Development of the Detection Method for Aflatoxin B1 Based on Nanobody-Nano Luciferase Fusion Proteins
Aflatoxin B1(AFB1)is a highly toxic and carcinogenic foodborne contaminant that seriously threatens food safety and public health.To develop a rapid bioluminescent enzyme immunoassay(BLEIA)for detecting AFB1,this study systematically evaluated the soluble expression,purification,and enzymatic activity of three anti-AFB1 nanobody-nano luciferase fusion proteins(G8-Nluc,Nluc-NB26,and Nluc-NB28).Based on the three fusion proteins,BLEIA assays were established,and Nluc-NB26 was selected for the analysis and validation of cereal samples.The results indicated that Nluc-NB26 exhibited the highest soluble expression level and better stability,followed by Nluc-NB28,while G8-Nluc was largely insoluble.Testing with various surfactants revealed that adding sodium lauroyl sarcosinate improved the solubility of G8-Nluc significantly.The purified fusion proteins all exhibited suitable enzymatic and antigen-binding activ-ities.BLEIA results based on the fusion proteins showed the IC50 values for detecting AFB1 were 4.213,1.697 and 2.169 ng/mL for the Nluc-NB28-BLEIA,G8-Nluc-BLEIA and Nluc-NB26-BLEIA systems,indicating that G8-Nluc-BLEIA had the highest sensitivity,comparable to Nluc-NB26-BLEIA,while Nluc-NB28-BLEIA had the lowest.Considering the soluble expression level,stability,and detection performance of the fusion proteins,Nluc-NB26-BLEIA was further ap-plied to analyze and validate cereal samples.The results demonstrated that this method achieved average recovery rates of 91.1％to 104.1％,comparable to commercial ELISA kits,but with significantly reduced detection time and reagent cost.These findings offer valuable insights for developing rapid and highly sensitive detection techniques for AFB1.
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