Development of a fluorescent latex immunoassay for detection of a spectinomycin antibiotic.
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 4000 g 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.
Medina
Marjorie B
MB
Microbial Biophysics and Residue Chemistry and Core Technologies Unit, Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA. mmedina@arserrc.gov
D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis*; D002417:Cattle; D015200:Fluoroimmunoassay / Q000379:methods*; D007668:Kidney / Q000737:chemistry; D007840:Latex; D000198:Spectinomycin / Q000032:analysis*
D000900:Anti-Bacterial Agents; D007840:Latex; D000198:Spectinomycin