Study And Application Of Anti-AFB₁ Immunoaffinity Column Based On Specific Directional Coupling

Aflatoxin is a carcinogenic,teratogenic and mutagenic substance produced by Aspergillus flavus and Aspergillus parasiticus through secondary metabolism,and aflatoxin B₁is classified as a class I carcinogen by the World Health Organisation due to its greater toxicity.The current conventional methods for detecting aflatoxin are high performance liquid chromatography and enzyme-linked immunosorbent assay.Immunoaffinity chromatography is a common method used in sample pretreatment to maximise the specificity of purification and concentration of target compounds and to reduce interference and error.Nanobodies are relatively single in structure compared to monoclonal antibodies consisting of only the variable region of the heavy chain antibody and have the advantages of high resistance to heat,easy expression,good specificity,high affinity and resistance to organic reagents.In this study,we used genetic engineering to fuse and express an anti-aflatoxin B₁nanobody(G8)with a self-recognition Halo-Tag to prepare an anti-aflatoxin B₁immunoaffinity column by one-step targeted coupling,and to optimise the preparation and use conditions of the immunoaffinity column.1.Fused expression and activity analysis of recombinant protein G8-HaloThe active recombinant protein G8-Halo was successfully obtained from E.coli BL21(DE3)host bacteria by transferring calcium into p ET-30a-G8-Halo prokaryotic expression vector using genetic engineering technology,and the optimal induction conditions(IPTG concentration 0.1 mmol/L,induction temperature 25?,induction time 6 h)were determined.The purity of recombinant protein G8-Halo was obtained by NI-NTA affinity chromatography,and the amount of purified recombinant protein was up to 4.4 mg/L.The IC₅₀was 2.27 ng/m L by indirect competitive ELISA analysis.The thermal stability test showed that G8-Halo still had 55%binding activity after 24 days at 37?.2.Preparation of anti-aflatoxin B₁immunoaffinity columnsA specific anti-aflatoxin B₁immunoaffinity column was successfully prepared by one-step self-coupling of recombinant protein G8-Halo and Halo-Tag ligand,and the optimal coupling time for this immunoaffinity column was 2 h.The preparation process and coupling time were greatly simplified and shortened.The immunoaffinity column exhibited a maximum adsorption of 1.5?g/m L of AFB₁when coupled to 5.48mg/m L of fusion protein,increasing the column capacity by at least 2.3-fold.Optimal loading buffer conditions(5%methanol-PBS buffer,ionic strength 0.1 mol/L,p H=7.4)were determined for the prepared anti-AFB₁immunoaffinity column,which fully eluted AFB₁at 70%methanol concentration.The immunoaffinity column was successfully prepared by coupling the protein crushing supernatant and was able to adsorb 43.35 ng and 70.89 ng of AFB₁at coupling volumes of 0.19 mg and 0.35 mg,respectively,which laid the foundation for the direct preparation of the immunoaffinity column without the protein purification step in the future.The spiked recoveries of both AFB₁standard and cereal samples were found to be in the range of80%-112%,which effectively removed the matrix effect and purified the concentrated AFB₁.The actual test of the self-made immunoaffinity column and the application sample of the anti-AFB₁immunoaffinity column purchased by the company was analyzed by paired t-test,P=0.309>0.05,there was no significant difference in the detected value.