Immunoassay Study Of β-Agonists Clenbuteol And Salbutamol

β-Agonists, including clenbuterol (CBL) and salbutamol (SAL), are banned in the livestock industry due to their toxic effects on consumer health. For the detection of P-agonists, a two-stage control program using enzyme linked immunosorbent assay (ELISA) for screening and GC-MS for conformation is applied. ELISA is particularly suitable for the fast screening of large quantities of samples owing to its timesaving analysis, high sensitivity and simple sample preparation.The aim of this study was to establish immunoassays of CBL and SAL, which were based on the preparation of their corresponding polyclonal antibodies (PcAbs), and to explore the possibility of developing a new solid-phase carrier for the detection of P-agonists by comparing the assay results obtained from magnetic particles and conventional microtitre plates as carriers respectively.CBL was conjugated with BSA and OVA respectively via diazotized method. Antiserum raised in rabbits by immunization against CBL-BSA complex was preliminarily purified by the precipitation of saturated ammonium sulfate (SAS) and then by the chromatographic separation of DEAE-cellulose anion exchange column. Further purification was made to remove the antibodies against protein carrier BSA by reacting with the BSA immobilized on the surface of GoldMag particles, and the purified anti-CBL antibodies was left in the liquid phase after magnetic separation. Preliminary research was performed for the indirect competitive enzyme immunoassay (IC-EIA) of CBL based on the purified anti-CBL PcAbs. The linear range of the assay was between 1.5 ng/mL and 121.5 ng/mL, and IC50 (Intercept at 50%) was 22 ng/mL.SAL succinate was prepared through the alcoholysis reaction between SAL free base and succinic anhydride. Then SAL succinate was conjugated with BSA and OVA via mixed anhydride method. Different from anti-CBL antiserum, the titer against protein carrier BSA was much lower than that against SAL in anti-SAL antiserumraised in rabbits by immunization against SAL-BSA complex. Therefore purified anti-SAL PcAbs could be simply prepared by the precipitation of SAS and then by the chromatographic separation of DEAE-cellulose anion exchange column.Using microtitre plates as carriers, IC-EIA of SAL was established based on the purified anti-SAL PcAbs. The linear range of the assay was between 0.5 ng/mL and 10 ng/mL, and IC50 was 5.29 ng/mL. CBL had a relatively high cross reactivity of 39.6% for anti-SAL PcAbs, causing the linear range of the assay for CBL was between 0.5 ng/mL and 50 ng/mL, and IC50 was 13.37 ng/mL. Using magnetic particles as carriers, a one-step IC-EIA of SAL was established based on the commercialized anti-SAL monoclonal antibodies. The whole assay could be finished in two hours and the linear range of the assay was between 0.5 ng/mL and 100 ng/mL, and IC50 was 11.19 ng/mL. Average recoveries for different porcine samples ranged from 84.3% to 118.6%. Compared with microtitre plates, magnetic particles as earners possess the advantages of broader assay range and shorter assay time.