Study On Preparation Of Highly Specific Monoclonal Antibody Against Triptolide And Rapid Immunoassay Technology

Objective:As Tripterygium wilfordii（T.wilfordii）is more and more widely used in clinical practice,and there are many fake products in the market,traditional methods can not fully identify the authenticity and evaluate the quality of T.wilfordii.Therefore,it is of great important to establish a simple,accurate,fast and convenient method for quality evaluation of T.wilfordii in order to realize on-site rapid screening of the quality of T.wilfordii and ensure the safety and effectiveness of clinical medication.Immunoassay is a new quality control technology for Traditional Chinese Medicine（TCM）,which has the advantages of simple and fast operation,low cost and high throughput.Based on the stipulation,triptolide is an index component to control the quality of T.wilfordii in the ministerial standards,the paper was prepared mAb of T.wilfordii with mAb preparation technology,ELISA and colloidal gold labelled antibody technology,and established an immunoassay method for T.wilfordii.The results were verified by molecular identification and chemical analysis methods.The rapid detection of the quality of T.wilfordii medicinal materials and preparations on the spot was realized,which can be used as the supplement of ministerial standard.Methods:（1）Hapten mTP was prepared by structural modification of 14 hydroxyl groups of triptolide（TP）,and triptolide antigen was synthesized by activated ester method.The antigen coupling results were identified by ultraviolet scanning,TNBS and MALDI-TOF-MS.After immunizing mouse with immune antigens for many times,the serum of mouse was taken for detection.The mouse with high titer and specificity were selected and their spleen cells were fused with myeloma cells.MAb preparation technology was used to screen specific monoclonal cell lines targeting the structure of triptolide.Ascites were induced in mouce.MAbs against triptolide were purified by saturated ammonium sulfate method and lyophilized.The properties of the mAbs were investigated.（2）IcELISA for triptolide was established based on the prepared complete antigen and mAbs to triptolide,which could be used to detect the content of TP in T.wilfordii and preparations rapidly and with high throughput.（3）Colloidal gold immunoassay strips were prepared by labeling mAbs with colloidal gold.The preparation process of colloidal gold strip was optimized and the properties of the strip were evaluated.The triptolide in T.wilfordii samples was detected,and the quality of T.wilfordii medicinal materials and preparations was quickly identified on the spot.UFLC-ESI-MS/MS was used to validate the results of immunochromatographic analysis.（4）In addition,based on DNA barcode and chemical instrumentation methods,the identification and quality evaluation of Tripterygium were carried out in this experiment.Results:（1）The conjugation was confirmed by ultraviolet scanning,TNBS and MALDI-TOF-MS.The coupling ratios of mTP-BSA and mTP-OVA were 18:1 and 7:1,respectively.Triptolide monoclonal cell line mAb 2D10 1H8 was obtained by cell fusion,screening and cloning.In the blank solvent,the IC₅₀0 of the antibody to TP was 0.28μg/mL and its titer was about 2.56×10⁵.The affinity constant was 2×10⁷ L/mol.The type of antibody was IgGl.The cross-reaction rates of TP mAb to triptonide,triptophenolide,tripterifordin and celastrol were 48.57%,20.47%,3.27%and 1.15%,respectively.The cross-reaction rates of TP mAb to wilforlide A,oridonin and demethylzeylasteral were all less than 1.0%.（2）The optimum working concentrations of antigen and antibody were determined by checkerboard method at 0.016 and 0.032μg/mL,respectively.Under those conditions,an ic-ELISA detection method was established.Its IC₅₀0 value was 0.28μg/mL,the minimum detection limit was 0.3 ng/mL,and the optimal detection range(IC₂₀^IC₈₀)was 0.09⁰.87μg/mL.The recovery was 90.05¹06.6%.The established icELISA method was used to determine the content of triptolide in 38 batches of samples.The results were basically consistent with those of UFLC-ESI-MS/MS.（3）Triptolide colloidal gold immunoassay strip was prepared by colloidal gold labeling antibody technology.The concentration of mTP-BSA was 0.3 mg/mL;the concentration of anti-mouse IgG was 0.5 mg/mL;the optimal concentration of conjugated gold labeled antibody was 40μg;the pH value of colloidal gold labeled antibody was 8.0;the tolerance of the strip to methanol concentration was less than 30%;and the detection limit of triptolide test strip was 1μg/mL.The cross-reaction of triptolide test strip to triptonide was high,and there were no cross-reaction totriptophenolide,tripterifordin,celastrol,wilforlideA,demethylzeylasteral,oridonin.According to the standards issued by the Ministry of Reference and the limitation of literature,66 batches of samples（root and preparation of T.wilfordii）were tested with the prepared test strip of T.wilfordii.38 batches of samples were dripped on the test strip without extinction,that is,the content of fake or triptolide was low.UFLC-ESI-MS/MS method was used to validate the results.The results were consistent with those of test strips.（4）The clustering of T.wilfordii and its adulterants was clear in the tree of NJ cluster,and 12of 27 samples were identified as true T.wilfordii.The chemical fingerprint spectrum research indicates that the feature one region can distinguish the false product of tripterygium glycosides more intuitively.The cluster analysis of HCA-thermal map showed that the contents of six active components of T.wilfordii from different habitats were significantly different,which could be used to evaluate the quality of T.wilfordii.Conclusion:The prepared mAb to triptolide has high specificity.The established icELISA method has the advantages of high sensitivity,specificity,rapidity and high throughput.It is suitable for the rapid determination of triptolide TCMs and preparations of triptolide.The established colloidal gold immunoassay strip has the advantages of fast detection,low cost,visualization and convenience.With the advantages of portability,There is broad application prospects in field rapid detection of T.wilfordii herbs and preparations.