Study On Immunochemistry For Analysis Of Clenbuterol

R- carboxymethyl ether (CL₁) , a derivative of clenbuterol (CL), was synthesized. was activated by N-Hydroxysuccinimide and conjugated with BSA and OVA to prepare artificial antigens I (CL₁- BSA) and coating antigen I (CL₁-OVA). CL was diazotized to be conjugated with BSA and OVA to prepare artificial antigens II (CL-BSA) and coating antigen II (CL-OVA). The conjugates were confirmed and the molar ratios of hapten to carrier protein were ensured by UV spectrophotometry. CL₁-BSAand CL-BSA were used to immunize New Zealand white rabbits respectively. Therefore, anti-BSI₁-BSA serum and anti-CL-BSA serum were obtained. Both the titers for anti-CL₁-BSA and anti-CL-BSA serums were determined to be 64:1 by the method of agar double immunodiffusion. The polyclonal antibodies were separated from antiserum by salting out method with 35% saturated ammonium sulfate and freeze-dried in a high vacuum.CL₁-OVA were used to coat micro-plate to establish indirect competitive inhibition ELISA of CL. The optimum concentration of CLi-OVA and anti- CL₁-BSA antibody (antibody I ) selected were by phalanx test determined to be lμg/mL and 1.25μg/mL respectively. The effects of pH, ionic strength and organic solvent on coating antigen-antibody I affinity were evaluated. It was shown that the suitable pH value was 7.6 for the coating antigen-antibody I reaction. The affinity of the antibody I to CL could be improved when the ionic strength was properly increased. Methanol with a proper concentration can accelerate the combination of coating antigen and antibody, but high concentration of methanol would bring on a high background, which made the test result impractical. Under optimized conditions, the standard ELISA inhibition curve of CL was established and the regression equation was I=18.90LogC+97.28(r=0.9915). the half-maximal inhibition (IC50) was 3.15ng/mL and the limit of detection (IC₂0) was 0.081ng/mL, the relative standard deviation (CV%) was 8.03%(n=6).CL-OVA were used to coat micro-plate to establish another indirect competitive inhibition ELISA of CL. By phalanx test, the optimum concentration of CL-OVA and anti-CL-BSA antibody (antibody II )were selected to be 6μg/mL and 3μg/mLrespectively. The regression equation of the standard ELISA inhibition curve was I=19.44LogC+ 72.07(r = 0.9905), IC50=73.23ng/mL , IC20=2.10ng/mL, RSD=5.40% (n=6).The standard ELISA inhibition curve of salbutamol (an analog) with antibody I and antibody II was established respectively. To antibody I, the regression equation of the standard ELISA inhibition curve was I=28.66LogC-22.58, the cross reaction(CR%) with salbutamol (an analog) was smaller than 0.0006%, to antibody II, the regression equation of the standard ELISA inhibition curve was I =16.87LogC + 48.24, CR%=5.7 %. The results showed that the indirect competitive ELISA established by coating antigen I and antibody I was superior to that established by coating antigen II and antibody II in sensitivity and specificity, which can overcome the usual problem of non-specific response in practical test.