Preparation Of Polyclonal Antibody Against Gentiopicroside And Establishment Of ELISA For Gentiopicroside

Gentiopicroside(GTP)is the main active ingredients of medicinal plant Gentiana,as well as the quality control index.Gentiopicroside has choleretic,analgesic,stomachic,anti-inflammatory,anti-tumor,anti-hypertensive,antiviral and other pharmacological effects and has been widely applied in clinic.With the study of pharmacological effects of gentiopicroside of Gentiana gentiopicrin in depth,the rising demand resulted in a shortage of the wild medicinal plant Gentiana resources.Thus,Gentiana has been listed as the national three endangered medicinal materials,leading to the appear of counterfeiting of Gentiana macrophylla in the market driven by the interest.Therefore,it is particularly important to establish gentiopicroside detection technology of a low-cost,simple and efficient.Enzyme linked immunosorbent assay(ELISA),as a new technique of detecting small molecular compounds,has attracted growing concern focusing on the application in the detection of active ingredients of Chinese medicine.Compared with the traditional detection methods,ELISA has advantages of high-specificity,high-sensitivity,simple operation,simple sample pretreatment process and without the use of precision instrument and equipment,plus the skill requirements for operators,thereby making it very suitable for the rapid detection of grassroots.The results are as follows:(1)The succinic anhydride method was applied to modify the gentiopicroside structure to introduce the carboxyl groups that can be coupled directly with carrier protein,leading to obtaining carboxylated gentiopicroside.The structure of modified gentiopicroside were analyzed using TLC.The results showed that the emergence of a new compound in thin layer electrophoresis,and the purity of the material is high.And the structure of modified gentiopicroside were analyzed using MS.The results showed that the molecular weight of modified gentiopicroside is consistent with its theoretical value.(2)Artificial antigen GTP-BSA and GTP-OVA were prepared successfully from BSA and OVA covalently bound to carboxylated gentiopicroside by using the mixed anhydride method couple,respectively.A combination of TLC with IR,UV,SDS-PAGE and TNBS was applied to analyze the artificial antigens of GTP-BSA and GTP-OVA.The results showed that artificial antigen GTP-BSA and GTP-OVA have been successfully prepared.Then the conjugation ratio was analyzed using TNBS method,the results showed that the conjugation ratio of GTP-BSA and GTP-OVA are 9:1 and 6:1,respectively.(3)Immune experiment was performed based on the artificial antigen GTP-BSA and GTP-OVA.New Zealand white rabbits were used as immune object,where the basic diet and the basic diet with anti-stress glutathione feed were fed,respectively.The results showed that body weight,mental state,activity,fur color and appetite of the animal in the glutathione anti-stress feed group were better than those of basic diet group.These results showed that glutathione can used be stress response to alleviate effectively the immune stress of animal generated in the immune process,which provides a new idea for animal immunization.(4)The indirect GTP-ELISA method was established and optimized.The results showed that CBS is the best coating liquid,goat serum is the best sealing liquid,PBS+0.5%BSA is the best antibody diluent,optimal working concentrations of coating antigen is 1:2000,the optimal concentration of secondary antibody is 1:3000.The titer of rabbit immune serum were determined by indirect GTP-ELISA,the titer of rabbits were above 1:12800,and GTP-BSA as the immune serum titer of immune antigen produced significantly higher than that of the GTP-OVA immune serum titer of immune antigen produced.(5)The indirect competitive GTP-ELISA method was established and optimized.The results showed that the best competitive reaction time was 40 min,the best organic solvent was 10% methanol solution,and the optimum p H value of washing solution PBST was 7.5.The sensitivity(IC50),Cross-reactivity and specificity of antibody were detected by indirect competitive GTP-ELISA method.The results show that 4 of the experimental group were 95.2 ng/mL,38.9 ng/mL,41.5 ng/mL and 10.9 ng/mL,respectively.The competitive inhibition rate of linear equation of standard curve was y=18.666x-22.452(R~2=0.9722),y=19.07x-18.3315(R~2=0.9658),y=16.872x-12.188(R~2=0.9714)and y=15.523x-1.4934(R~2=0.9688).The cross reactivity rate of 4 experimental groups for gentiopicroside was 100%,and the cross reactivity rate for swertiamarin and sweroside were less than 0.5%,and the cross reactivity rate of jasmine,loganin and oleuropein were less than 0.1%.The results showed that the specificity of the 4 experimental groups were good,especially no cross reaction with jasmine loganin and oleuropein.