 μg/kg (R2 > 0.99) 甲基对硫磷parathion-methyl 0.39~25 μg/kg (R2 > 0.99) 0.10~25 μg/kg (R2 > 0.99) 杀螟硫磷fenitrothion 0.39~25 μg/kg (R2 > 0.99) 0.10~25 μg/kg (R2 > 0.99)
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表 4 CLEIA法对有机磷农药的IC50及交叉反应率
Table 4. IC50 and cross reactivity of CLEIA against organophosphorus pesticides
农药Pesticide ic-CLEIA dc-CLEIA IC50/(μg/kg) 交叉反应率CR/% IC50/(μg/kg) 交叉反应率CR/% 对硫磷parathion 5.57 100.00 5.43 100.00 甲基对硫磷parathion-methyl 2.30 242.17 1.34 405.22 杀螟硫磷fenitrothion 2.62 212.60 1.24 437.90 倍硫磷fenthion > 1 000 < 1 > 1 000 < 1 辛硫磷phoxim > 1 000 < 1 > 1 000 < 1 水胺硫磷isocarbophos > 1 000 < 1 > 1 000 < 1 毒死蜱chlorpyrifos > 1 000 < 1 > 1 000 < 1 三唑磷triazophos > 1 000 < 1 > 1 000 < 1
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表 5 有机磷农药多残留检测的ELISA、CLEIA分析方法比较
Table 5. ELISAs and CLEIAs for multi-residue detection of organophosphorus pesticide
检测方法Detection methods 抗体类型Type of antibody 检测分析物的IC50/(μg/kg) IC50 of analytes/(μg/kg) 参考文献References 对硫磷parathion 甲基对硫磷parathion-methyl 杀螟硫磷fenitrothion icELISA PAb > 1 000 483.90 324 [21] icELISA PAb NDa 447 1 023 [22] icELISA PAb 348 ND ND [23] icELISA MAb 64.20 78.40 43.90 [24] icELISA MAb 20.32 21.44 42.15 [25] icELISA MAb 1 430 2 150 ND [26] dcELISA MAb 1 × 105 2 × 106 ND [26] icELISA MAb 1.53 197.96 1 133.80 [27] icCLEIA PAb 1.14 7.05 321.43 [34] icCLEIA MAb 5.57 2.30 2.62 dcCLEIA MAb 5.43 1.34 1.24 ND: not detected.
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表 6 采用dc-CLEIA方法添加3种有机磷农药的回收试验结果(n=3)
Table 6. Recovery test of three organophosphorus pesticides in samples (n=3)
样品Sample 添加水平Spiked level/(μg/kg) 对硫磷parathion 甲基对硫磷parathion-methyl 杀螟硫磷fenitrothion 检出量Found/(μg/kg) 回收率Recovery/% RSD/% 检出量Found/(μg/kg) 回收率Recovery/% RSD/% 检出量Found/(μg/kg) 回收率Recovery/% RSD /% 苹果Apple 20 20.85 104 3.35 16.28 81 6.08 18.54 93 7.22 100 101.63 102 9.45 91.04 91 8.97 94.13 94 9.80 白菜Chinese cabbage 20 15.14 76 4.02 19.19 96 5.83 14.51 73 5.24 100 96.05 96 9.92 100.66 101 1.70 86.72 87 6.12 糙米Rice 40 42.47 106 6.40 34.55 86 11.18 34.92 87 10.12 200 189.30 95 7.50 235.74 118 3.38 236.48 118 2.98
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