Device for detecting neurotoxins and process for manufacture thereof
A neurotoxin and in vitro detection technology, applied in the monitoring of freshwater reservoirs and seafood monitoring, can solve problems such as expensive, low detection sensitivity, and complexity
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Embodiment 1
[0170] Example 1: Method of Manufacturing a Device for Detecting Neurotoxins
[0171] In the following examples, the products and equipment used are as follows:
[0172] Biotinylated α-bungarotoxin (Molecular Probes, Invitrogen, USA)
[0173] Alkaline phosphatase conjugated to streptavidin (Streptavidin-AP, Promega, Madison, WI, USA)
[0174] α-Bungarotoxin (Sigma-Aldrich, USA)
[0175] TBS buffer (150mM NaCl, 50mM Tris-HCl, pH 7.5)
[0176] TBS-BSA buffer (150mM NaCl, 50mM Tris-HCl, 0.5% BSA, pH 7.5)
[0177] Western Blue (registered trademark) (Promega, WI, USA)
[0178] Filter paper glass fiber GF / C (Whatman, Maidstone Kent, UK)
[0179] Filter paper cellulose (1 mm thick, Whatman, UK)
[0180] headband double sided tape
[0181] Toxin standards: Toxoid-a (Tocris), 13-desmethylspironolactone C (NRC-IRAP Halifax, NS, Canada), α-bungarotoxin (Sigma-Aldrich, USA), in Professor Armen Zakarian ray toxin-G (University of California, Santa Barbara, California, USA) and 13,...
Embodiment 2
[0193] Example 2: Detection of neurotoxins using a device according to the invention
[0194] The device used was that manufactured according to Example 1 above.
[0195] In this example, several experiments were carried out, experiments without any toxin as control experiments and experiments with toxins ie α-bungarotoxin, spironolactone, ray toxin, toxoid-a.
[0196] a) Control experiment:
[0197] 150 μL of TBS-BSA (50 mM Tris-HCl, 150 mM NaCl, 0.5% bovine serum albumin, pH 7.5) was added to the sample well.
[0198] Then add 150 μL for alkaline phosphatase Western (Promega).
[0199] The results obtained demonstrate that the "control" line is positive, which means and demonstrates that in the absence of toxin, streptavidin conjugated to alkaline phosphatase has been complexed with [nicotinic acetylcholine receptor-α- Bungarotoxin-Biotin] binds to the biotin group and is immobilized on the "control" thread. Hydrolysis of the Western Blue (registered trademark) substra...
Embodiment 3
[0208] Example 3: Detection of neurotoxins with a device using two test lines
[0209] The device used was manufactured according to Example 1 above, except that two test lines were used instead of one test line and one control line. The control line is used to test whether the control strip is working properly. The inventors controlled that all lateral flow test devices functioned well and performed multiple tests without toxin as a control (Ctrl) that the device was working as intended (Figure 4).
[0210] In this example, several experiments were performed using different concentrations of different toxins. With the two test lines specified, we tested the potency of the toxin at a given concentration.
[0211] In particular, different doses of the snake toxin α-bungarotoxin ( Figure 4A ). For this purpose, prepare 1 mL of α-bungarotoxin at a concentration of 10 μM from commercially available α-bungarotoxin (Sigma; 250 μM) using TBS-BSA buffer (150 mM NaCl, 50 mM Tris-H...
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