Chapter 14
Particle Concentration Fluorescence Assays for Rapid Detection of Trace Levels of Antibiotics Downloaded by PENNSYLVANIA STATE UNIV on September 10, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0636.ch014
Marjorie B. Medina Eastern Regional Center, Agricultural Research Service, U.S. Department of Agriculture, 600 E. Mermaid Lane, Philadelphia, PA 19118 New approaches for detection of spectinomycin and penicillin G are presented in these studies. An aminoglycoside-binding protein (ABP) immobilized on polystyrene latex particles and spectinomycin labeled with fluorescein isothiocyanate (FITC) were utilized for the spectinomycin assay. A penicillin binding protein (PBP) labeled with FITC, and a betalactam covalently bound to particles were used for detection of penicillin G. The antibiotics in the samples were pre-incubated with the binding proteins prior to addition of spectinomycin-FITC or betalactam-particles. The excess reagents were drained and after washing the particles, the fluorescent labeled compounds captured by the particles were measured. The assays were designed for detection of spectinomycin at 0-50 ppb (parts per billion) and 0-25 ppb for penicillin G. These techniques can provide rapid and sensitive biochemical methods to detect antibiotics in foods of animal origin. Rapid methods are needed to screen for the presence of trace levels of specific antibiotics or a class of antibiotics in biological fluids and tissues. Enzyme immunoassay techniques have been used only to a limited extent for detection of veterinary drugs due to lack of sensitivity needed for detection at action levels. The high specificity of immunoassays also limits the number of compounds that can be analyzed compared to a broad spectrum detection obtainable using microbial inhibition assays. Production of antibodies with desired specificities and affinities is time consuming and requires specialized facilities not available to most analysts. Due to these difficulties, the use of proteins with binding properties to antibiotics were explored and characterized. The binding proteins used in this research had been utilized for affinity chromatography (7), a rapid assay (2) and for screening of betalactams using an enzyme tracer in SNAP test (Idexx Laboratories). In general, the sensitivity of assays can be improved by increasing the surface area for the capture molecules and by improving the signal generating tracer. The use of latex particles provide a larger This chapter not subject to U.S. copyright Published 19% American Chemical Society
In Veterinary Drug Residues; Moats, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
Downloaded by PENNSYLVANIA STATE UNIV on September 10, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0636.ch014
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Particle Concentration Fluorescence Assays for Antibiotics
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surface area than microliter wells. In addition, the capture molecule can be covalently attached to latex particles in contrast to passive adsorption onto microliter wells. Higher signals are generated by fluorescence labels than chromophores generated by enzymes and thefluorescentsignals are measured directly, thus, eliminating the development step necessary for enzyme labels. In addition, high sensitivity fluorescence detectors for microliter wells have recently become available. There are very few methods for the analysis of spectinomycin in foods. Chromatographic methods utilizing an ion pair solid phase extraction, HPLC separation followed by post column derivatization and fluorescent detection of its 2-naphthalenesulfonyl chloride (NSC1) derivatives were reported for quantification of spectinomycin. The sensitivity of the liquid chromtographic method was 4 ng per sample load (3) and 50 ppb spectinomycin was detectable in swine and chicken plasma (4). The official method (5) for detection of spectinomycin is a microbial turbidimetric assay (
