Fundamental Study On Immuoassays For The Determination Of Salbutamol And Paclobutrazol

Immunoassay has the characteristics of highly sensitive and highly specificity. It can quickly detect a large number of samples. Immunoassay provides a reliable analysis method for the detection of pesticide and veterinary drug residues in environment. In this paper, the indirect competitive Enzyme-linked Immunosorbent Assay(ic-ELISA) and Time-resolved Fluorescence Immunoassay(TRFIA) for the determination of salbutamol and paclobutrazol had been developed.The haptens for salbutamol and paclobutrazol were conjugated to bovine serum albumin(BSA) for immunogens by carbodiimide method(DCC/NHS) and to ovalbumin(OVA) for coating antigens by carbodiimide method(EDC/NHS),respectively. The conjugates were confirmed by UV spectra. The conjugation molar ratios were 11:1 and 7:1 for the immunogen and coating antigen of salbutamol and that were 12:1 and 8:1 for the immunogen and coating antigen of paclobutrazol.Polyclonal antibodies of salbutamol and paclobutrazol were generated by immunizing New Zealand rabbits with the immunogens. The titers of the antibodies were 4.0×104and 6.4×105, respectively.The effects of organic solvent, Na+ and pH value on the ic-ELISA of salbutamol were evaluated. The resulted showed that 10% methanol, 0.2 mol/L Na+, pH 7.5 were determined to optimum assay conditions. Under the optimized conditions, the ic-ELISA was developed for detecting salbutamol, IC50 of which was 0.43 μg/L and the limit of detection(LOD) was 0.0056 μg/L, the linear concentrations was kept well from 0.04 μg/L to 3.85 μg/L. The cross-reactivity was not obvious for ractopamine and it was 68.75% for clenbuterol. In river water and Swine urine, the recoveries of salbutamol were from 93.98% to 107.66%, with coefficient of variations being from2.04% to 7.43%. The TRFIA had been also established for salbutamol. Under the optimized conditions(concentration of coating antigen was 1.0 mg/L, dilution ratio of polyclonal antibody and Eu3+-antibody were 40000 and 2000, 10% methanol, 0.2mol/L Na+, pH 7.5), the linear concentrations ranged from 0.004 to 5.02 μg/L, the LOD was 0.66 ng/L, IC50 was 0.11 μg/L for the determination of salbutamol. The cross-reactivity was unconspicuous for ractopamine and it was 44% for clenbuterol.In river water and Swine urine, the recoveries of salbutamol were from 86.98% to106.75%, with coefficient of variations being from 4.27% to 10.0%. Compared with the sensitivity of ic-ELISA, that of TRFIA improved 3.8 times.The ic-ELISA had been established for paclobutrazol. Under the optimized conditions(10% methanol, 0.2 mol/L Na+, pH 7.5), the linear concentrations ranged from 1.86 to 310.9 μg/L, the LOD was 0.43 μg/L, IC50 was 21.35 μg/L for the determination of paclobutrazol. The cross-reactivity was all unconspicuous for some structurally related compounds. In soil and river water, the recoveries of paclobutrazol were from 89.39% to 109.25%, with coefficient of variations being from 4.24% to7.32%. The TRFIA had been also established for paclobutrazol. Under the optimized conditions(concentration of coating antigen was 1.0 mg/L, dilution ratio of polyclonal antibody and Eu3+-antibody were 80000 and 2000, 10% methanol, 0.2mol/L Na+, pH 7.5), the linear concentrations ranged from 0.066 to 22.84 μg/L, the LOD was 0.012 μg/L, IC50 was 1.09 μg/L for the determination of paclobutrazol. The cross-reactivity was under 0.011% for some structurally related compounds. In soil and river water, the recoveries of paclobutrazol were from 96.85% to 102.48%, with coefficient of variations being from 1.07% to 6.25%. Compared with the sensitivity of ic-ELISA, that of TRFIA had obvious improve for the determination of paclobutrazol and it improved 19.6 times.