46I-11 |
Development of a fluorescent latex immunoassay for detection of spectinomycin antibiotic |
M. B. MEDINA, USDA-ARS-Eastern Regional Research Center, 600 E. Mermaid Ln., Wyndmoor, PA 19038-8598 Spectinomycin is an aminocyclitol antibacterial agent used to treat infections caused by Gram negative and Gram postiive microorganisms in poultry and swine production. There is a need to develop a rapid and sensitive method to detect spectinomycin residues in animal tissues. A competitive immunoassay was designed using reagents specially developed for this assay. Sheep polyclonal spectinomycin antibody was produced against spectinomycin conjugated to keyhole limpet hemocyanin (KLH). The spectinomycin immunoglobulin (IgG) was purified through Protein G affinity column and was immobilized onto latex particles. The spectinomycin was labeled with 5-([4,6-dichlorotriazin-2-YL]amino)-fluorescein (DTAF). The optimum assay conditions consisted of pre-incubating the latex-IgG with spectinomycin standards and/or spectinomycin spiked in bovine kidney extracts for 15 min at room temperature (RT). The DTAF labeled spectinomycin was added to compete for the antibody binding sites and the mixture was further incubated for 20 min at RT. The bound spectinocymin-DTAF-IgG-latex complex was separated by centrifugation at 4000 x g for 10 min. Aliquots of the supernate containing the unbound spectinomycin-DTAF were transferred to microtiter wells and the fluorescence signals were measured at 485 nm excitation and 535 nm emission. The signals were inversely proportional to the concentration of spectinomycin in the samples. The immunocompetive assays detected spectinomycin at 0 to 100 ppb with minimum detectability of 5 ppb. Plotting the % bound complex against the spectinomycin concentration, the regression correlation values (R^2) were 0.976, 0.938, 0.974, 0.859 (mean=0.936). Detection of spectinomycin spiked in kidney extract had a lower R^2 (0.816) which suggests interferences in the tissue extract and refinement of the tissue sample preparation is needed. This assay is more sensitive and rapid than traditional microbiological and chromatographic methods for the detection of spectinomycin.
Session 46I, Toxicology & Safety Evaluation
|