Development Of Anti-Idiotypic Nanobody And Its Non-toxic Immunoassays For Aflatoxin M₁

Aflatoxin M1(AFM1)is the hydroxylated metabolite of aflatoxin B1(AFB1).When lactating mammals are fed with AFB1-contaminated feed,AFB1 is metabolized in the liver into AFM1 then passed to the animals’ milk.AFM1 is toxinogenic,carcinogenic,mutagenic,and is listed as a Class Ⅰ carcinogenic compound by the IARC.With the people’s longing for a better life in the new era,our country’s diet structure and level are developing towards nutrition and balance.Dairy products have become an indispensable part,and the quality and safety of dairy products are also related to the health of the masses and industries’ development.Many countries of the world have set strict limit standards for AFM1 in milk and dairy products.Therefore,the high-sensitivity detection technology of AFM1 in milk and dairy products is urgently needed for ensuring food safety and breaking international technical trade barriers.At present,AFM1 detection methods mainly include instrumental analysis and immunological analysis.Instrumental analysis methods mainly include high performance liquid chromatography(HPLC),liquid chromatography-tandem mass spectrometry(LC-MS),and thin-layer chromatography(TLC).But there are some problems such as expensive equipment,cumbersome maintenance,and complicated pre-processing.The immunological analysis method has the advantages of high sensitivity and specificity.These methods have attracted the attention and application of the majority of scholars.High-quality antibodies are the core material for establishing immunological detection technology.However,all AFM1 immunological detection methods require use of a large number of toxic antigens and standards,which endanger the health of operators and environmental safety.Therefore,there is an urgent need to develop a safe,non-toxic,environmentally friendly,and highly sensitive detection technology to detect AFM1 in milk and dairy products.The main contents of this research are as follows:1.The phage-displayed immune library was successfully constructed.An anti-idiotypic nanobody against AFM1 was screened to provide core identification materials to establish non-toxic immunoassay technology.In this study,the monoclonal antibody(mAb)2C9 against AFM1 was used as the immunogen to immunize the alpaca.When the alpaca’s immune response reached its peak,RNA from the alpaca’s peripheral blood was extracted and reversed transcription to cDNA.Two different subtypes(IgG2 and IgG3)of heavy chain antibody were amplified by PCR.Then the amplified VHH gene fragment was ligated with the plasmid vector pComb3X and transferred into E.coli ER2738.After rescuing by helper phage M13KO7,a phage-displayed library was constructed.The library capacity was 4.56×107 pfu after measurement.The random sequencing results showed that the insertion rate was 100%and the gene sequences were different,which indicated that the constructed phage-displayed library has a large size and good diversity.The established phage-displayed library was subjected to four rounds of biopanning using the improved high pressure competition method.The anti-idiotypic nanobody against AFM1,named VHH C4,that could specifically bind to the mAb 2C9 was successfully screened.2.Successfully expressed and purified VHH C4.The competitive ELISA using VHH C4 as a coating antigen was established,providing a reference for non-toxic detection technologies for the other small molecule hazards in dairy products.The plasmid of VHH C4 was extracted and transferred into nonsuppressor E.coli TOP 10F’ for independent expression.After purification by a Ni-NTA affinity column,the soluble anti-idiotypic nanobody VHH C4 was obtained.A competitive ELISA was used to detect the VHH C4 specificity,the results showed that VHH C4 has no cross-reactivity with the other aflatoxin analogs.The independently expressed VHH C4 was used as a substitute antigen for AFM1 to establish a competitive ELISA for monitoring the AFM1 in milk and dairy products.Through parameter optimization,an ELISA detection method based on VHH C4 instead of antigen was established.The established ELISA’s sensitivity to AFM1 is 0.25 ng/mL,and the linear range is 0.10-0.60 ng/mL.The recovery results of three kinds of sample including milk,yogurt,and milk powder were 84.1%-119.1%.The established VHH C4 based ELISA and HPLC were used to detect the same samples collected from farms and supermarkets.The results have high consistency,which proves that the established VHH C4 based ELISA can be used to detect AFM1 in milk and dairy products.3.Using the independently expressed nanobody VHH C4 as a substitute standard for AFM1,and establish a VHH C4 based ELISA immunoassay method to provide a new idea for the determination of AFM1 in milk and dairy products.In this study,a non-competitive ELISA was used to determine the affinity of VHH C4 to be 4.02×107 L/mol.The thermal stability of VHH C4 and mAb 2C9 was compared by indirect non-competitive ELISA.The results showed that VHH C4 still maintained 70%immune activity after heating at 90℃ for 5 min,while mAb 2C9 was completely lost immune activity after heating at 80℃ for 5 min.Independent expressed VHH C4 was used as a substitute for AFM1 standard substances to establish a competitive ELISA to detect AFM1 in milk and dairy products.By using the concentration of VHH C4 as x axis,the value of B/B0 as y axis,a standard curve was developed.The concentration of VHH C4 showed excellent linear relationship with AFM1 concentration at the same inhibition value.A linear quantitative relationship equation was established between the competition inhibition curve set based on VHH C4 as the standard substance and the competition inhibition curve established by the traditional AFM1 standard substance.The content of AFM1 was determined by substitution method.The concentration of VHH C4 was determined and calculated,and convert into the AFM1 concentration through a quantitative equation.Milk,yogurt,and milk powder were selected as the representative samples,and the recovery of picked AFM1 was 84.8-94.2%;20 real samples collected or purchased from supermarkets and farms were compared with the HPLC-MS/MS results,which showed a good agreement.