Immunoassay Of Nanobody Against Aflatoxin B₁ And Its Modification By Site-directed Mutagenesis In Vitro

Aflatoxin B₁ is a secondary metabolite produced by flavus and is commonly found in cereal grains. It is harmful for humans and animals to absorb food which contaminated by AFB₁. For the toxigenecity, carcinogenicity, teratogenicity of AFB₁,many researchers have developed kinds of methods for detecting AFB₁. Among them,immunoassay methods which based on antibody-antigen interactions are specific to analytes. It has several advantages such as high sensitivity, fast detection, cost effective, etc. In the same time, immunoassays are suitable for high-throughput screening. The high quality of antibody is the key to high sensitivity immunoassay. In this study, nanobody was applied for detecting AFB₁, which is derived from the heavy chain variable of heavy chain antibody in alpacas, it has characters of easy production,good water-solubility and good stability, and it has been widely used for food safety analysis, medical diagnosis. To improve the immunoassay sensitivity, the most direct approach is producting high-affinity antibody by gene modification. In this work, the production of nanobody against AFB₁ and its modification in vitro were described.The essential results are shown as follows:1. Biopanning, expression and activity analysis of AFB₁ nanobodyArtificial antigen AFB₁-OVA worked as target site, the phage display immuned library was applied to four rounds of biopanning using Gly-HCl method and competition method. In total, sixteen unique sequences were obtained after identification and DNA sequencing analysis. Among them, p HEN1-G8 has the lowest half inhibitory concentration?IC50?, which was 0.98 ng/m L, the linear range was 0.21 4.64 ng/m L. Then gene fragment encoding p HEN1-G8 was subcloned to p ET25b?+?vector, then the recombinant vector p ET25b?+?-G8 was transformed into E.coli BL21?Rosetta, DE3?, after IPTG induced expression and purification. Immunoassay showed IC50 of nanobody Nb-G8 was 4.61 ng/m L, and the linear range was 1.14 ¹8.59 ng/m L.2. Prokaryotic expression and purification of p ET25b?+?-G8-AP and activity analysisThe gene fragment encoding pET25b?+?-AP was subcloned to pET25b?+?-G8 to construct fusion vector p ET25b?+?-G8-AP. The fusion vector was expressed in E.coli BL21?Rosetta, DE3? as soluble protein, and was purified by immobilized metal affinity chromatography. The purified G8-AP exhibited AP activity of 364.51±8.34U/mg. ELISA showed that G8-AP was also capable of recognizing AFB₁, with neglible cross activity with other six common mycotoxins. Under optimized condition?methanol 2.5 %, Na Cl 20 mmol/L, p H7.4?, a one-step ELISA was established to detect AFB₁ with a half inhibitory concentration?IC50? of 5.25 ng/m L, with the liner range of 1.02 26.98 ng/m L.3. Molecular docking and alanine-scanning mutagenesisMolecular docking of Nb-G8 and AFB₁, several sites that play a key function on binding were predicted. Six of binding sites were implaced by alanine-scanning mutagenesis to confirme their influence on binding. Mutants were selected by sequencing and the activity of mutants were detected by indirect competitive phage-ELISA. one of six mutants, T32 A still has the same character with wild type?WT?. But the half inhibitory concentration of other five mutants?I33A, F49 A,W54A, Y106 A, V112A? improved, and Y106 A almost completely lost its binding activity.4. Construction of mutated libraryby site-saturation mutagenesis and panningTen residues of Nb-G8 were changed into NNS to alter its binding activity. For inducing mutation, three saturation mutagensis libraries were constructed by over-lap PCR. After panning, mutants were selected by indirect competitive phage-ELISA and sequencing. M1-4-12 lost binding activity; M3-3-8, M3-3-12 and M3-4-18 can not been cut off by AFB₁; the sensitivity of M3-4-10 and M3-4-13 improve 15 % than wild type Nb-G8.