Flow cytometric immunoassay for sulfonamides in raw milk

Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this automated FCI, a previously developed biotinylated multi-sulfonamide mutant antibody (M.3.4) was applied in combination with fluorescent beads, directly coated with a sulfathiazole derivative, and streptavidin–phycoerythrin (SAPE) for the detection. With this FCI, at least 11 different sulfonamides could be detected (more than 50% inhibition at the 100 ng mL⁻¹ level) and, after an incubation of 1 h, measurements were rapid (10 s per sample). For the application with raw milk, a 96-well microplate-based filtration step was included into the protocol to remove disturbing milk fat particles. Because of differences in sensitivity towards different sulfonamides, the FCI was considered and validated as a qualitative screening assay. For sulfadoxine, the most applied sulfonamide in Dutch dairy cattle, the detection capability (CCβ) was <50 μg L⁻¹ and this level seems feasible for five other sulfonamides. For sulfadiazine, the CCβ was <200 μg L⁻¹ and this level seems feasible for four other sulfonamides. A major advantage of the applied xMAP-technology, with its 100 different color-coded bead sets, is the possibility to develop multiplex immunoassays for the simultaneous detection of several antibiotics.     

Simultaneous Screening for Five Veterinary Drug Residues by a Developed Multiplexed Luminex Assay

Abstract

The development of sensitive and rapid methods for simultaneous determination of multiple residual veterinary drugs in foods is increasingly important. In this study we reported a new method to simultaneously detect five veterinary drug residues, including ractopamine, clenbuterol, furazolidone, chlortetracycline and qlaquindox. The experimental results indicated that the developed multiplex assay enable to detect all the five drugs at the concentration of 0.1 ng/mL, with desirable specificity, suggesting that it could be applicable for rapid screening of five residual veterinary drugs in a faster turnaround time and higher throughput.     

Development of a sensitivity enhanced multiplexed fluorescence covalent microbead immunosorbent assay (FCMIA) for the measurement of glyphosate,atrazine and metolachlor mercapturate in water and urine

Body burdens from exposures to pesticides may be estimated from urinary analyses of pesticide parent/metabolite concentrations. Pesticide applicators and others are often exposed to numerous unrelated pesticides, either sequentially or simultaneously. Classically, body burdens of pesticides are analyzed using chemical/instrumental analysis (CIM) or enzyme immunoassays (EIAs). Both of these technologies can usually be used to quantitate one analyte (or closely related groups of analytes) per analysis. Alternatively, multiple analytes can be measured simultaneously using a multiplexed fluorescence covalent microbead immunoassay (FCMIA). We developed a multiplexed FCMIA to simultaneously measure glyphosate (Gly), atrazine (Atz), and metolachlor mercapturate (MM) in water and urine. The assay had least detectable doses (LDDs) in water/diluted urine of 0.11/0.09 ng/ml (Gly, water/urine LDD), 0.10/0.07 ng/ml (Atz), and 0.09/0.03 ng/ml (MM). The sensitivity for the measurement of Gly was enhanced by derivatization. All assays gave linear responses from the LDDs for each respective pesticide to 300 ng/ml. There was no cross-reactivity between the three analytes. Using a 96-well microplate and an autosampler, as many as 288 separate analyses can be completed in ~120 min with precision, sensitivity, and specificity equivalent to, if not better, than that found when these same analytes are measured by CIM or EIA.     

Acoustic trapping as a generic non-contact incubation site for multiplex bead-based assays

In this study, we show a significantly reduced assay time and a greatly increased bead recovery for a commercial Luminex-based multiplex diagnostic immunoassay by performing all liquid handling steps of the assay protocol in a non-contact acoustic trapping platform.     

Improving the Sensitivity of Fluorescent Microsphere Immunoassays for the Simultaneous Detection of Foodborne Pathogens Using Dimethylacetamide

The liquid-chip assay is a new method for detecting bacterial surface antigens. This assay conjugated self-prepared monoclonal antibodies against Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica serovar Typhi with carboxylated fluorescent microspheres based on the double-antibody sandwich principle. This experiment used dimethylacetamide (DMAC) as the solvent to dissolve 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (S-NHS) during conjugation. The modified liquid-chip assay was used to simultaneously detect the four foodborne pathogens. The sensitivity of the assay using the new conjugation method was also evaluated. The limits of detection for E. coli O157:H7, L. monocytogenes, S. aureus, and S. typhi during multiplex detection using the improved method were 0.25, 0.25, 0.5, and 0.25 cfu/mL, respectively, whereas those using the traditional method were 0.5, 0.5, 1, and 0.5 cfu/mL, respectively. Therefore, the improved method is reliable and effectively improves the detection sensitivity of liquid-chip assays.     

Bead-Based Fluid Array Detection of Pentaerythritol Tetranitrate: Comparison of Monoclonal vs. Llama Polyclonal Antibodies

In today's security conscious environment rapid detection of explosives, such as pentaerythritol tetranitrate (PETN), the high energy material common in plastic explosives, is of critical importance. We evaluated two monoclonal antibodies and a rabbit polyclonal antibody, developed for the detection of PETN. Although these antibodies showed binding to bioconjugates of PETN as well as the hapten, PETN-succinate, we did not observe binding by free PETN. For demonstration of a competitive bead based fluid array immunoassay for PETN detection, we developed llama polyclonal antibody for the detection of free PETN. Our ultimate goal is developing anti-PETN single domain antibodies.