Preparation Of Aflatoxin B₁ Monoclonal Antibody And Its Application In Rapid Detection Of Contamination Of Traditional Chinese Medicines

Chinese herbal medicines（CHM）are mainly derived from medicinal plants.When they are not dried in time,stored improperly or affected by other external conditions,they are prone to mildew and contaminated by aflatoxin B₁（AFB₁）.AFB₁ is extremely carcinogenic which can lead to liver toxicity,nephrotoxicity,and immunosuppression,and cause great damage to human health.Therefore,in order to effectively control the AFB₁ pollution in CHM and ensure the quality and safety of Chinese medicines,it is important to develop efficient and sensitive rapid detection methods.At present,the traditional detection methods for AFB₁ in CHM are mainly laboratory instrument based on analysis methods,including high-performance liquid chromatography and liquid chromatography-mass spectrometry.However,the sample preparation process is usually cumbersome,time-consuming,and requires high operation technology,which has limitations in the application of AFB₁ screening of large quantities of CHM.The immunological detection technology（ELISA,GICA,etc.）based on the specific recognition of antigen and antibody has the characteristics of simple operation,high throughput and on-site detection,and can be used as an effective supplement to laboratory instrument analysis and detection technology.Whereas,due to the complex matrix of CHM,it requires higher detection sensitivity and stability in the detection of CHM.Based on this,the following studies were carried out:Highly sensitive antibodies is the most important prerequisite for the establishment of immunological detection technology.Firstly,after modifying AFB₁ by an improved oximation method,the AFB₁ was coupled with BSA and OVA to successfully obtain the complete AFB₁antigen.The reaction did not require heating,and no toxic organic solvents such as benzene are used,which was beneficial to environmental protection.In addition,the Balb/c female mice were immunized by increased immunization dose,a combination of subcutaneous multi-point and intraperitoneal injection.The method added an immune site to make the immunization more adequate.After cell fusion and multiple clonal screening,three stable AFB₁ monoclonal cell lines1E5,1E6 and 1F7 were obtained,and the 1F7 cell line with the best effect of inhibiting AFB₁ was selected to obtain a large number of AFB₁ monoclonal antibodies by inducing ascites and purification.Immune characteristics were identified,and the results showed that the AFB₁monoclonal antibody was an IgG_2b antibody with a sensitivity(IC₅₀)of 0.15μg/L and an affinity constant of 2.81×10⁸ L/mol.The cross-reaction rates with AFB₂,AFG₁,AFG₂,and AFM₁ were35.07%,8.75%,1.15%,and 36.75%,and there was almost no cross-reaction with other mycotoxins,indicating that the antibodies prepared in this study had high sensitivity while ensuring high specificity and high affinity,and could be used for the development and production of AFB₁ rapid detection methods.For the purpose of high-throughput rapid detection,based on the prepared AFB₁ monoclonal antibody,with the easy-to-contaminate Chinese medicinal material Semen Ziziphi Spinosae as the research object,with systematic optimization and matrix matching standard curve to eliminate matrix effect interference,a highly sensitive enzyme-linked immunosorbent assay（ELISA）for rapid detection of Chinese herbal medicine AFB₁ was established.The detection limit could reach1.69μg/kg,with a good linearity in the range of 0.05⁰.58μg/L（R²=0.992）,and the spike recovery rate was 88%¹19%.The established method was used to determine AFB₁ in 33 batches of Semen Ziziphi Spinosae,and the results were compared and confirmed by HPLC-MS/MS.The results showed that there were no false positives and false negatives in the ELISA test results.The measured content data was linearly fitted,and the correlation coefficient R was 0.996,indicating that the ic-ELISA method established in this study was accurate and reliable,and could be used for rapid screening of actual Semen Ziziphi Spinosae samples,and could provide a reference for the detection and monitoring of AFB₁ in other Chinese medicines.In addition,for the purpose of rapid on-site detection,the prepared AFB₁ monoclonal antibody was used in the study to develop two different detection forms of colloidal nano-gold probe immunochromatographic test strips according to whether the gold-labeled antibody was immobilized,that were a wet method（W-GICA）and a dry method（D-GICA）.The research focused on the key parameters in the construction of immunochromatographic test strips,including the pH of antibody-colloid gold coupling,antibody coupling amount,and antigen concentration.After optimization,the visible detection limit of the AFB₁ standard solution by W-GICA was 0.25ng/mL,and the sensitivity is 0.5 ng/mL,while the visible detection limit of D-GICA could only reach 1 ng/mL.Therefore,the more sensitive and simpler W-GICA method was finally selected for the detection of aflatoxin B₁ in CHM.In this study,Semen Ziziphi Spinosae,which was easy to be polluted and rich in pigments,was selected as the model research sample to test the applicability of the method.The results showed that the sample only needs one-step dilution after simple extraction,and its visual detection limit could reach 5μg/kg,which could meet the current Chinese Pharmacopoeia and the European Union’s testing requirements for the AFB₁contamination limit standard level in CHM.In the 33 batches of Semen Ziziphi Spinosae samples screened by W-GICA,12 batches exceeded the standard,and the results were consistent with HPLC-MS/MS verification.The method effectively avoids the sample concentration step involved in many quick detection methods,and at the same time,it could also reduce the interference of the matrix by diluting the sample extract,which could realize the rapid screening of CHM in the initial processing of the production place and in the storage process.