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Eu3+‐labeled IgG‐based time‐resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1 in feed

Hu, Xiaofei, Yao, Jingjing, Wang, Fangyu, Yin, Mengqi, Sun, Yaning, Hu, Mei, Shi, Qiaoqiao, Zhang, Gaiping
Journal of the science of food and agriculture 2018 v.98 no.2 pp. 674-680
aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin M1, cell fusion, cross reaction, detection limit, employment, enzyme-linked immunosorbent assay, feeds, fluorescent antibody technique, high performance liquid chromatography, immunization, immunoglobulin G, inhibitory concentration 50, monoclonal antibodies, toxicity
BACKGROUND: Aflatoxin B₁ (AFB₁) is a kind of toxic and carcinogenic mycotoxin. A time‐resolved fluoroimmunoassay (TRFIA) was established for quantitative detection of AFB₁ in feed using Eu³⁺‐labeled IgG as tracer. RESULTS: Monoclonal antibody (McAb) against AFB₁ (9B11‐D7) was prepared through immunization and cell fusion and was identified as high affinity, specificity and sensibility by enzyme‐linked immunosorbent assay (ELISA). The 50% inhibition value (IC₅₀) was 0.81 ng mL⁻¹, the limit of detection (LOD) was 0.10 ng mL⁻¹ and detection range was 0.10–3.94 ng mL⁻¹. Goat anti‐mouse immunoglobulin G (IgG) was modified by Eu³⁺‐DATT, generating Eu³⁺‐labeled IgG. Under optimal assay conditions, TRFIA was shown to be highly sensitive and specific in detection of AFB₁. The IC₅₀ and LOD were 94.73 pg mL⁻¹ and 3.55 pg mL⁻¹, respectively, and detection range was 3.55–1.11 × 10³ pg mL⁻¹. Cross‐reactivity with AFM₁, AFB₂, AFG₁ and AFG₂ was 31.26%, 37.6%, 127.46% and 35.74%, respectively, but zero with other analogues. In determination of AFB₁ spiked in feed sample, TRFIA showed high accuracy and precision. The average recoveries ranged from 93.71% to 97.80%, and coefficient of variation was 1.25–3.73%. Good correlation between TRFIA and HPLC was demonstrated for determination of AFB₁ in feeds, confirming the reliability of the developed method. CONCLUSION: The developed TRFIA exhibited good potential for employment in the ultrasensitive detection of AFB₁ in feed and could be used to determine total aflatoxins. © 2017 Society of Chemical Industry