ZHANG Xun,et al Green and Nontoxic Enzyme-Linked Immunosorbent Assay for Aflatoxin B 1 Detection Research Mini-Review Article (Aflatoxins,AFs) 2.2 (As

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1 勋, 等绿色无毒黄曲霉毒素 B 1 免疫检测方法 B 1 勋, 匡华 *, 徐丽广, 马伟, 刘丽强, 胥传来 (, ) 建立了一种基于抗独特型抗体的绿色无毒的 ELISA 方法, 并用于检测面粉中黄曲霉毒素 B 1 通过酶解 2 种抗黄曲霉毒素小鼠单克隆抗体 (11A9 和 1G3) 制备 F(ab')2 片段, 并将其免疫新西兰大白兔制备抗独特型抗体最终得到两种抗独特型抗体, 并对抗独特型抗体和 AFB 1 进行相关性分析通过实际样品添加回收, 其批内回收率为 %~121.88%, 变异系数在 5% 以内 ; 而批间回收率为 %~126.98%, 变异系数低于 8% 分析结果准确可靠, 表明此无毒绿色 ELISA 是一种安全可靠的 AFB 1 分析方法黄曲霉毒素 B 1 ; F(ab')2 片段 ; 抗独特型抗体 ; 无毒 ELISA TS A (2013) Green and Nontoxic Enzyme-Linked Immunosorbent Assay for Aflatoxin B 1 Detection ZHANG Xun, KUANG Hua *, XU Li-guang, MA Wei, LIU Li-qiang, XU Chuan-lai (School of Food Science and Technology,JiangNan University,Wuxi ,China) Abstract A environmental -friendly and nontoxic enzyme -linked immunosorbent assay (ELISA) for the detection of aftoxin B 1 (AFB 1 ) in wheat flour sample was developed. In this method,the standard substance was substituted by the anti-idiotype antibody (anti-id antibody). The anti-id antibodies were prepared by immunizing the rabbits with the F (ab')2 fragments that were obtained by digesting the monoclonal antibodies (11A9 and 1G3) against AFB 1. Two kinds of anti -id antibodies was prepared and both could mimic the three-dimensional structural of AFB 1 and caused specific inhibition to the 11A9 mab. The correlation of the AFB 1 and anti -id antibodies were analyzed by regression. The green ELISA based on anti-id antibody was applied in the analysis of spiked wheat flour sample. The recovery intra -and inter -assay was between % and %,respectively,while the CV less than 10%. The results indicated that the environmentalfriendly and nontoxic ELISA could be used for actual samples. Keywords Aflatoxin,F(ab')2 fragments,anti-id antibody,green ELISA (2012BACD1807,2012BAD29B04); 863 (2012AA06A303) * (1981 ),,,,,, khecho@163.com

2 ZHANG Xun,et al Green and Nontoxic Enzyme-Linked Immunosorbent Assay for Aflatoxin B 1 Detection Research Mini-Review Article (Aflatoxins,AFs) 2.2 (Aspergillus flavus) (Aspergillus [1] parasiticus),, 2 mg/, 10,, 3, B 1 (AFB 1 ), 7, [2],, 0.5 mg A, - AFB1, 20, 2.3 ELISA [3-7], AFB 1 -BSA 0.15 μg/ml (Anti-idiotype,Anti-Id Ab2) [8],, 0.05% PBST 3,, AFB 1 0.2% CBS 1 1.1, ; HRP,100 μl/,37 30 min 3,3,5,5- (TMB), 3 ; 100 μl TMB,, Sigma ; A,GE ; HRP- IgG,Jackson ;, 450 nm, 1.2 Thermo MK3,,, AFB 1 HH-S, ; PH050A, 20%~80%, 5% [9] ;96,Corning F(ab )2 11A9 1G3 (1) IC 50AFB1 A, ph A9 IC 50, IC 50 6 h, 2 mg/ IC 50 ml ph mg/ml 2.5 HPLC- 1 1,37 MS/MS, 5 g, 1 mol/l Tris, ng AFB 1 A,, 50 ml - ( 70 30), SDS-PAGE,-20 F(ab )2,100 μl/,37 2 h,,200 μl/,4, 3 50 μl AFB 1 (0~1 ng/ml) (0~100 μg/ml),,37 30 min,3 ; min, 50 μl 2 mol/l 2.4 AFB 1,, CR CR=(IC 50AFB1 /IC 50 ) 100% (1), 30 min, 1294 Journal of Food Science and Biotechnology Vol.32 No

3 勋, 等绿色无毒黄曲霉毒素 B 1 免疫检测方法 PBST( 0.05% Tween20 PBS) 10, ELISA 3 d ELISA F(ab )2 IgG,,,,, F(ab )2,, ph A9 ( 1), ph 3.8, 5 h,, A, F(ab )2 Fig ~51~5 h; MMarker 11A9 Digestion of mab 11A9 at ph3.8 for different times1~5 h;mmaker 3.2 ELISA Fig. 2 Standard curve of 11A9 mab AFB 1 11A9-Ab2 1G3-3.3 Ab2 ELISA, AFB 1 ( 2), 11A9, 2 AFB 1 11A9 (a)(b)(c), IC 50, 20%~80% ng/ml ng/ml ng/ml,,1g3, 5%, 3 3 1G3-Ab2, 11A9,, AFB 1 11A9-Ab2, AFB 1, AFB 1 y= x (11A9-Ab2) (2) y= x (AG3-Ab2) (3) 2 11A

4 ZHANG Xun,et al Green and Nontoxic Enzyme-Linked Immunosorbent Assay for Aflatoxin B 1 Detection Research Mini-Review Article x,y AFB 1, R , AFB 1,,, 1% , ELISA, % ~ %, 5%; %~126.98%, 8% 1 Table 1 Analysis of wheat flour samples spiked with AFB 1 / (ng/g) wheat flour (n=3) CV/ % /% (n=3) CV/ % /% Fig. 3 Regression analysis 3.4, 8%,, AFB 1 F (ab )2 AFB 1,, AFB 1 AFB 1 ELISA,, %~126.98% [ 1 ] Reddy K R N,Raghavender C R,Salleh B,et al. Potential of aflatoxin B 1 production by Aspergillus flavus strains on commercially important food grains[j]. International Journal of Food Science & Technology,2011,46(1) [ 2 ] Ghali R,Hmaissia-Khlifa K,Ghorbel H,et al. Incidence of aflatoxins,ochratoxin A and zearalenone in tunisian foods [J]. Food Control,2008,19(9) [ 3 ] Rodríguez-Cervantes C H,Ramos A J,Robledo -Marenco M L,et al. Determination of aflatoxin and fumonisin levels through ELISA and HPLC,on tilapia feed in Nayarit,Mexico[J]. Food and Agricultural Immunology,2012 (ahead-of-print)1-10. [ 4 ] Khayoon W S,Saad B,Yan C B,et al. Determination of aflatoxins in animal feeds by HPLC with multifunctional column cleanup[j]. Food Chemistry,2010,118(3) [ 5 ] Nonaka Y,Saito K,Hanioka N,et al. Determination of aflatoxins in food samples by automated on -line in -tube solid -phase microextraction coupled with liquid chromatography-mass spectrometry [J]. Journal of Chromatography A,2009,1216(20) [ 6 ] Wang J J,Liu B H,Hsu Y T,et al. Sensitive competitive direct enzyme -linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk[j]. Food Control,2011,22(6) Journal of Food Science and Biotechnology Vol.32 No

5 勋, 等绿色无毒黄曲霉毒素 B 1 免疫检测方法 [ 7 ] Bircan C. The determination of aflatoxins in spices by immunoaffinity column extraction using HPLC[J]. International journal of Food Science & Technology,2005,40(9) [ 8 ] Jerne N K. Towards the network theory of the immune system[j]. Ann ImmunolInst Pasteur,1974, [ 9 ] Guan D,Li P,Cui Y,et al. A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk [J]. Analytica Chimica Acta,2011,703(1) , (EFSA) ( spinosad), (EC)No396/2005 6, (MRL), (EC)No396/2005 8,,, /(mg/kg) [ ] WTO. [EB/OL]. ( ). http//www. xmtbt-sps.gov.cn/detail.asp?id= /(mg/kg) / / 0.02*/ ( ) 0.3 / ( ) 0.3 / / ( ) 0.02*/ Azarole( )( ) 0.3 / ( /,,,, ) 0.3 / */ / / / / / / / / / , 0.05/2 0.3, 2/ / / , 0.2/1 0.2() , 1/1 1() , 0.2/ () , 0.2/ () / / ()

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较