A competitive immunoassay with a surrogate calibrator curve for aflatoxin M₁ in milk

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• 作者：Di Guan^a ; ^b ; ^c ; ¹ ; Peiwu Li^a ; ^b ; ^c ; ¹ ; ^{peiwuli@oilcrops.cn"" rel=""nofollow} ; ^{peiwuli@public.wh.hb.cn"" rel=""nofollow} ; Yehan Cui^d ; Qi Zhang^a ; ^c ; ^{zhangqi521x@yahoo.cn"" rel=""nofollow} ; Wen Zhang^a ; ^b
• 关键词：Aflatoxin M₁ ; Anti-idiotype antibody ; Surrogate ; Enzyme-linked immunosorbent assay
• 刊名：Analytica Chimica Acta
• 出版年：2011
• 出版时间：3 October 2011
• 年：2011
• 卷：703
• 期：1
• 页码：64-69
• 全文大小：627 K

文摘

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M₁ (AFM₁) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM₁ surrogate, was generated by immunizing rabbits with F(ab′)₂ fragments from the anti-AFM₁ monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM₁ mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM₁ and the anti-Id antibody (y = 31.91x − 8.47, r = 0.9997). The assay was applied to analyze AFM₁ in spiked milk samples. The IC₅₀ value of the surrogate calibrator curve was 2.4 μg mL⁻¹, and the inter-assay and intra-assay variations were less than 10.8 % ; recovery ranged from 85.2 to 110.9 % . A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y = 0.81x + 9.82, r = 0.9922).