Rapid and Sensitive Nano-Immunosensors for Botulinum.

Botulinum is a deadly bacterial toxin that causes neuroparalytic disease. However, appropriate tools to detect trace toxic proteins are scarce. This study presents a bead-based diffusometric technique for the rapid, simple, and quantitative detection of biological toxins. Functionalized particles called nano-immunosensors were fabricated by forming sandwiched immunocomplexes comprising Au nanoparticles (AuNPs), toxic proteins, and antibodies on fluorescent probe particles. Particle diffusivity tended to decline with increasing concentration of the target proteins. Calibration curves of purified botulinum toxins (0.01-500 ng/mL) were obtained from whole milk and bovine serum, and results suggested that measurement was independent of the background matrix. The activity of botulinum toxin was evaluated by coating synaptosomal-associated protein 25 (SNAP-25) on fluorescent probe particles. AuNP-conjugated antibodies attached to the probe particles when SNAP-25 proteins were cleaved by active botulinum. Thus, toxicity could be detected from slight changes in diffusivity. A short measurement time of 2 min and a limit of detection of 10 pg/mL were achieved. The nano-immunosensors demonstrated rapid biosensing capability and met the demands of onsite screening for food safety, medical instrument hygiene, and cosmetic surgery products.

Immuno-detection of cleaved SNAP-25 from differentiated mouse embryonic stem cells provides a sensitive assay for determination of botulinum A toxin and antitoxin potency.

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products.

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E.

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

A Highly Specific Monoclonal Antibody for Botulinum Neurotoxin Type A-Cleaved SNAP25.

Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A's location, distribution, and movement within a cell. Due to BoNT/A's high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25197) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25197, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25197 rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25197 in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25197-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.

Acute treatment with diphenyl diselenide inhibits glutamate uptake into rat hippocampal slices and modifies glutamate transporters, SNAP-25, and GFAP immunocontent.

Diphenyl diselenide (PhSe)(2) is a selenium organic compound that has been described to inhibit glutamate binding at synaptic membranes and uptake into cortical slices, but there are no studies about its effects on glutamate transporters and related synaptic proteins. Hippocampal slices from rats treated acutely with (PhSe)(2) (1, 10, and 100 mg/kg, oral route) were evaluated on glutamate uptake, redox state, the immunocontent of glial (glutamate/aspartate transporter [GLAST] and glutamate transporter type I [GLT1]), neuronal (excitatory amino acid carrier 1 [EAAC1]), and vesicular (vesicular glutamate transporter 1 [VGLUT1]) glutamate transporters. Besides, cell viability was evaluated by glial fibrillar acid protein (GFAP) and synaptosomal-associated protein 25 (SNAP-25) immunocontent and 4', 6-diamidino-2-phenylindole (DAPI) and Fluoro Jade C staining. Hippocampal slices from rats treated with (PhSe)(2) exhibited a nondose-dependent inhibition of glutamate uptake (53, 38, and 45%, respectively). All doses increased EAAC1, decreased SNAP-25, did not modify GLT1 immunocontent, and there was no evidence of oxidative stress. (PhSe)(2) (100 mg/kg) increased 32% GLAST, decreased 34% VGLUT1, and 21% GFAP immunocontent. Besides, (PhSe)(2) (100 mg/kg) decreased by 25% GFAP-stained astrocytes and 27% DAPI-stained cells in the CA1 subfield. Our results suggest that the increase of EAAC1 and GLAST immunocontent by (PhSe)(2) might be a compensatory mechanism by surviving cells in order to reduce extracellular glutamate levels, avoiding possible neurotoxic effects. The impairment of glutamate uptake by the highest dose of (PhSe)(2) seems to be related to a decrease on VGLUT1, SNAP-25, and damage to astrocytes. Since there were no signs of oxidative stress, our findings revealed that depending on the dose, acute administration of (PhSe)(2) causes modifications in important synaptic-related proteins and damage to the astrocytes, and these events must be taken into account in its pharmacological properties.

N-terminal acetylation of the neuronal protein SNAP-25 is revealed by the SMI81 monoclonal antibody.

The monoclonal antibody SMI81 binds SNAP-25, a major player in neurotransmitter release, with high affinity and has previously been used to follow changes in the levels of this protein in neuropsychiatric disorders. We report here that the SMI81 epitope is present at the extreme N-terminus of SNAP-25 and, unusually, cannot be recognized when present as an internal sequence. Although it is known that SNAP-25 can be palmitoylated and phosphorylated in brain, we now reveal the existence of a third modification, acetylation of the N-terminus. This acetylation event greatly increases the efficiency of SMI81 antibody binding. We show that this highly specific antibody can be used for studying brain function in many vertebrate organisms.

Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, E and C1 intra-cellularly cleave SNAP25 resulting in a flaccid paralysis. As a consequence, various different endopeptidase assays have been developed to specifically detect the toxins enzymatic activity, however, many of these suffer from variability, low sensitivity or unwanted interference exerted by product specific excipients. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and specific antibody to either the SNAP25(190-197) or (173-180) octapeptide epitopes that become exposed following cleavage by toxin types A or E respectively. Assay sensitivity was increased 50 fold by the use of an optimal 0.5% Tween 20 concentration in tandem to 0.1% albumin together with an improved, simplified assay design without a pre-activation / reduction step. Sensitivities capable of detecting 0.01 LD50/ml (40fg/ml or 0.3fM) of type A toxin was achieved with a linear dose response between 0.1 and 1 LD50/ml. This provides sufficient sensitivity and precision (inter assay GCV of < 2%) for monitoring activity within any current or newly marketed therapeutic products containing less units per vial and may also make it applicable for other applications. Both purified haemagglutinin free and complexed toxins could be detected equally. Unlike type A, type E activity could unexpectedly be detected in the complete absence of reducing conditions and the optimal assay had a limit of detection of 0.2LD50/ml (4.8pg/ml) with a linear dose response between 1 and 10LD50/ml. The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to other specific enzyme cleavage reactions in the future.

Galphao2 regulates vesicular glutamate transporter activity by changing its chloride dependence.

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by alpha subunits of trimeric G-proteins, including Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.