Construction Of Anti-aflatoxin B₁ Nanobody Phage Display Library And Its Detection Technology

Purpose:Aflatoxin B₁（AFB₁）has been classified as a Group Ⅰ carcinogen by the World Health Organization’s Agency for Research on Cancer because of its toxicity and carcinogenicity.Due to the lack of efficient means of detoxification at this stage,food chain accumulation has become the main pathway for AFB₁ to threaten human health.To ensure food safety,analytical methods such as thin-layer chromatography and mass spectrometry are commonly used for AFB₁ detection.However,this type of method has the disadvantages of complicated sample pre-processing,cumbersome operation steps and time-consuming detection,which is difficult to meet the needs of on-site,immediate detection and analysis.Therefore,this study is devoted to the development of analytical methods for AFB₁ detection.Methods:1.Anti-AFB₁ nanobody phage display library construction and VHH screening.In this study,healthy camel was immunized with aflatoxin B₁-bovine serum albumin（AFB₁-BSA）complete antigen.Peripheral blood lymphocytes from immunized camels were used as the starting material,and then simplified primers were designed to obtain heavy chain antibody gene fragments,which were used as templates to obtain VHH gene fragments.The nanobody phage display library was constructed by optimizing the gene fragment to plasmid ligation ratio and electro-transformation process.The phage display VHH screening technique was used to effectively enrich positive clones of phage specifically recognizing AFB₁.Afterwards,the VHH gene was cloned into the plasmid and the recombinant plasmid was adopted to achieve soluble expression of VHH through the prokaryotic expression system.The screening VHH gene sequences were entered into Rosstta DOCK software for computerized molecular docking validation.2.Development of a rapid and stable magnetic separation chemiluminescence immunoassay for AFB₁ detection of cereals and their products based on the screening of specific high affinity VHH.The primers were designed to pick up the E.coli Alkaline Phosphatase gene fragment,which was cloned into the plasmid with the VHH gene fragment by flexible peptide ligation,and ALP-VHH fusion protein was prepared by the prokaryotic expression system.Preparation of AFB₁-BSA-modified immunomagnetic beads based on the ring-opening reaction of epoxy groups with sulfhydryl groups.Optimize the workload of fusion protein,the size of immunomagnetic beads,the reaction time,the p H and salt ion concentration of the reaction buffer and other detection conditions to improve the sensitivity and stability of the assay.Under the optimal reaction conditions,the competitive standard curve of magnetic separation chemiluminescence immunoassay was established,the specificity was assessed by structural and functional analogues of AFB₁,and finally,spiked samples of maize,oats,etc.were selected to verify the analytical capability of the actual sample assay.3.Development of nanobody lateral flow immunoassay test strips（VHH-LFIA）based on Avi/streptavidin（Avi/SA）oriented coupling strategy for AFB₁ detection.The tolerance to organic solvents,acidity and temperature of VHH and mouse anti-AFB₁ m Ab was compared by ELISA.Primers were designed to clone the VHH gene fragment and the Avi Tag gene fragment into the plasmid by flexible peptide splicing,and the VHH-Avi Tag fusion protein was prepared by the prokaryotic expression system.AFB₁-BSA was sprayed as Test line,and Biotin-BSA was sprayed as Control line of the test strips.VHH-Avi/SA@QDs immunoassay probes were prepared using quantum dots（QDs）as signal elements,and VHH@QDs immunoassay probes based on carbodiimide（EDC/NHS）random coupling method were prepared to compare the detection and analysis ability of both.Establishing a grayscale comparison signal output strategy through smartphone photography.The sensitivity and stability of the method were optimized from two perspectives:quantitative analysis by grayscale comparison and qualitative analysis by naked eye observation,and the detection conditions such as particle size of QDs,working concentration of AFB₁-BSA on T-line,p H,methanol concentration and Tween-20 concentration of reaction buffer were optimized.Under the optimal reaction conditions,the competitive standard curve of VHH-LFIA was established,the specificity was assessed by structural and functional analogues of AFB₁.To evaluate the applicability of the Avi/SA oriented coupling strategy,VHH-Avi/SA@Au NPs immunodetection probes were prepared using Au NPs as signaling elements instead of QDs.The stability of VHH-LFIA was evaluated by accelerated aging assay,and compared with methanol,temperature and salt ion tolerance of commercial AFB₁ assay strips based on m Ab.Finally,oat spiked samples were selected to verify the analytical ability of the actual samples tested.Result:1.Successful construction of phage display VHH library.The experimental results showed that construction of phage-displayed VHH library with calculated library capacity of6×10⁷ CFU/mL,meanwhile,the library has well diversified by gene sequencing analysis.Three specific anti-AFB₁ VHH,named VHH_C8,VHH_E10 and VHH_E12,were successfully screened and prepared,and the standard curves of the VHH were established respectively.The experimental results showed that VHH_C8 and VHH_E10 exhibited high sensitivity to AFB₁,IC₅₀ were 0.969ng/mL or 1.349 ng/mL,and linear ranges were 0.286～4.185 ng/mL and 0.33～4.487 ng/mL,respectively;the sensitivity of VHH_E12 was lower than VHH_C8 and VHH_E10,IC₅₀ was 4.069ng/mL,but its linear range was wider,ranging from 1.00 to 11.14 ng/mL,which can be applied to the development of analytical methods to cope with different concentrations of AFB₁ limit standards.Molecular docking results showed that AFB₁ was in the active pocket composed of the CDR2 and CDR3 regions of VHH_C8,and interaction analysis showed that the high specificity and affinity were determined by the Pi-PiT-type interaction force formed by AFB₁ with TRP113,and the conventional hydrogen bond formed with GLY114/GLY115/SER117/SER118;AFB₁ was stably identified with the active pocket by the combined effects of Pi-PiT-type binding with PHE49 of VHH_E10,the presence of van der Waals forces with CYS45/MET46/GLY47/GLY59/ALA61/ILE63/TYR71/TYR72/ALA73/CYS121/PRO122/GLU127,the formation of hydrocarbon bonds with GLN124/GLY62,and Pi-Alkyl binding with TYR128.2.Establishment of a rapid and stable magnetic separation chemiluminescent immunoassay assay.The experimental results showed that compared with the conventional one-step chemiluminescence ELISA based on ALP-VHH,the sensitivity and LOD of the magnetic separation chemiluminescence immunoassay assay were significantly improved by about 3-fold and 5-fold,respectively,and the total detection time could be reduced from 120 min to 30 min.Under the optimal conditions,the method achieved a sensitive detection of AFB₁ with LOD of0.743 pg/mL and IC₅₀ of 0.33 ng/mL and linear range of 7.23 pg/mL～12.38 ng/mL,showing strong tolerance and practicality for sample detection in complex matrix environments.This method may provide a new avenue for the rapid and sensitive detection of AFB₁ and may expand the application of VHH.3.Establishment of a VHH lateral flow immunoassay test strip detection technique based on Avi/SA oriented coupling strategy.The experimental results showed that compared with random modification,the oriented coupling strategy can greatly ensure VHH binding activity,and after optimization,the detection limit of AFB₁ for VHH-LFIA was 0.095 ng/mL,the IC₅₀was 0.847 ng/mL,and 1.25 ng/mL AFB₁ could be achieved qualitatively under naked eye conditions.The detection of AFB₁ in actual samples of oats was validated and the results showed that VHH-LFIA exhibited excellent analytical performance for real samples.Compared with conventional m Ab-based LFIA,VHH-LFIA exhibits higher stability and tolerance to temperature and methanol,showing better potential for field detection in complex environments.Conclusion:1.In this work,genetic sequences distinct from the previously reported anti-AFB₁nanobodies were screened from a newly constructed immune nanobody library.2.An ultra-rapid one-step detection method for AFB₁ without immobilization and multi-step washing was developed based on magnetic separation technology and ALP-VHH fusion protein.And the problem of reduced VHH activity in the fusion protein was solved by changing the sequence of fusion protein expression in the experiment.This method can provide a new avenue for rapid and sensitive detection of AFB₁,and can expand the application of VHH.3.In this study,a VHH-based Avi/streptavidin oriented coupling strategy was used to develop a VHH-LFIA for AFB₁ detection.This strategy not only provides a convenient and sensitive tool for AFB₁ detection,but also will open up a feasible path for efficient application of nanobodies.