6 7 Vol. 6 No. 7 2015 7 Journal of Food Safety and Quality Jul., 2015 ELISA B 1 谢 体 波 1, 刘 红 1*, 陆 苇 1, 易 重 任 1, 张 磊 1, 罗 贵 昆 1, 何 方 洋 (1., 550009; 2., 100012) 1, 2 摘 要 : 目 的 B 1,, ELISA 方 法 AFB 1 结 果 ELISA HPLC 87.1% 92.7% 85.8% 100.8%; 76.0% 92.8% 76.3% 92.9%; 82.6% 92.7% 82.7% 106.4%, 6 RSD 1.29% 2.46% 5.15% 8.70%, 11 2 (r 2 =0.995) 结 论 ELISA HPLC, ELISA B 1,, 关 键 词 : B 1 ; ; Determination of aflatoxin B 1 in cooking oil, peanut, and grain by using direct competitive ELISA kit XIE Ti-Bo 1, LIU-Hong 1*, LU Wei 1, YI Zhong-Ren 1, ZHANG Lei 1, LUO Gui-Kui 1, HE Fang-Yang 1, 2 (1. Guizhou Kwinbon Food Safety Science-Technology Co., Ltd., Guiyang 550009, China; 2. Beijing Kwinbon Biotechnology Co., Ltd., Beijing 100012, China) ABSTRACT: Objective To establish a set of rapid, simple, and accurate method for aflatoxin B 1 in food. At the same time, to further verify the test effect of aflatoxin ELISA kit developed by Beijing Kwinbon biotechnology company. Methods To investigate the occurrence of aflatoxin B 1 in cooking oil, peanut, and grain by using direct competitive enzyme-linked immunosorbent assay (ELISA) kit and high-performance liquid chromatography (HPLC). Results The recovery rates of AFB 1 from the cooking oil spiked by ELISA and HPLC were 87.1%~92.7% and 85.8%~100.8%, respectively. The recovery rates of AFB 1 from the peanut spiked by ELISA and HPLC were 76.0% 92.8% and 76.3% 92.9%, respectively. The recovery rates of AFB1 from the grain by ELISA and HPLC were 82.6% 92.7% and 82.7% 106.4%, respectively. The RSD of 2 methods from the peanut sample by 6 parallel determinations were 1.29%~2.46% and 5.15%~8.70%. By 11 positive samples 基 金 项 目 : 2012 ([2012HK]209-40) 2013 ([2013]209-01) 2013 ([2013]5043/GZK-01) Fund: Supported by the 2012 Guiyang Innovative Talents Scheme ([2012HK]209-40), the 2013 Guiyang Innovative Talents Scheme ([2013]209-01), the 2013 Guizhou Innovative Talents Scheme ([2013]5043/GZK-01) * 通 讯 作 者 :,, E-mail: 1114358560@qq.com *Corresponding author: LIU Hong, Intermediate Engineer, Guizhou Kwinbon Science and Technology for Food Safety Co., Ltd, Guiyang 550009, China. E-mail: 624188083@qq.com
7, : ELISA B 1 2835 results showed that those two methods had a very good consistency (r=0.995). Conclusion It showed that the linear calibration for ELISA and HPLC were good. The measurement results of these methods were close with two methods. The ELISA kit which developed by Beijing Kwinbon Biotechnology company could simultaneously determine aflatoxin B 1 in food, and had advantages of short period and high efficiency of analysis. KEY WORDS: aflatoxin B 1 ; enzyme-linked immunosorbent assay kit; high-performance liquid chromatography 1 引 言 B 1 (aflatoxin, AFB 1 ) (Aspergillus flavus)(aspergillu sparasiticus), AFB 1, ( ),,, [1-3],, B 1,,, [4], 25% [5],,,,, [6] [7], FB1, ELISA [8-12],,,, ELISA B 1, 2 材 料 与 方 法 2.1 材 料 2.1.1 样 品 与 试 剂 ( ) ( ) ( ): ; B 1 ELISA :, 96, 0 0.02 0.06 0.18 0.54 1.62 μg/l, : A B 20 2 ; ( ) ( ) ( ) ( )( ) ( ); (Milli-Q50 ) 2.1.2 仪 器 与 设 备 (CJ-04, ), (MK3, ), (DSY-III, ), (AL204, ), (CHZ-82A, ), (GT16-3A, ), (QL-901, ), ( 20~200 μl 100~1000 μl, 250 μl)(thermo), (Anglent1200), (RF-10AL, ) 2.2 方 法 [5] 2.2.1 样 品 处 理 方 法 (1) 200 μl 2 ml, 1 ml, 0.5 ml, 5 min(20~25 ) 4096 r/min 5 min, 50 μl (2) ; (2.0±0.05) g 50 ml
2836 6 ( 3.0 ml 3.0 ml ; 4.0 ml, 2.0 ml ), 5 min, 4096 r/min 5 min 3.0 ml 50 ml ( 4.5 ml ; 6 ml ), 5 min, 4096 r/min 5 min, 3 ml 10 ml, 50~60 1 ml, 30 s, 1 ml, 30 s, ( ),, 50 μl 2.2.2 样 品 检 测 方 法,,, 3, 50 μl 50 μl 50 μl,, 25 30 min,, 250 μl/, 10 s, 4~5, 50 μl/ A B,, 25 ; 15 min 50 μl/,, 450 nm 5 min OD, 2.2.3 免 疫 试 剂 盒 的 工 作 参 数 1:1 10 4, 1:2 10 4 : 50 μl 50 μl 50 μl : 25 30 min 15 min: OD 1.5~2.0, 70%~80% 2.2.4 标 准 曲 线 的 建 立 (1) ELISA, ELISA 0 0.02 0.06 0.18 0.54 1.62 μg/l 6 GB/T 18979-2003 [13],,, 1; B 1, logity, logity=a+blogc, logity=ln[b/(b 0 -B)], 2 1 B 1 ELISA Fig. 1 The standard curve of AFB 1 2 Fig. 2 Standard curve and equation of AFB 1 3 结 果 与 讨 论 3.1 免 疫 试 剂 盒 最 低 检 出 限 20 3, 1 LOD X 3 2 2 n X ( X ) nn ( 1) X, n 20,,
7, : ELISA B 1 2837 表 1 Table 1 空 白 食 用 油 (ng/l) 花 生 谷 物 (ng/kg) 中 AFB1 含 量 测 定 结 果 The AFB1 content of edible oil (ng/l), peanuts and grain (ng/kg) 1 2 3 4 5 6 7 8 9 10 50.6 27.3 0.8 11.5 34.2 9.6 ND 48.9 16.4 21.8 11 12 13 14 15 16 17 18 19 20 3.2 10.5 31.4 70.8 ND 67.3 25.4 13.6 34.9 7.5 1 2 3 4 5 6 7 8 9 10 103.4 ND 85.7 46.9 5.2 63.5 141.6 32.8 71.5 18.4 11 12 13 14 15 16 17 18 19 20 51.8 37.5 9.3 128.1 90.6 27.0 7.4 ND 45.3 23.6 1 2 3 4 5 6 7 8 9 10 15.1 0.8 7.4 30.9 16.2 24.7 ND 9.3 35.1 6.9 11 12 13 14 15 16 17 18 19 20 ND 26.5 31.6 16.4 8.7 10.8 29.1 11.8 20.7 14.2 88.82 176.56 48.51 : ND 88.82 ng/l, 176.56 ng/kg, 48.51 ng/kg 3.2 方 法 的 回 收 率 和 精 密 度 B 1 100 200 400 600 800 ng/l 5 ; 200 400 800 ng/kg 3 ; 50 100 200 400 800 ng/kg 5, ELISA 87.1%~92.7%, HPLC 85.8%~100.8%; ELISA 76.0%~92.8%, HPLC 76.3%~92.9%; ELISA 82.6%~92.7%, HPLC 82.7%~106.4%( 2), 3 6, RSD 1.29%~2.46%, HPLC RSD 5.15%~8.70%( 3) 3.3 ELISA 方 法 和 HPLC 方 法 的 比 较 分 析 ELISA HPLC ELISA HPLC, 20,, 2, 60 B1 3.7~40.5 ng/l, 2 r 2 =0.995, ELISA HPLC, [14,15] 3.4 样 品 污 染 状 况 60 ELISA,, HPLC, 4 ELISA 12, HPLC 11 ELISA 4.2~36.7 ng/l, HPLC 3 3.7 40.5 ng/l, 97% (<30 ng/l) 12, 5, 42%, 25%; 4, 33%, 18%; 3, 25%, 15%, B 1,,, B 1,,,
2838 6 表 2 样 品 加 标 回 收 率 ( 10-2 ) Table 2 The recovery rate of the sample ELISA HPLC 100 90.3 2.80 90.3 200 173.4 5.48 86.7 200 174.2 1.85 87.1 600 604.8 4.88 100.8 400 370.8 2.03 92.7 800 686.7 6.83 85.8 200 152.1 1.76 76.0 200 152.7 5.01 76.3 400 371.4 2.59 92.8 400 371.9 4.25 92.9 800 619.2 1.45 77.4 800 600.7 4.28 75.1 50 41.3 1.29 82.6 100 106.4 2.83 106.4 100 92.7 2.78 92.7 200 185.2 4.01 92.6 200 178.4 2.62 89.2 400 330.8 4.98 82.7 Table 3 表 3 方 法 精 密 度 (ng/kg, n=6) The precision of both ELISA and HPLC (ng/kg, n=6) ELISA HPLC 200 400 800 200 400 800 x 179.2 367.5 729.2 174.6 348.8 711.2 s 4.40 4.76 12.00 15.18 20.42 36.63 RSD 2.46 1.29 1.66 8.70 5.86 5.15 Table 4 表 4 食 用 油 花 生 谷 物 食 品 中 AFB1 的 污 染 状 况 和 污 染 水 平 Levels of AFB1 contamination and of edible vegetable oil, peanuts and grain (ng/l ng/kg) ( 10-2 ) ELISA HPLC ELISA HPLC 20 4 3 18 4.5 36.7 3.8 40.5 20 5 5 25 5.3 8.9 4.8 8.5 20 3 3 15 4.2 10.8 3.7 11.3 60 12 11 19 4.2 36.7 3.7 40.5 4 结 论 B 1, HPLC,, ELISA HPLC 2 AFB1, 2, ELISA ELISA, 参 考 文 献 [1] Safara M, Zaini F, Hashemi SJ. Aflatoxin detoxification in rice using citric acid [J]. Iranian J Public Health, 2010, 39(2): 24 29. [2] Mobeen AK, Aftab A, Asif A, et al. Aflatoxins B 1 and B 2 contamination of peanut and peanut products and subsequent
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