Structure-activity Relationship Of Antigen And Antibody Against Four Triphenylmethane Compounds By Molecular Simulation

With the rapid development of the aquaculture industry in China, the relateddiseases have increased year by year. Malachite green and crystal voiet were widelyused for resisting bacteria, fungus and parasite. However, the related studies havefound that malachite green, crystal voiet and their metabolites have somedisadvantages such as high toxic, high residual, carcinogenic, teratogenic,mutagenicand so on,and they are prohibited in the aquaculture in many countries. The detectionof triphenylmethane compounds plays an important role on the quality of aquaticproducts. The immunoassay methods have been widely used in the analysis ofmalachite green drug residue, which is a convenient and fast modern detectionmethod.However, the current researches about immunoassay methods of malachite greencompounds at home and abroad mostly focus on the analysis of a single compound,but the simultaneous detection on four determinands such as malachite green,leucomalachite green, crystal violet and leucocrystal violet is still a challenge. At thesame time, the application of molecular simulation technology provides a new wayfor multi-residue determination of triphenylmethane compounds in immunoassay.Therefore, hapten was designed and optimized based on molecular simulation,polyclonal antibody was prepared, and the structure-activity relationship of antigenand antibody was studied in this paper. The study can give help in improvingantibodies with purpose, and it is the theoretical foundation for improvingimmunoassay method against a number of similar drugs in aquatic products further toensure the healthy development in aquaculture industry.The main research results are as follows：1. By MOE software and density function （B3LYP/6-31G*） of Gaussian03quantum mechanics calculation method, malachite green,crystal violet and theirmetabolites were analysised in three dimensional conformation, then they wereoptimized in structure and were calculated in the charge distribution. The resultsshowed that the space overlap was good, and their structures were greatly similar. But there existed obvious differences in atomic charge distribution of malachite green,crystal violet and their metabolites. So we can conclude that there is a certainrelationship between cross response rate of prepared antibody and charge distributionof commonly used antigen, considering the results that there was regularity betweenthem Then charge distributions of modified haptens were analysised and the haptenwith charge at about0.174that named N⁺was chosen for the following experimentswith their commercial information and reaction information in CAD database andSciFinder database, eventually.2. With leucocrystal violet as raw materials, the hapten containing-N⁺group andfive carbon atoms connecting arm was synthesized by two step chemical reaction, andwere analysised and identified through the thin layer chromatography（TLC）,TOF-mass spectrometry and nuclear magnetic resonance （NMR） of one-dimensionalspectrum（1H NMR and13C NMR） as well as two-dimensional spectrum(1H^-1HCOSY、HMBC and HSQC), Firstly, the TLC results showed that newsubstances were formed, and were purified by normal phase silica gel column;secondly, the TOF-mass spectrometry results showed that ESI positive ion detectionof the intermediate and target product were M+=488.1and M+=474.3, separately,which were consistent with the theoretical value. As a result, they were synthesizedsuccessfully by preliminary analysis; thirdly, the NMR results showed that targetproduct had consistent characteristics of NMR signal with the intermediate, withoutmethoxy spectrum signal, so the target structure was synthesized successfully.3. Antigen that synthesized were conjugated to carrier protein KLH and BSAusing earbodiimide method, synthesizing immune antigen and envelope antigenrespectively, and they were identified by the UV scanning, SDS-PAGE and MALDI-TOF-MS analysis.The results showed that envelope antigen were successfullycoupled with three methods, and found that three methods were same in some degree.So we chose the UV scanning method to identify the big molecular carrier proteinKLH with small molecular drugs, and it demonstrated that conjugate was successful.Polyclonal antibodies were raised through immunizing New Zealand whiterabbits and BALB/C mice with immune antigen, and the titers of antiserum hadreached1:51200and1:12800separately. The optimal working concentration wasoptimized by ELISA as follows: the concentration of envelope antigen was1μg.mL^-1，the dilution ratios of Goat anti Rabbit IgG secondary antibody and Goat anti mouse IgG secondary antibody was5000and10000separately. Indirect competitive ELISAresults found that two antibodies had specificity recognization with synthetic antigen（-N⁺）, and half inhibition rates were in the range of10^-100ng.mL–1. But antibodieshad little recognization rate with malachite green, leucomalachite green, crystal violetand leucocrystal violet, and the trend of recognization rate was LCV＞LMG＞CV＞MG.Then the structure-activity relationship of antigen and antibody was drew withfour drugs difference degree of charge distribution compared to target product as theabscissa and similarity of inhibition rate under concentration of104ng.mL^-1as theordinate in10⁴ng.mL^-1concentration.The results showed that there was a good linearcorrelation with R2=0.8378, and the overall trend was correspond with molecularsimulation prediction model of charge distribution, so the model will have certainguiding significance on antibody design.