An electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of Clostridium botulinum outbreak strains associated with type A botulism.

BACKGROUND: Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products. RESULTS: The limit of detection of this ECL assay is 40 pg mL⁻¹ for BoNT/A complex, 10 pg mL⁻¹ for SEB and 40000 CFU mL⁻¹ for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. CONCLUSION: The results of the study indicate that this ECL assay is very sensitive, rapid (<6 h) and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays.

Detection and confirmation of Clostridium botulinum in water used for cooling at a plant producing low-acid canned foods.

Our laboratory tested water samples used for cooling low-acid canned foods at a canning facility under investigation by the U.S. Food and Drug Administration. We used an enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies (DIG-ELISA) and real-time PCR as screening methods and confirmed the presence of neurotoxin-producing Clostridium botulinum in the samples by mouse bioassay.

Structural differences of proteins between solution state and solid state probed by attenuated total reflection Fourier transform infrared spectroscopy.

A Fourier transform infrared (FT-IR) spectroscopic method combined with an attenuated total reflection (ATR) sampling technique has been developed to analyze protein secondary structure in both solid and solution states. The method has been applied to analyze the protein structural differences between solution state and solid state. For alpha-helix dominant proteins, beta-sheet structures increase significantly in the solid state, with significant decrease in alpha-helical structures. For beta-sheet dominant proteins, beta-sheet structures increase only moderately in the solid state. When proteins are re-dissolved in solution, their structures are re-natured to their native structures, as suggested by the fact that their structures in solution state are similar to those determined by X-ray crystallography or other spectroscopic methods in solution state. The ATR sampling technique avoids the high pressure and chemicals that are needed for the conventional potassium bromide (KBr) disc method for solid samples in FT-IR spectroscopy. Our approach from this study demonstrated that ATR sampling is more appropriate for analysis of protein structures in the solid state.

Evaluation of an enzyme-linked immunosorbent assay (ELISA) kit for the detection of botulinum neurotoxins A, B, E, and F in selected food matrices.

The mouse bioassay (MBA) is the only accepted standard method for detection of botulinum neurotoxins (BoNTs) in foods. The ELISA method has several advantages over the MBA and is therefore widely used for in vitro detection of BoNTs. The US Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) conducted a precollaborative study to evaluate the applicability of Botulinum Toxin ELISA kits for the detection of BoNT serotypes A, B, E, and F in a variety of food matrices. In this study, food samples (e.g., broccoli, salami, smoked salmon, green beans, orange juice, tomato juice, low-fat plain yogurt, whole milk, liquid infant formula milk, and liquid eggs) were spiked with high, medium, and low concentration BoNT serotypes A, B, E, and F. Samples (unspiked and spiked) were tested at both laboratories by the ELISA kits. All food samples were positive for BoNTs by ELISA in both laboratories at medium and high spiking levels; a positive ELISA result in low spiked samples was both serotype and laboratory dependent. Overall, the ELISA method appears to be an effective preliminary screening method for BoNT detection in food matrices.

Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium Botulinum Strain PS-5.

Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography-Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.

Simultaneous determination of creatinine and uric acid in human urine by high-performance liquid chromatography.

An environmentally friendly reversed-phase HPLC method for simultaneous determination of creatinine and uric acid in human urine samples has been developed. Human urine samples were pretreated by dilution, protein precipitation, centrifugation and filtration, followed by HPLC separations using a reversed-phase C(18) column with an aqueous mobile phase of phosphate buffer. The retention loss of a C(18) column when using the highly aqueous mobile phases was avoided by employing a gradient elution using a small volume (<0.23 mL) of acetonitrile and phosphate buffer at pH 4.75. This developed method provides a simple, rapid separation and sensitive detection for the species of interest in 10 min with UV detection at 205 nm. Quantitation was carried out by relating the peak areas of the identified compounds to that of hypoxanthine as an internal standard. The detection limits for creatinine and uric acid were 0.045 and 0.062 microg mL(-1), respectively. The recoveries of the standards added to urine samples were 87.3 - 102.2% for creatinine and 97.3 - 108.6% for uric acid, and the relative standard deviation for both analytes was less than 1.0%. This method has been successfully applied to estimating of creatinine and uric acid in human urine.