Broad-Specificity Immunoassay For Salbutamol And Study Of Antibody Recognition Mechanism

?-agonists were often therapeutically used as broncho-dilating agents.In addition,some?-agonists can result in muscle hypertrophy and reduce carcass fat content,so they have been used as“lean meat agents”in illegally way in meat and poultry production,which causes serious impact on people's health.Many countries have made laws to forbid using?-agonists as feed additives one by another.immunoassays for?-agonists are sensitive,rapid,and simple to operate,so they have broad development prospectin food safety field.But most previous studies built specific method,can not realize detect multiple?-agonists in a single test.So it is necessary to develop a broad-specific immunoassay for?-agonists.In this study,one of?-agonists,salbutamol,were chosen as study object,and Rac-salbutamol and R-salbutamol were derivated and immuned to obtain polyclonal antibody,respectively.Indirect competitive enzyme-linked immunoassay?ciELISA?and fluorescence polarization immunoassay?FPIA?were build with high sensitivity.Cross reaction was conducted between antibody and small molecules,and the data was analysed by three-dimensional quantitative structure-activity relationship.This study demonstrate the recognition mechanism between salbutamol antibody and small molecules,and raise suggestion on hapten design.?1?Florescence Polarization Immunoassay for the Detection of SalbutamolSalbutamol were synthesized with different length florescein,EDF and HDF,and obtained 4 tracer.Via optimation to find the best condition and build the calibration curve.For Rac-sal-suc-EDF,the intercept at 50%(IC₅₀)was 5.81 ng/mL ng/mL,linear detection range is from 1.43-23.56 ng/mL ng/mL.For Rac-sal-suc-HDF,the intercept at 50%(IC₅₀)was 25.87 ng/mL ng/mL,linear detection range is from 7.77-86.24 ng/mL ng/mL.For R-sal-suc-EDF,the intercept at 50%(IC₅₀)was 85.80 ng/mL,linear detection range is from 29.49-249.68 ng/mL ng/mL.And for R-sal-suc-EDF,the intercept at 50%(IC₅₀)was 197.98 ng/mL ng/mL,linear detection range is from99.94-392.17 ng/mL ng/mL.Four FPIA method can recognize 26,24,25 and 21?-agonists,respectively.?2?Indirect competitive enzyme-linked immunoassay for salbutamolCoating antigen and immunogen was conjuncted by mixed anhydride method.Two healthy female New Zealand white rabbits were immunized subcutaneously with two immunogen,then obtained two antibody.Finally,two ciELISA method were built.For R-salbutamol antibody,IC₅₀ was 0.5 ng/mL ng/mL,linear detection range is from0.05-4.00 ng/mL ng/mL,and limit of detection is 0.04 ng/mL.For Rac-salbutamol antibody,IC₅₀was 2.10 ng/mL,linear detection range is from 0.11 to40.86 ng/mL,and limit of detection is 0.02 ng/mL.R-salbutamol antibody can recognize 33?-agonists.Rac-salbutamol antibody can recognize 29?-agonists and In recovery test,the urine was spiked with salbutamol,clenbuterol,brombutero and cimbuterol,respectively.The recovery was from 81.3 to 118.3,and the coefficient of variation was lower than 15%.Compared with HPLC-MS,the result of two method obtained a good correlation,which can prove the ciELISA for?-agonists is precise and reliable.?3?The recognition mechanism study of the broad-specificity between salbutamol antibody and small moleculeWhen analysed the cross activity of salbutamol antibody,a comparative molecular field analysis?CoMFA?and a comparative molecular similarity indices analysis?CoMSIA?were used for the investigation.The result of the model indicated that there were multiple binding site when antibody reacted with small molecule.The group at C-2 and the substituent tertamyl at C-2?was influenced by steric force,moderately increasing the volume of group is beneficial to the antibody recognition.Negative charge decreased at C-1 and C-6 position and negative charge increased at C-2 could increase the affinity of antibody.The group at C-1?and the substituent tertamyl at C-2?was impacted by hydrophobic force,which also could influence recognition of the antibody.In addition,the QSAR revealed that the change of the direction of hydroxyl at the chiral center C-1?of S-salbutamol would alter strength of hydrophobic force.When it inserted into hydrophobic pocket,it would final affected class specificity of the chiral salbutamol antibody,which leaded to the antibody against R-salbutamol show better broad-specificity than that against Rac-salbutamol.Based on the analysis about essential binding site of salbutamol antibody in this study and other report analysis of cross reactivity conducted by antibody immuned by hatpen with various arm binding site,when small molecules derivate arms,recognition site should be exposed as much as possible.It might be favorable to increase the broad specificity of the antibody.Through this study,two sensitive and broad-specific immunoassay for?-agonists were built,and the broad-specificity recognition mechanism between salbutamol antibody and small molecule were studied,the rules for broad-specific hapten design were discussed,which offer necessary reference for broad-specific hapten design and immunoassay development.