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 (, 214122) 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 115.60%~121.88%, 变异系数在 5% 以内 ; 而批间回收率为 111.89%~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 201.6 A 1673 1689(2013)12 1293 05 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 214122,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 111.89% and 126.98%,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 2013-07-30 (2012BACD1807,2012BAD29B04); 863 (2012AA06A303) * (1981 ),,,,,, E-mailkhecho@163.com 2013 32 12 1293
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 2 2.1 F(ab )2 11A9 1G3 (1) IC 50AFB1 A, ph 3.8 11A9 IC 50, IC 50 6 h, 2 mg/ IC 50 ml ph 3.8 0.1 mg/ml 2.5 HPLC- 1 1,37 MS/MS, 5 g, 1 mol/l Tris, 10 25 50 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 ; 1 3 000 37 15 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.12 2013
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 3 3.1 F(ab )2 IgG,,,,, F(ab )2,, ph 3.8 11A9 ( 1), ph 3.8, 5 h,, A, F(ab )2 Fig. 1 1 1~51~5 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% 0.057 ng/ml 30 145 ng/ml 18 458 ng/ml,,1g3, 5%, 3 3 1G3-Ab2, 11A9,, AFB 1 11A9-Ab2, AFB 1, AFB 1 y=0.554 4x-3.711 3 (11A9-Ab2) (2) y=0.501 2x-3.389 6 (AG3-Ab2) (3) 2 11A9 2013 32 12 1295
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 2 0.994 9 0.998 7, AFB 1,,, 1% 3.5 1 1, ELISA, 115.60% ~ 121.88%, 5%; 111.89%~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/ % /% 2 0.024 4.65 119.01 0.025 6.88 126.98 5 0.058 4.25 115.60 0.056 1.50 111.89 10 0.122 3.05 121.88 0.113 7.95 112.59 4 3 Fig. 3 Regression analysis 3.4, 8%,, AFB 1 F (ab )2 AFB 1,, AFB 1 AFB 1 ELISA,, 111.89%~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)161-165. [ 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)921-924. [ 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)882-886. [ 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) 4416-4422. [ 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)964-969. 1296 Journal of Food Science and Biotechnology Vol.32 No.12 2013
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)929-934. [ 8 ] Jerne N K. Towards the network theory of the immune system[j]. Ann ImmunolInst Pasteur,1974,125373-389. [ 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)64-69. 2013 11 4, (EFSA) ( spinosad), (EC)No396/2005 6, (MRL), (EC)No396/2005 8,,, /(mg/kg) [ ] WTO. [EB/OL]. (2013-11-6). http//www. xmtbt-sps.gov.cn/detail.asp?id=43711. /(mg/kg) 0154010 0.4/- 1.5 0154020 / 0.02*/- 1.5 0154030 ( ) 0.3 /- 1.5 0154040 ( ) 0.3 /- 1.5 0154050 0.3 /- 1.5 0154060 ( ) 0.02*/- 1.5 0154070 Azarole( )( ) 0.3 /- 1.5 0154080 ( /,,,, ) 0.3 /- 1.5 0154990 0.02*/- 1.5 0243020 2/2 2 1011010 0.05 /2 0.1 1011020 1/2 2 1011030 0.5/0.5 0.7 1011040 0.3 /0.5 0.5 1012010 0.3/3 0.3 1012020 3/3 3 1012030 2/2 2 1012040 1/1 1 1013010 1014010 1013020 1014020 1013030 1014030 1013040 1014040, 0.05/2 0.3, 2/2 3 0.5/0.6 1.5 0.5/0.5 0.5 101610, 0.2/1 0.2() 1016020, 1/1 1() 1016030, 0.2/0.2 0.2() 1016040, 0.2/0.2 0.2() 1020000 0.5/0.2 0.2 1030000 0.2/0.2 0.2() 2013 32 12 1297