Enzyme immunoassays for the analysis of streptomycin in milk, serum and water: development and assessment of a polyclonal antiserum and assay procedures using novel streptomycin derivatives

Abstract

A polyclonal antiserum to streptomycin was generated in sheep using a streptomycin-bovine serum albumin conjugate as immunogen. Streptomycin was linked to the carrier protein with cyanuric chloride using a new two-step conjugation method. Plate coating antigen conjugates of streptomycin and gelatine were prepared using either cyanuric chloride (homologous bridge) or 1,4-butanediol diglycidyl ether to provide an heterologous complex. The reagents enabled the generation of a specific antiserum with a titre of 1/40,000 and the development of a sensitive ELISA method suitable for the measurement of streptomycin sulfate in milk, serum and water samples. A minimum detection value of 1 ng mL(-1) and a dynamic range of 1 to 200 ng mL(-1) were demonstrated in the three matrices. No detectable cross reactivity with any of the common aminoglycosides was found except the related dihydrostreptomycin which gave a 75% cross reaction value. The details of the preparation of the hapten-protein conjugates, characterisation of the antiserum and assay construction and assessment methods are presented. The introduction of new coupling methods and antibody assessment provide improved basic methodologies necessary for the advancement of immuno-analysis of streptomycin, one of the most widely used antimicrobial substances.