Study Of The Recognition Mechanisms Of Aristolochic Acid Ⅰ Antibody And Its Application In Rapid Immunoassay

Aristolochic acids（AAs）are a mixture of structurally related nitrophenanthrene carboxylic acids,present in plants of the genera Aristolochia and Asarum,which have irreversible nephrotoxicity,carcinogenic and mutagenic effects,and have been classified as class I carcinogens by the International Agency for Research on Cancer.Aristolochic acids exposure,in particular,has been linked to liver cancer.Recent studies on the origin of nephropathy in the Balkans have found that aristolochic acids enter the soil as plant rot and are absorbed by the plants that are subsequently grown in the soil.Therefore,Aristolochic acids has become a new worldwide public health problem.So,the establishment of AAs detection methods to effectively monitor the AAs content in drug homologous and healthy foods is necessary for the health of humans.At present,the detection methods of AAs mainly include high performance liquid chromatography（HPLC）,high performance liquid chromatography-mass spectrometry（HPLC-MS）,and capillary electrophoresis（CE）.Although these methods have high detection sensitivity,they need to purchase professional equipment being operated by professional technicians,which make them only suitable for laboratory detection and analysis.In contrast,immunoassays based on the specific recognition of antigens and antibodies have the advantages of strong specificity,high sensitivity,and fast detection speed,which is suitable for rapid screening AAs of a large number of samples in laboratory and field.Therefore,in this study,monoclonal antibodies were generated using the cell fusion technique with the most toxic and highest content of AAI monoclonal antibody as the target,and enzyme-linked immunosorbent assay（ELISA）,fluorescence quenching immunoassay（FIA）methods and immunochromatographic assay（ICA）based on monoclonal antibodies to AAI were established.The method is appropriate for the rapid detection of aristolochic acids in a variety of scenarios.The main results obtained are as follows:（1）Preparation and recognition characteristics of AAI monoclonal antibodyIn this experiment,the immune antigen GCA-BSA synthesized by the active lipid method was used to immunize female Balb/C mice.The immune antigen AAI-KLH synthesized by the carbodiimide method was used to immunize Balb/C mice,through cell fusion,a hybridoma cell strain with high sensitivity and stable secretion of AAI monoclonal antibody was obtained,named AAI-KLH-3A3.An indirect competitive ELISA was established to identify the performance of the antibody.The results showed that the half inhibitory concentration(IC₅₀)of the antibody was 2.4 ng/m L,the detection linear range(IC₂₀～IC₈₀)was 0.2～3.1 ng/m L,and the detection limit(IC₁₀)was 0.1ng/m L.The cross-reactivity with AAⅡwas 86.2%,and the cross-reaction rate with other structural analogs and compounds with relatively high content in Aristolochiaceae plants were all less than 10%.Computer molecular simulation results revealed that the hydroxyl group at position 7 of AAs was the key group influencing monoclonal antibody recognition of aristolochic acid compounds.The ic ELISA recovery rate were between 88%and 110%,with coefficients of variation less than or equal to 14%for the Chinese medicinal materials pseudobulbus cremastrae seu pleiones,the Chinese patent medicine Shedanchuanbei oral liquid,and weight loss tea colon cleansing tea.After comparing the detection results with UPLC-QQQ-MS/MS,it showed that they had a good correlation（R~2=0.997）.（2）Study on Indirect Competitive Chemiluminescence enzyme immunoassay based on AAI monoclonal antibodyIt was investigated that the chemiluminescence enzyme-linked immunosorbent assay（CLEIA）based on the Luminol System.By optimizing the key parameters such as the working concentration of the coated antigen and antibody,the ionic concentration of operating solution,p H,Tween-20 and methanol content,the established CLEIA detection method has an IC₅₀ of1.8 ng/m L and the detection linear range(IC₂₀～IC₈₀)was 0.7～4.4 ng/m L,the detection limit（LOD）was 0.4 ng/m L,which showed a good specificity.The detection results were compared with UPLC-QQQ-MS/MS,and the results showed a good correlation（R~2=0.999）.Compared with the traditional ic ELISA method,the ic CLEIA method had better sensitivity and wider linear range for the detection of AAI.（3）Study on r CDs-based FIATo further improve the sensitivity of the detection method,an FIA based on fluorescence quenching of r CDs was studied.A CD with red fluorescence at 610 nm was prepared under excitation at 540 nm,and oxidized 3,3’,5,5’-tetramethylbenzidine hydrochloride（ox TMB）was quenched by internal filtration.The emission of r CDs converts the chromogenic signal into a fluorescence quenching signal,which further improves the sensitivity of the immunoassay method.Using the prepared AAI monoclonal antibody,a r CDs-based FIA based on CDs sensitization was established.The IC₅₀value of the method reached 0.41 ng/m L,the linear range was 0.14～1.12 ng/m L,and the detection limit（LOD）was 0.08 ng/m L.Compared the detection results with UPLC-QQQ-MS/MS,it showed that they had a good correlation（R~2=0.999）.The sensitivity of this approach to detect AAI was nearly 6 times higher than did ordinary ic ELISA.（4）Study on gold immunochromatographic assay（GICA）based on AAI monoclonal antibodyTo achieve the spot rapid detection,a rapid detection test strip based on colloidal gold was investigated.Colloidal gold particles with an average particle size of about 43 nm were prepared,and AAI monoclonal antibody was labeled with colloidal gold by electrostatic adsorption,and an immunochromatographic detection test strip was constructed.Key parameters like optimal p H for colloidal gold labeling,optimum dosage of labeled antibody,coating concentration of C-line and T-line were optimized.As per the result,it showed that the test strip had a limit of 50 ng/m L under naked eye detection,detection time of 5～10 min.It had good specificity,high repeatability and stable performance,the test results were compared with UPLC-QQQ-MS/MS,which had the same qualitative judgment results,and it was applicable to rapid spot screening of large quantities of samples.（5）Study on Time-Resolved Fluorescence Immunochromatography（TRFIA）based on AAI monoclonal antibodyTo improve the accurate quantification of ICA,a time-resolved fluorescence-based immunochromatographic detection method（TRFICA）was further investigated.Fluorescent probes were prepared by labeling antibodies on fluorescent microspheres through carboxyl activation,and TRFICA based on AAI monoclonal antibodies was established.The method had an IC₅₀ of 8.5 ng/m L,a quantitative range(IC₂₀～IC₈₀)of 3.9～18.5 ng/m L,and the detection limit（LOD）of 2.5 ng/m L.The method had good specificity.The detection results of spiked recovery were compared with UPLC-QQQ-MS/MS,and the results showed a good correlation（R~2=0.990）.In conclusion,the hybridoma cell strain AAI-KLH-3A3 with high sensitivity and stable secretion of monoclonal antibody was prepared in this study.Based on the AAI monoclonal antibody,ic ELISA,CLEIA and r CDs-based FIA suitable for laboratory detection,and GICA and TRFICA suitable for field detection were established respectively.r CDs-based FIA enjoyed the highest sensitivity,followed by ic CLEIA,which had better sensitivity and wider linear range than did traditional ic ELISA.GICA was applicable to rapid spot qualitative screening,which could be determined by the naked eyes;TRFICA could be used for rapid spot qualitative and quantitative detection only with the help of a cheap and portable fluorescence quantifier.The immunoassay method established in this study met the needs of different situations,providing a rapid detection method for monitoring the utilization of Aristolochiaceae plants and products using Aristolochiaceae plants as raw materials,and offered a key technical support for decreasing the exposure of AAs.