Enzyme-linked immunosorbent assay (ELISA) has the advantages of high throughput, simple operation and obvious results, which makes it a more ideal immunoassay technique for detecting fungal contaminants in food.However, traditional ELISA methods have major limitations, such as the use of antibodies susceptible to temperature, easy denaturation, short storage period, susceptible to false positives or false negatives due to the interference of other substances in the detection environment, etc.In this experiment, we used the anti-AFM1 nanobody (Nb-M4) obtained in the laboratory in the previous stage to fuse with the C-terminal fragment of C4-binding protein (C4 bpα) to form a self-assembled heptameric fusion protein (Nb-M4-C4 bpα), and based on this heptamer, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) applied to AFM1 in dairy products.Under the optimal experimental parameters, the IC50 of AFM1 was 0.038 ng/mL and the LOD was 0.009 ng/mL, which were 8.63-fold and 5.56-fold higher than those of monomeric Nb-M4.Negligible cross-reactivity with AFM1 analogs and other common mycotoxins and good recoveries and reproducibility in spiked milk samples were obtained.In addition, the developed method was applied to the analysis of AFM1 in real samples, and the results correlated well with those of HPLC (R2=0.995).This work shows that self-assembled heptamerized nanobodies are an effective strategy for improved affinity and signal amplification, which could be used in the future for sensitive, selective and rapid detection of mycotoxins and other small molecule contaminants in food.
LIU Yingda
,
LIU Haiyuan
,
SU Na
,
LI Qinghui
,
LIU Jia
,
TUYATSETSEG Jambel
,
YI Li
,
JI Rimutu
. Sensitivity improvement of indirect competitive ELISA for the detection of aflatoxin M1 based on nanobody multimerization assay[J]. Food and Fermentation Industries, 2024
, 50(18)
: 291
-299
.
DOI: 10.13995/j.cnki.11-1802/ts.038416
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