Development of a Fluorescent Latex Immunoassay for Detection of a Spectinomycin Antibiotic

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Abstract
There is a need to develop a rapid and sensitive method to detect spectinomycin residues in animal tissues. A latex fluorescent immunoassay was designed using reagents developed for this assay. The spectinomycin antibody was produced in sheep, and the immunoglobulin (IgG) was purified through a Protein G affinity column and was immobilized onto latex particles. Spectinomycin was labeled with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF). The optimum assay conditions consisted of preincubating the latex−IgG with spectinomycin in buffer solutions or in bovine kidney extracts. DTAF−spectinomycin was added and was further incubated. The bound spectinomycin−DTAF/IgG−latex complex was separated by centrifugation at 4000g for 10 min. The fluorescence signals of the unbound spectinomycin−DTAF in the supernatant were measured at 485/535 nm excitation/emission. The measured signals were directly proportional to the concentration of spectinomycin in the samples, and spectinomycin was detected at 0−100 ppb with minimum detectability of 5 ppb. The mean regression correlation of four trials in buffer was 0.936 when the % bound complex vs spectinomycin concentration was plotted. Analysis of the kidney extract spiked with 0−100 ppb spectinomycin had a regression correlation of 0.959. This assay provides a rapid screening method for low ppb detection of spectinomycin. Keywords: Spectinomycin; fluorescent immunoassay; latex immunoassay

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