Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Quantitative detection of staphylococcal enterotoxin B by resonant acoustic profiling.

A rapid and sensitive detection of staphylococcal enterotoxin B (SEB) was developed using a novel acoustic sensing technique: Resonant Acoustic Profiling (RAP), which utilizes high-frequency piezoelectric quartz resonators for monitoring biomolecular interactions. An automated four-channel instrument consisting of acoustic sensors covalently conjugated with anti-SEB antibodies was used. As the samples flowed across control and active sensors simultaneously, binding was measured as a change in the resonant frequency. The lower limit of detection (LLOD) for the label free direct format was 25 ng/mL. Detection sensitivity was increased by adding mass sequentially to the captured SEB on the sensor in the form of sandwich antibodies and biotin-avidin-based gold nanoparticles. The LLOD for the mass enhanced formats were 5 and 0.5 ng/mL of SEB, respectively. The lowest sensitivity corresponds to 1.3 fM in a 75 microL sample. The total assay time including the enhancement steps was less than 10 min. SEB was detected in both neat urine and PBS buffer-spiked samples, with linear correlations between resonant frequency signals and SEB concentrations (R(2) of 0.999 and 0.998, respectively). No significant cross-reactivity was observed with homologue toxins SEA, SED, and TSST, but some cross-reactivity was observed with the closely related toxin SEC(1) when we used a polyclonal antibody in the assay. SEC(1) cross-reactivity was not observed when a SEB-specific monoclonal antibody was employed in the assay. Thus the specificity of the assay presented here was dependent on the quality of the antibodies used. In addition to detection, we evaluated RAP's ability to measure the toxin in unknown samples rapidly by measuring the initial binding rate of the interaction, thereby further shortening the assay time to 6 min.

Identification of the RNA N-glycosidase activity of ricin in castor bean extracts by an electrochemiluminescence-based assay.

A simple electrochemiluminescence-based assay for RNA N-glycosidase activity has been modified to permit its use with authentic extracts of Ricinus communis (castor beans) and Abrus precatorius (jequirity seeds)--the natural sources of ricin and abrin. Modifications include the addition of an RNase inactivator to the reaction mixture, elimination of a signal-enhancing monoclonal antibody, and optimization of the incubation temperature. Concurrent testing with two substrates provides a diagnostic tool enabling castor bean toxins to be differentiated from a larger selection of N-glycosidase toxins than was previously examined.

An electrochemiluminescence-based competitive displacement immunoassay for the type-2 brevetoxins in oyster extracts.

A new competitive electrochemiluminescence-based immunoassay for the type-2 brevetoxins in oyster extracts was developed. The assay was verified by spiking known amounts of PbTx-3 into 80% methanol extracts of Gulf Coast oysters. We also provide preliminary data demonstrating that 100% acetone extracts, aqueous homogenates, and the clinical matrixes urine and serum can also be analyzed without significant matrix interferences. The assay offers the advantages of speed ( 2 h analysis time); simplicity (only 2 additions, one incubation period, and no wash steps before analysis); low limit of quantitation (conservatively, 50 pg/mL = 1 ng/g tissue equivalents); and a stable, nonradioactive label. Due to the variety of brevetoxin metabolites present and the lack of certified reference standards for liquid chromatography-mass spectrometry confirmation, a true validation of brevetoxins in shellfish extracts is not possible at this time. However, our assay correlated well with another brevetoxin immunoassay currently in use in the United States. We believe this assay could be useful as a regulatory screening tool and could support pharmacokinetic studies in animals and clinical evaluation of neurotoxic shellfish poisoning victims.

A quantitative electrochemiluminescence assay for Clostridium perfringens alpha toxin.

Described is a rapid direct sandwich format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru)-labeled detection antibodies. The ruthenium chelate of detection antibodies chemically reacted in the presence of tripropylamine and upon electronic stimulation emitted photons (electrochemiluminescence) that were detected by the photodiode of the detector. Elevated toxin concentrations increased toxin immunoadsorption and the specific immunoadsorption of Ru-labeled antibodies to alpha toxin, which resulted in increased dose-dependent electrochemiluminescent signals. The standardized assay was rapid (single 2.5-h coincubation of all reagents), required no wash steps, and had a sensitivity of about 1 ng/ml of toxin. The assay had excellent accuracy and precision and was validated in buffer, serum, and urine with no apparent matrix effects.

An activity-dependent assay for ricin and related RNA N-glycosidases based on electrochemiluminescence.

Synthetic biotinylated RNA substrates were cleaved by the combined actions of ricin holotoxin and a chemical agent, N,N'-dimethylethylenediamine. The annealing of the product with a ruthenylated oligodeoxynucleotide resulted in the capture of ruthenium chelate onto magnetic beads, enabling the electrochemiluminescence (ECL)-based detection of RNA N-glycosidase activities of toxins. ECL immunoassays and the activity assay exhibited similar limits of detection just below signals with 0.1 ng/ml of ricin; the ECL response was linear as the ricin concentration increased by two orders of magnitude. Activities were detected with other adenine-specific RNA N-glycosidases, including Ricinus communis agglutinin (RCA), saporin, and abrin II. The substrate that provided the greatest sensitivity was composed of a four-residue loop, GdAGA, in a hairpin structure. When the 2'-deoxyadenosine (dA) was substituted with adenosine (A), 2'-deoxyinosine, or 2'-deoxyuridine, toxin-dependent signals were abolished. Placing the GdAGA motif in a six-residue loop or replacing it with GdAdGA or GdAAA resulted in measurable activities and signal patterns that were reproducible for a given toxin. Data indicated that saporin and abrin II shared one pattern, while ricin and RCA shared a distinct pattern. A monoclonal antibody that enhanced the activities of ricin, RCA, and abrin II to different extents, thus improving the diagnostic potential of the assay, was identified .

Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection.

Sensitive and specific electrochemiluminescence (ECL) assays were used to detect Clostridium botulinum neurotoxins serotypes A, B, E, and F in undiluted human serum, undiluted human urine, assay buffer, and selected food matrices (whole milk, apple juice, ground beef, pastry, and raw eggs). These novel assays used paramagnetic bead-based electrochemiluminescent technology in which biotinylated serotype-specific antibodies were bound to streptavidin-coated paramagnetic beads. The beads acted as the solid support and captured analyte from solution. Electrochemiluminescent detection relied on the use of ruthenium chelate-labeled anti-serotype antibodies and analysis with a BioVeris M-Series M1R analyzer. The sensitivities of the assays in clinically relevant matrices were 50 pg/ml for serotypes A and E, 100 pg/ml for serotype B, and 400 pg/ml for serotype F. The detection limits in selected food matrices ranged from 50 pg/ml for serotype A to 50 to 100 pg/ml for serotypes B, E, and F. The antibodies used for capture and detection exhibited no cross-reactivity when tested with the other serotypes. When purified native toxin was compared with toxins complexed to neurotoxin-associated proteins, no significant differences in assay response were noted for serotypes A, B, and F. Interestingly, the native form of serotype E exhibited reduced signal and limit of detection compared with the complexed form of the protein. We suspect that this difference may be due to trypsin activation of this particular serotype. The assays described in this article demonstrate limits of detection similar in range to the gold standard mouse bioassay, but with greatly reduced time to data. These rapid sensitive assays may have potential use in clinical settings, research studies, and screening of food products for botulinum toxins.

Multi-Laboratory Study of Five Methods for the Determination of Brevetoxins in Shellfish Tissue Extracts.

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.

An enzymatic electrochemiluminescence assay for the lethal factor of anthrax.

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.

Sensitive and specific colorimetric ELISAs for Staphylococcus aureus enterotoxins A and B in urine and buffer.

We present here simple, sensitive and accurate colorimetric capture ELISAs for staphylococcal enterotoxins A and B. Standard curves were linear over the range 0.5-1 ng/mL, and toxins could be accurately measured at 0.5 ng/mL in assay buffer or 0.1 ng/mL in human urine. Cross-reactivity between serotypes was negligible. Detection in serum was complicated by the presence of specific antibodies to SE's in most normal sera. These assays offer a viable, cost-effective method for analysis of these ubiquitous toxins. Further, their sensitivity in undiluted urine makes them ideal candidates for evaluating human exposure.

Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F.

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.