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Abstract: Development of a microsphere-based competitive fluorescence immunoassay for the determination of hazardous low-molecular-weight compounds in food is described. In this method, antigens are covalently bound to carboxy-modified microspheres to compete monoclonal antibody with low-molecular-weight compounds in food samples; mouse IgG/fluorescein isothiocyanate conjugate is used as the fluorescent molecular probe. Thus, the hazardous low-molecular-weight compounds are quantified using a multiparameter flow cytometer. This method has been evaluated using clenbuterol as a model compound. It has a sensitivity of 0.01ng/mL with dynamic range of 0.01–100ng/mL, and the concentration of clenbuterol providing 50% inhibition (IC50) is 1.1ng/mL. The main advantages of this method are its high efficiency, biocompatibility, and selectivity, as well as ultralow trace sample consumption and low cost. [Copyright &y& Elsevier]