Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/3/2003
Publication Date: 3/4/2004
Citation: Medina, M.B. 2004. Development of a fluorescent latex immunoassay for detection of spectinomycin antiobiotic. Journal of Agricultural and Food Chemistry. p. 3231-3236. Interpretive Summary: Veterinary antibiotics are used to protect our food from animal diseases. Spectinomycin is used to treat infections caused by Gram negative and positive microorganisms in poultry and swine production. However, improper use of veterinary drugs can leave chemical residues in food products. Laboratories of the regulatory agencies need rapid methods to detect trace amounts of drugs. We developed a method utilizing antibodies as "probes" to capture and detect spectinomycin residue in low parts per billion. In this study, we produced a specific spectinomycin antibody and attached a fluorescent signaling compound to the antibiotic for the development of a sensitive and effective assay. Utilizing this assay, spectinomycin added to kidney tissue extract was detected in low parts per billion. Forty samples in duplicate can be analyzed in 1.5 hours compared to existing techniques which have less sensitivity and will take hours and days to analyze the same number of samples. This method can be used to detect the spectinomycin compound in meat and poultry currently not identified by the FSIS official methods using the Seven Plate assay or the FAST screening test. Reliable methods which can detect illegal drug residues can prevent animal foods with forbidden chemical residues from entering the market, thus, assuring the consumers with a safe food supply.
Technical Abstract: Spectinomycin is an antimicrobial agent used to treat infections caused by Gram negative and positive microorganisms in poultry, swine and non-lactating cattle. 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. Sheep polyclonal 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 signaling molecule was spectinomycin 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 or spectinomycin spiked in bovine kidney extracts for 15 min. at room temperature. DTAF-spectinomycin was added and a second incubation for 20 min. at room temperature followed. The bound spectinomycin-DTAF-IgG-latex complex was separated by centrifugation at 4000 x g for 10 min. The fluorescence signals of the supernatant containing the unbound spectinomycin-DTAF were measured at 485 nm excitation and 535 nm emission. The signals were directly proportional to the concentration of spectinomycin in the samples. This immunoassay detected spectinomycin at 0 to 100 ppb (parts per billion) with minimum detectability of 5 ppb. The mean regression correlation of four trials 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 R^2 0.959. This assay provides a rapid screening method for low ppb detection of spectinomycin.