Method for manufacturing an analysis substrate, and use thereof for detecting toxins
10073089 ยท 2018-09-11
Assignee
Inventors
- Romulo Araoz (Gif-sur-Yvette, FR)
- Hoang-Oanh Nghiem (Paris, FR)
- Jordi MOLGO (ANTONY, FR)
- Luis Botana (Lugo, ES)
- Natalia Vilarino (Lugo, ES)
Cpc classification
C40B40/10
CHEMISTRY; METALLURGY
G01N33/543
PHYSICS
G01N33/554
PHYSICS
International classification
G01N33/543
PHYSICS
G01N33/554
PHYSICS
G01N33/50
PHYSICS
G01N33/53
PHYSICS
C40B40/10
CHEMISTRY; METALLURGY
Abstract
The prsent invention relates to a method for manufacturing an analysis device including torpdo membrane fragments immobilized at the surface thereof, to the resulting analysis device, and to the use of said device for detecting, purifying, and characterizing molcules acting on nicotinic acetylcholine receptors. The prsent invention is useful in the field of monitoring seafood, monitoring neurotoxic phytoplankton for the shellfish industry, monitoring the quality of bathing waters along tourist beaches, the field of monitoring fresh water reserves, the field of mdical research, the field of the biological analysis and characterization of molcules, e.g., non-radioactive assays of the movement of the ligand-receptor bond on an ELISA-type microplate, thereby enabling comptitive agonists and antagonists of targets to be detected, e.g., of highly sensitive receptors.
Claims
1. A method for manufacturing an analysis device for detection, quantification, and physicochemical identification of neurotoxins, the analysis device comprising Torpedo membrane fragments immobilized at the surface thereof, the method comprising the steps of: a. isolating Torpedo electrocyte cell membranes, b. fragmenting said isolated membranes, c. incorporating the membrane fragments obtained in step b into a solution with a pH from 6.5 to 10 and ionic strength from 0.1 mol/L to 0.7 mol/L, d. attaching said membrane fragments to the surface of the device by non-covalent and non-ionic interaction between the membrane fragments and the surface, wherein the surface of the device is made of plastic, and wherein the membrane fragments are not chemically modified before attaching to the surface.
2. The method according to claim 1, wherein the method comprises, before the step d of immobilizing the membrane fragments on the device, a step c of diluting the membrane fragments.
3. The method according to claim 1, wherein the total protein concentration in said solution with a pH from 6.5 to 10 is from 5 to 200 g/ml.
4. The method according to claim 1, wherein the Torpedo is Torpedo marmorata or Torpedo californica.
5. The method according to claim 1, wherein the membranes are plasma membranes of electrocyte cells.
6. The method according to claim 1, wherein the surface is a microplate and/or strip well surface.
7. The method according to claim 1, wherein the membrane fragments are attached to the surface via non-covalent interactions and formation of hydrogen bridges between the membrane fragments and the surface.
8. The method according to claim 1, wherein the device is a 96- or 384-well ELISA plate, or a 4-, 8- or 12-well plate, or 6-, 8- or 12-well strips, and the amount of protein in the membrane fragments attached to the surface of the device is 0.5 to 5 g of protein per well.
9. The method according to claim 8, wherein the amount of protein is 1 to 2 g of protein per well.
10. The method according to claim 8, wherein the amount of protein is 1 to 1.5 g of protein per well.
11. The method according to claim 1, wherein the solution is selected from the group consisting of (a) a tris buffered saline (TBS) solution consisting of 150 mM NaCl and 50 mM Tris having pH of 7.5, (b) a phosphate buffered saline (PBS) S) comprising 130 mM sodium chloride, 10 mM sodium phosphate, having pH of 7.0, and (c) a carbonate/bicarbonate buffer comprising 150 mM sodium chloride, 100 mM sodium carbonate, having a pH of 9.5.
12. An analysis device obtained by the method according to claim 1, wherein the surface of the device is made of plastic.
13. The device according to claim 12, wherein the membrane fragments are attached to the surface of wells.
14. The device according to claim 12, wherein the device is a 96- or 384-well ELISA plate, or a 4-, 8- or 12-well plate, or 6-, 8- or 12-well strips.
15. The device according to claim 14, wherein the amount of protein in the membrane fragments attached to the surface of the device is 0.5 to 5 g of protein per well.
16. The device according to claim 12, wherein the device is configured for detecting and quantifying neurotoxins.
17. The device according to claim 16, wherein the neurotoxins act on nicotinic acetylcholine receptors.
18. The device according to claim 12, wherein the device is configured for detecting and quantifying nicotinic acetylcholine receptor ligands.
19. The device according to claim 12, wherein the device is configured for the high-throughput screening of nicotinic acetylcholine receptor ligands.
20. The device according to claim 12, wherein the device is configured for the purification and physicochemical characterization of nicotinic acetylcholine receptor ligands.
21. A method for manufacturing an analysis device for detection, quantification, and physicochemical identification of neurotoxins, the analysis device comprising Torpedo membrane fragments immobilized at the surface thereof, the method comprising the steps of: a. isolating Torpedo electrocyte cell membranes, b. fragmenting said isolated membranes, c. incorporating the membrane fragments obtained in step b into a solution with a pH from 6.5 to 10 and ionic strength from 0.1 mol/L to 0.7 mol/L, d. attaching said membrane fragments to the surface of the device by non-covalent and non-ionic interaction between the membrane fragments and the surface, e. capturing the neurotoxins contained in a sample by incubating the sample with the analysis device; f washing the analysis device with a washing buffer; g. recovering the captured neurotoxins by eluting them with an eluting solution, wherein the surface of the device is made of plastic, and wherein the membrane fragments are not chemically modified before attaching to the surface.
22. The method of claim 21, wherein the method further comprises the step: h. analyzing the eluted neurotoxins by mass spectrometry.
23. The method of claim 21, wherein the washing buffer is a Tris buffered saline (TBS) containing 0.1% Tween 20.
24. The method of claim 21, wherein the eluting solution comprises methanol.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1: Method for Detecting the Interaction Between Nicotinic Acetylcholine Receptors (nAChRs) and Toxins
Products Used
(18) In the example below, the products and devices used were the following:
(19) The flat-bottomed Maxisorp ELISA plates were purchased from the company Nunc (Kamstrupvej, Denmark).
(20) The coating buffer was the following: tris buffered saline (TBS) (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5).
(21) Washing buffer: TBS containing 0.1% Tween 20.
(22) Blocking buffer: TBS containing 0.5% bovine serum albumin (BSA).
(23) A 10-times concentrated TBS buffer stock solution at pH 7 was prepared and was autoclaved and stored at ambient temperature, namely 25 C., for preparing said buffers.
(24) The biotinylated -bungarotoxin (biotin--BgTx) was purchased from the company Molecular Probes (Eugene, Oreg., USA).
(25) The Complete Protease Inhibitor mix was purchased from the company Roche Diagnostics GmbH, Mannheim, Germany. The bovine serum albumin (BSA) came from the company Sigma-Aldrich (St. Louis, Mo., USA). The peroxidase-coupled streptavidin was purchased from the company Sigma-Aldrich. The o-phenylenediamine dihydrochloride tablets were purchased from the company Dako (Glostrup, Denmark). The -bungarotoxin was purchased from the company Sigma-Aldrich. The 13-desmethyl spirolide C was purchased from the company NRC-CNRC (Institute for Marine Biosciences, National Research Counsel, Halifax, NS, Canada). The gymnodimine A was purchased from the company NRC-CNRC. The 13,19-didesmethyl spirolide C was purchased from the company CIFGA (Lugo, Spain). The anatoxin-a came from the company Tocris (Ellisville, Mo., USA).
(26) Protocol:
(27)
(28) Coating with the Membranes
(29) A Nunc Maxisorp 96-well flat-bottomed ELISA plate was coated with 100 l per well of Torpedo electrocyte membranes, the total protein concentration of which was 13.5 g/ml in TBS coating buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, i.e. 1.35 g of protein per well). The plates were closed and incubated at ambient temperature (25 C.) for 2 hours or overnight at 4 C.
(30) The stock solution of membranes was diluted 200-fold in TBS buffer before the coating of the plates. The coating was carried out with an eLINE multichannel electronic pipetting device (Biohit, Helsinki, Finland) which made it possible to accelerate the loading of the membranes. Three wells were left without coating with membranes so that they could be used as negative control wells (plate signal).
(31) Blocking
(32) The excess membrane was removed. The residual buffer on the plate was removed with absorbent paper.
(33) Without washing, the wells were filled with 250 l of blocking buffer (TBS, 0.5% BSA) per well. The plate was closed and incubated for one hour at ambient temperature, namely 25 C.
(34) The plates were stored at 4 C. for subsequent use. Parallel experiments were carried out with plates coated six months beforehand and with plates coated on the previous day: no significant difference was observed between the various plates for the ligand binding dose-response curves (results not represented).
(35) Incubation with the Toxins/Extracts
(36) The blocking buffer was removed and the residual liquid on the top of the plate was absorbed with absorbent paper. Without washing, the plates were loaded either with a) 100 l of standard toxins prepared in blocking buffer or with b) 100 l of blocking buffer containing up to 10% of extracts (volume/volume). Each toxin or sample concentration was tested 3 times. The plate was closed and incubated for three hours with constant shaking at ambient temperature (25 C.) or overnight at 4 C. for maximum sensitivity.
(37) Six wells coated with Torpedo membranes were left free of toxin or of extract so as to use them as negative controls (100% signal). In addition, three wells coated with Torpedo membranes were left free of toxin or extract in order to use them as a nonspecific inhibition control. Said wells are incubated with 1 M -bungarotoxin (100% inhibition).
(38) The methanol concentration was kept below 1% in order to avoid any interference with the binding of the ligand to the nAChR.
(39) Biotin-BgTx Displacement:
(40) Without removing the toxins and/or the extracts, 50 l of biotin--BgTx (810.sup.8 M, final concentration) were added to each well. The plate was closed and the whole was incubated for 30 minutes with constant shaking at ambient temperature (25 C.)
(41) The unbound toxins, extracts or biotin--BgTx were removed and the wells were washed three times with 250 l of washing buffer (TBS, 0.1% Tween 20). The residual buffer was absorbed with absorbent paper.
(42) Streptavidin-Biotin Reaction
(43) An amount of 100 l of peroxidase-coupled streptavidin (diluted 5000 times, 220 ng/nl protein, Sigma) was added to each of the wells with an eLINE (registered trade mark) multichannel electronic pipetting device (Biohit). The plate was closed and incubated for 30 minutes at ambient temperature (25 C.) with constant shaking, i.e. shaking of approximately 50 revolutions per minute.
(44) The multichannel electronic pipetting device makes it possible to minimize the differences in exposure times to the reaction between the biotin part of the tracer and the streptavidin part of the enzyme. However, owing to the high affinity between biotin and streptavidin (K.sub.d=10.sup.14 M), a reaction of 30 minutes gives a negligible effect linked to the difference in exposure time for each row of wells. The reaction time between the biotin--BgTx and the streptavidin-peroxidase was determined experimentally, namely Torpedo electrocyte membranes immobilized on wells of the Maxisorp microplate were incubated with 810.sup.8 M biotin--BgTx for 30 minutes at 25 C. After washing, 100 l of peroxidase-coupled streptavidin (diluted 5000-fold) were added to each well. The incubation time was 0, 1, 2.5, 5, 10, 15 and 30 min. The wells were washed and the peroxidase activity was detected as described below.
(45) Colorimetric Detection
(46) The excess streptavidin-peroxidase was removed and the wells were washed three times with 250 l of washing buffer (TBS, 0.1% Tween 20). The residual buffer on the plate was removed with absorbent paper. An amount of 100 l of OPD peroxidase substrate (Dako, Glostrup, Denmark) was added to each well with a multichannel electronic pipetting device. When a suitable color developed, i.e. after approximately 5 minutes of incubation, the enzymatic reaction was stopped by adding 100 l of 0.5 M H.sub.2SO.sub.4 to each well with a multichannel electronic pipetting device.
(47) The use of a multichannel electronic pipetting device makes it possible to avoid variations in the development of the color due to the difference in exposure time. The stopping solution was added in the same order as the peroxidase substrate (OPD) solution was added in order to minimize the differences due to the incubation time.
(48) The peroxidase substrate (OPD) was prepared according to the supplier's recommendations. Four OPD tablets were dissolved in 12 ml of deionized water at ambient temperature (25 C.), following which, 5 l of 30% hydrogen peroxide were added.
(49) The plate was recorded with an ELISA reader (Genios Pro, Tecan), at an absorbance of 492 nm (
(50) The percentage inhibition was calculated according to the following formula:
% inhibition=[100(100% signalsample signal)/(100% signal(100% inhibitionplate signal))].
(51) The 100% signal was obtained in wells in which Torpedo membranes were applied and incubated without toxins/extracts; the plate signal was obtained from the wells in which the coating with the Torpedo membranes was omitted. Independent experiments were carried out at least twice with triplicates.
(52) The percentage inhibition was also calculated according to the following formula:
% inhibition=[100(100% signalsample signal)/(100% signal100% inhibition)].
(53) The 100% signal was obtained in wells in which Torpedo membranes were applied and incubated without toxins/extracts; the 100% inhibition signal (100% inhibition) was obtained from the wells in which the Torpedo membranes were incubated with -bungarotoxin (1 or 10 M). Independent experiments were carried out at least twice with triplicates.
(54) The results showed that nAChR antagonists, namely: -bungarotoxin, 13-desmethyl spirolide C, 13,19-didesmethyl spirolide C, gymnodimine, d-tubocurarine (curare), and nAChR agonists, namely: anatoxin-a and homoanatoxin-a, inhibit the interaction between -BgTx and Torpedo nAChR in a concentration-dependent manner, with different affinities (
(55) This system is also usable with alkaline phosphatase-coupled streptavidin in addition to the horseradish peroxidase-coupled streptavidin, and, as agents for visualizing the enzymes, substrates either producing a colorimetric reaction (OPD, nitro blue tetrazolium chloride) or which are a substance detectable by chemiluminescence (ECL+) (Aroz R, Herdman M, Rippka R, Ledreux A, Molg J, Changeux J P, Tandeau de Marsac N, Nghim H O. (2008). A non-radioactive ligand-binding assay for detection of cyanobacterial anatoxins using Torpedo electrocyte membranes. Toxicon 52:163-174 [14]). The list of visualizing agents is not limiting.
(56) TABLE-US-00001 TABLE 1 Sensitivity of the nonradioactive test for ligand-receptor binding displacement on an ELISA microplate for detecting -bungarotoxin (-BgTx), 13-desmethyl spirolide C (13-SPX C), 13,19-didesmethyl spirolide C (13,19-SPX C), gymnodimine A (GYM A) and anatoxin-A (ANTX) -BgTx 13-SPX C 13,19-SPX C GYM A ANTX IC.sub.50 (95% CI) 0.27 6.84 3.10 74.23 68.81 (0.18- (5.07- (1.98- (62.4- (35.3- 0.41) 9.22) 4.85) 88.3) 34.2) LOD (nM) 0.02 1 0.8 2 2 LOQ (nM) 0.20 4 2.0 20 30 IC.sub.90 (nM) 70 60 20 2000 2000
(57) The IC.sub.50 represents the neurotoxin concentration at which there is 50% inhibition of the binding between the biotin--BgTx and the Torpedo nAChRs; the 95% CI is the 95% confidence interval of the corresponding IC.sub.50 value; the LOD is the limit of detection of the method, the LOQ is the limit of quantification of the method; IC.sub.90 represents the neurotoxin concentration required to inhibit 90% of the binding between the biotin--BgTx and the Torpedo nAChRs.
(58) The nonradioactive test for ligand-receptor binding displacement on an ELISA microplate is a functional method based on the mechanism of action of competitive nAChR ligands. This method makes it possible to detect competitive nAChR agonists and antagonists with great sensitivity.
(59) The sensitivity of the method using the devices of the present invention was compared with ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS/MS) for the detection of 13-SPX C, 13,19-SPX C and GYM A [Aroz et al., manuscript in preparation]. The LOD and LOQ values obtained by UPLC-MS/MS show that the sensitivity of the nonradioactive displacement method using the device of the present invention is in the same order of magnitude as that of UPLC-MS/MS (table 2).
(60) TABLE-US-00002 TABLE 2 Sensitivity of UPLC-MS/MS for detecting 13-SPX C, 13,19-SPX C and GYM A 13-SPX C 13,19-SPX C GYM A LOD (nM) 4.78 1.66 1.52 LOQ (nM) 13.1 5.55 4.17
(61) As demonstrated in this example, the use of the device obtained by means of the method of the invention makes it possible to detect the interaction between nAChRs and ligands with a sensitivity which is as high as the most widely specified techniques of the prior art, without the use of a complex and/or expensive device and in parallel on a large number of samples (96) for the simultaneous detection of the multitude of nAChR ligands/agonists/antagonists, namely spirolides, gymnodimines, pinnatoxins, anatoxins, curare, etc. (the list is not limiting).
Example 2: Method for Detecting 13-Desmethyl Spirolide C, 13,19-Didesmethyl Spirolide C and Gymnodimine A from Seafood Extracts and Anatoxin-A from Cyanobacterial Extracts
(62) In this example, the following solutions and protocols were used: Coating buffer: TBS (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5). Dilution of membranes: 200-fold. A volume of 50 l of Torpedo membrane (2.7 mg.Math.ml.sup.1 of protein in 5 mM of glycine) was diluted in 10 ml of TBS. Support: Maxisorp flat-bottomed ELISA plates (Nunc). Coating volume: 100 l of Torpedo membranes diluted 200-fold. Coating temperature and time: ambient temperature (25 C.) for 2 hours or 4 C. overnight. Control seafood (Queen scallops, oysters, mussels and scallops) extracts and seafood (Queen scallops, oysters, mussels and scallops) extracts doped with 13-desmethyl spirolide C, 13,19-didesmethyl spirolide C and gymnodimine A. Extracts of cyanobacteria producing anatoxin-a and homoanatoxin-a.
(63) A Maxisorp flat-bottomed 96-well ELISA plate (Nunc) was coated with 100 l per well of a solution of Torpedo electrocyte membranes (13.5 g/ml total protein, i.e. 1.35 g of membrane protein per well) in TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5) using a multichannel pipetting device. The plate was closed and incubated at ambient temperature (25 C.) for 2 hours or at 4 C. overnight.
(64) Detection of Phycotoxins in Seafood Extracts
(65) Extraction of the Lipophilic Toxins from Seafood:
(66) The preparation of the crude extracts from seafood samples was carried out as follows: Queen scallop, oyster, mussel or scallop flesh (100 g) was homogenized in a knife mill at maximum power and divided up into 10 aliquots and kept at 80 C.
(67) 50 l of standard 13-desmethyl spirolide C, 50 l of standard 13,19-didesmethyl spirolide C or 50 l of standard gymnodimine A, all three at a concentration of 10 M, were added separately to 1 g of each seafood homogenate.
(68) In order to extract these lipophilic phycotoxins, 4 ml of acetone were added to each of the doped homogenates. The mixtures were vortexed for 10 seconds and mixed on a rotary mechanical shaker for 15 minutes at ambient temperature (25 C.). The homogenates were then centrifuged at 3500 g for 15 minutes at 4 C. and the supernatants were recovered, whereas the pellets were re-extracted twice with 4 ml of acetone as has just been described. The supernatant pools were evaporated and the residues were resuspended in 4 ml of water. 4 ml of hexane were added to the aqueous extracts and left for 30 min at ambient temperature (25 C.) in order for the chemical partitioning to take place.
(69) Alternatively, the homogenates were centrifuged at 3500 revolutions per minute for 15 minutes at 4 C. and the supernatants were recovered, whereas the pellets were re-extracted twice with 4 ml of acetone as has just been described. The supernatant pools were evaporated and the residues were resuspended in 4 ml of water. 4 ml of hexane were added to the aqueous extracts and left for 30 min at ambient temperature (25 C.) in order for the chemical partitioning to take place.
(70) The aqueous phases were extracted three times with chloroform (1:1.5 v/v). The chloroform layers were combined and evaporated. The residues obtained were resuspended in 100 l of methanol and stored at 80 C. until they were used.
(71) 1 g of homogenate of each bivalve (Queen scallops, oysters, mussels and scallops) was subjected to the same treatment in order to serve as control.
(72) Plate Preparation:
(73) Serial dilutions of standard toxins, i.e. of 13-desmethyl spirolide C (1.710.sup.5-3.510.sup.12 M), 13,19-didesmethyl spirolide C (3.310.sup.7-2.110.sup.12 M) and gymnodimine A (1.710.sup.5-3.110.sup.11 M) (350 l) were prepared with blocking buffer (TBS, 0.5% BSA). In the same way, each seafood extract, control or doped with said toxins, was diluted 100-fold and 200-fold (350 l) in blocking buffer (TBS, 0.5% BSA). Each dose was analyzed in triplicate (100 l per well).
(74) 13-Desmethyl spirolide C, 13,19-didesmethyl spirolide C and gymnodimine A are distributed commercially in methanol. The methanol concentrations were kept to a level below 1% in order to avoid any interference with the method.
(75) Preparation of the Cyanobacterial Extracts
(76) The crude extracts of cyanobacteria producing anatoxin-a were prepared as follows:
(77) The biological material, i.e. cyanobacterial filaments, were harvested by centrifugation (3000 revolutions per minute at 15 C. for 10 minutes) from a culture of cyanobacteria. The cyanobacterial pellet was resuspended in distilled water and recentrifuged under the same conditions and lyophilized. The lyophilized cells were resuspended in 50 mM of acetic acid (10 mg: 2 ml) and lyzed by sonification (5 cycles of 1 minute with gaps of 3 minutes in ice) with a Branson 250 sonicator. The homogenate obtained was stirred for 4 hours in the dark and then centrifuged at 10 000 revolutions per minute for 30 minutes at 15 C. The supernatant was collected and then passed through a filtration unit (pore size 0.45 m) and stored at 20 C. until use.
(78)
(79)
(80) Furthermore,
(81) The absorption data (OD.sub.492 nm) were expressed as percentage inhibition using the following formula: % inhibition=100[(100% signalsignal of the sample)/(100% signal(100% inhibitionplate signal))] or with the following formula: % inhibition=[100(100% signalsignal of the sample)/(100% signal100% inhibition)]. An inhibition curve for each of the toxins tested was constructed from the inhibition data obtained (
(82) The results obtained demonstrate that the nonradioactive biotin--bungarotoxin displacement method is applicable for the detection and quantification of marine phycotoxins, namely, for example, 13-desmethyl spirolide C, 13,19-didesmethyl spirolide C and gymnodimine A from contaminated seafood samples (
(83) The sensitivity of the method depends on the affinity of the toxin for the receptor. As demonstrated in this example, the detection threshold of this nonradioactive method is very low, for example 54 picograms of 13,19-didesmethyl spirolide C, 69 picograms of 13-desmethyl spirolide C and 101 picograms of gymnodimine A.
(84) Furthermore, the method requires a very small amount of electrocyte membrane, for example from 1 to 2 g of Torpedo electrocyte membrane proteins per well in this example (
(85) Very important: microplates coated with Torpedo electrocyte membranes, when they are stored at 4 C. in 200 l of blocking buffer (TBS, 0.5% BSA, pH 7.5), can be used for up to six months after manufacture thereof. There is no significant difference between plates coated the day before and plates coated six months beforehand for the detection of nAChR ligands. This proof of the principle is very useful for designing the manufacture of ready-to-use plates pre-coated with Torpedo electrocyte membranes.
(86) The plates obtained in this example are denoted Torpecfo-MicroReceptor Plate.
(87) As demonstrated in this example, the use of the device obtained by the method of the invention makes it possible to detect the interaction between nAChRs and competitive ligands directly on methanolic extracts of seafood and aqueous extracts of cyanobacterial filaments. In addition, the devices of the present invention, namely the ready-to-use plates pre-coated with Torpedo electrocyte membranes, could be transported and distributed by mail in order to be used in other countries/continents for the detection of competitive nAChR ligands.
Example 3: High-Throughput Screening of Nicotinic Acetylcholine Receptor Competitive Ligands/Toxins
(88) The microplates coated with Torpedo electrocyte membranes which are stored at 4 C., obtained in Example 1, are suitable for high-throughput methods.
(89) A/ Use of the Liquidator 96 96-Tip Manual Pipetting Device (Rainin):
(90) a) 96-Tube Plates to be Prepared Before Beginning the Detection Method:
(91) A plate plan is prepared beforehand as represented in
(92) The plate of
(93) Rectangle I [A1-C3], CYA01; Rectangle II [D1-F3], CYA02; Rectangles III
(94) G1-G3 and [A4-B6], CYA03; Rectangle IV [C4-E6], CYA04; Rectangles V
(95) [F4-G6] and A7-A9, CYA 05; Rectangle VI [B7-D9], CYA06; Rectangle VII
(96) [E7-G9], CYA07; Rectangle VIII [A10-G12], 13,19-didesmethyl spirolide C.
(97) According to the plate plan, solutions of toxin or of extract in blocking buffer identical to that of example mentioned are prepared and distributed in 96-tube plates capable of containing up to 1 ml of solution per tube: TOXIN/EXTRACTS PLATE. For each standard toxin or sample to be tested in triplicate, the minimum volume to be prepared is 350 l per tube.
(98) A second 96-well plate containing a solution of 2.410.sup.7 M biotin--BgTx in blocking buffer (TBS, 0.5% BSA), BIOTIN--BgTx PLATE, is prepared. The volume to be prepared depends on the number of plates to be tested (one 96-well microplate consumes 50 l96=4.8 ml). This solution is freshly prepared.
(99) A third 96-tube plate, PEROXIDASE PLATE, containing peroxidase-coupled streptavidin (D.sub.5000 220 ng/nl protein in blocking buffer) is prepared. This solution is freshly prepared. The volume to be prepared depends on the number of plates to be tested (one 96-well microplate consumes 100 l96=9.6 ml).
(100) A fourth 96-tube preparation plate indicated OPD PLATE, containing the commercial substrate OPD, at least 120 l per well, in order to produce a screening device, is prepared. This solution is freshly prepared and must be protected from light. The volume to be prepared depends on the number of plates to be tested (one 96-well microplate consumes 100 l96=9.6 ml). The OPD solution is prepared according to the supplier's protocol: 4 OPD tablets are dissolved in 12 ml of deionized water, to which 5 l of hydrogen peroxide (H.sub.2O.sub.2) at 30% are added.
(101) 50 ml of stop solution for the peroxidase reaction (0.5 M H.sub.2SO.sub.4) are prepared in a rectangular dish suitable for the 96-channel manual pipetting device, denoted STOP PLATE.
(102) b) Procedure
(103) The pre-coated screening device obtained according to the method described in Example 1, indicated Torpedo-MicroReceptor Plate hereinafter, is taken out of the refrigerator. The plate is left on a shaker at ambient temperature (25 C.) for 30 min. Once the screening device is at temperature, the adhesive film is removed, the blocking buffer is removed and, after having dried its surface, the plate is placed on the pipetting zone.
(104) Incubations with Toxins/Extracts:
(105) The 96-channel manual pipetting device is loaded with new tips and set to 100 l, and the TOXIN/EXTRACTS PLATE is placed on a loading zone.
(106) 100 l from the TOXIN/EXTRACTS PLATE containing toxin standards, samples, extracts, synthetic products or negative and positive controls, are sampled and introduced into the Torpedo-MicroReceptor Plate placed in the pipetting zone.
(107) The screening device is sealed with an adhesive film (sealing tape, Costar) and left to incubate the immobilized Torpedo electrocyte membranes with the test samples for 3 h with shaking. Preferably, the incubation is carried out overnight at 4 C.
(108) When the incubation is carried out at 4 C., the Torpecfo-MicroReceptor Plate is placed on a shaker at ambient temperature (25 C.) for 30 min before being placed again in the pipetting zone of the Liquidator 96.
(109) Incubation with the Biotin--BgTx Tracer
(110) New tips are loaded, the pipetting device is set to 50 l and the BIOTIN--BgTx PLATE is placed on the loading zone. 50 l of each tube of the BIOTIN--BgTx PLATE are sampled and introduced directly into the Torpecfo-MicroReceptor Plate. The resulting plate corresponds to the screening device and is incubated for 30 min on a shaker at ambient temperature (25 C.), after which time the solution is removed and the plate is again placed in the pipetting zone.
(111) Washing
(112) New tips are loaded and 200 l of washing solution (TBS, 0.1% Tween 20) are sampled and introduced into the Torpedo-MicroReceptor Plate wells. The washing solution is removed and the Torpedo-MicroReceptor Plate is again placed in the pipetting zone. This step is repeated three times.
(113) Incubation with HRP-Streptavidin/Washing
(114) 100 l of each tube of the PEROXIDASE PLATE are sampled and introduced directly into the screening device. The plate is incubated for 30 min on a shaker at ambient temperature (25 C.). The Torpecfo-MicroReceptor Plate is washed as previously described and is again placed in the pipetting zone.
(115) Developing the Peroxidase Reaction and Stopping the Reaction
(116) 100 l of each well of the OPD PLATE are sampled and introduced into the screening device. The Torpedo-MicroReceptor Plate is incubated until an optimum coloration is obtained, i.e. approximately 5 minutes, and 50 l of the stop solution (0.5 M H.sub.2SO.sub.4) are introduced using the 96-tip pipetting device.
(117) Reading and Analysis of Data
(118) The Torpecfo-MicroReceptor Plate is read on the ELISA reader (GeniosPro, Tecan) and the results are analyzed.
(119) The results are shown in
(120) B/ Use of an Automated Pipetting Device:
(121) The following solutions: TOXIN/EXTRACTS SOLUTION, BIOTIN--BgTx SOLUTION, PEROXIDASE SOLUTION, OPD SOLUTION and STOP SOLUTION are prepared as previously indicated.
(122) A 96-well model plate is prepared for the samples of toxins/extracts/controls according to the test plate plan. The automated pipetting device is capable of automating the preparation of the toxin/sample dilutions, for the model plate.
(123) The steps are similar to the abovementioned steps.
Example 4: Method for Manufacturing Analysis Devices
(124) The solutions, membranes and experimental conditions are identical to those of Example 2 mentioned above.
(125) A dilution of the membrane stock solution, 2.7 mg of proteins per ml, prepared according to the method described in Examples 1-4 mentioned above, was diluted 200-fold in TBS buffer at a pH of 7.5.
(126) 100 l of the 200-fold-diluted Torpedo membrane solution: 13.5 g.Math.ml.sup.1 total protein, i.e. 1.35 g of membrane protein per well, were distributed into the wells of a Nunc Maxisorp (registered trade mark) flat-bottomed microplate, as represented in
(127) An adhesive film was placed over said plate and the plate was incubated at 4 C. overnight, i.e. 12-18 hours.
(128) The excess membranes were removed by manually inverting the plate and the edges of the wells were cleaned with absorbent paper.
(129) Finally, 200 l of TBS blocking solution containing 0.5% BSA were introduced into each well. An adhesive film was placed over the plate termed Torpecfo-MicroReceptor Plate and stored at 4 C. until it was used.
(130) The analysis device thus prepared is ready for use and in particular ready for transport.
Example 5: Comparison of the Known Detection Methods with the Method of the Invention
(131) In this example, the detection results obtained in the following prior art documents: Fonfria et al. [12], Vilarino et al. [13] and Aroz et al. [14] were compared with those obtained with the method and device of the present invention (Examples 1 and 2 above).
(132) Table 3 below represents the various results obtained, be they in terms of the sensitivity of the method according to the molecule to be detected.
(133) TABLE-US-00003 TABLE 3 comparison of the method of the invention with the known methods Fonfria et Vilarino Method of the al. Natalia et al. Aroz et al. invention Sensitivity 13,19-SPX C: 13-SPX C: 108.2 ANTX.sub.CHEM: 62 13,19-SPX C: 3.10 (concentration 63.60 3 11.9 ANTX.sub.VISUAL: 17 (1.98-4.85) in nM) GYM A: 391.0 13-SPX C: 6.84 55.3 (5.07-9.22) GYM A: 74.23 (62.43-88.28) ANTX: 68.81 (35.3-A34.2) Sensitivity 13-SPX C: 85 13,19-SPX C: (amount in g 0.051 of toxin in 13-SPX C: 0.069 the well) GYM A: 0.101 Limit of ANTX.sub.CHEM: 37 13,19-SPX C: 0.8 detection ANTX.sub.VISUAL: 9 13-SPX C: 1 (nM) GYM A: 2 ANTX: 2 Quantification 13,19-SPX C: 13-SPX C: 12.5- 13,19-SPX C: 2-20 range (nM) 30-150 125.2 13-SPX C: 4-60 GYM A: 12.5-500 GYM A: 20-2000 ANTX: 30-2000 Reading of Polarization Polarization Chemi- Microplate reader data fluorometer fluorometer luminescence detector/ visual Automation YES Transportable YES (tested by device rapid courier and (microplates) by airplane) Stability of 6 months (tested) the device (microplates)
(134) As demonstrated in this example, the use of the device obtained by means of the method of the invention makes it possible to detect the interaction between nAChRs and ligands with a much greater sensitivity than that of the prior art methods and devices.
(135) In addition, the device of the present invention, namely the ready-to-use plates pre-coated with Torpedo electrocyte membranes, can be transported and distributed, for example by mail, for their use in other countries/continents for detecting competitive nAChR ligands.
(136) Finally, as demonstrated in this example, the amounts of membrane fragments used for manufacturing the device of the invention are much smaller than the amounts required in the prior art methods. Thus, the method of the invention advantageously makes it possible to reduce the amount of membrane fragments to be used and thus reduces, compared with the prior art methods, the number of Torpedoes to be sacrificed while at the same time improving the sensitivity of detection of the device of the invention compared with the known devices/methods.
Example 6: Method for Eluting the Cyclized Imines having Reacted with the Torpedo Nicotinic Acetylcholine Receptors Immobilized on the Surface of the ELISA Plates
(137) Products Used:
(138) The same products as in Example 1. A ready-to-use analysis device obtained in Example 4 mentioned above, indicated Torpecfo-MicroReceptor Plate, i.e. a plate coated with Torpedo electrocyte membranes. Gymnodimine A, 13-desmethyl spirolide C and 13,19-didesmethyl spirolide C standards. Methanol. 2,5-Dihydroxybenzoic acid (DHB). Acetonitrile. Trifluoroacetic acid.
Equipment: ELISA microplate reader MALDI-TOF (Voyager-DETM STR Workstation, Applied BioSystems, Foster City, Calif., USA)
Procedure:
(139) The ready-to-use analysis device was placed at ambient temperature, namely 25 C., for 30 minutes.
(140) The blocking buffer was removed and, without washing, six wells were incubated with 100 l gymnodimine A, or 100 l of 13-desmethyl spirolide C, or with 100 l of 13,19-didesmethyl spirolide C, at a concentration of 1 M, prepared in blocking buffer (TBS, 0.5% BSA, pH 7.5).
(141) The device was incubated at 4 C. overnight.
(142) After incubation of the device overnight at 4 C., the plate was placed at ambient temperature, namely 25 C., for 30 min, after which time the wells are washed 5 times with 250 l of washing buffer comprising TBS, 0.1% Tween 20, pH 7.5.
(143) The elution of the gymnodimine A was carried out with 50 l of methanol added to each of the six wells previously incubated with this toxin. After 5 min of incubation at ambient temperature (25 C.), the methanol was recovered. The same procedure was applied for eluting 13-desmethyl spirolide C and 13,19-didesmethyl spirolide C.
(144) The eluates were then analyzed by MALDI-TOF. For this, the matrix was prepared as follows: 10 mg of 2,5-dihydroxybenzoic acid were dissolved in 1 ml of a water-acetonitrile (1:1) mixture containing 0.3% (v/v) of trifluoroacetic acid.
(145) For the analysis by MALDI-TOF, 10 l of standard toxin (1 M) were mixed with 10 l of matrix prepared as previously described, using a vortex. One microliter of this mixture was deposited on the surface of a stainless steel sample plate (Perseptive Biosystems, Framingham, Mass., USA).
(146) In the same way, 10 l of each eluate were mixed with 10 l of matrix prepared as previously described, using a vortex. One microliter of this mixture was deposited on the surface of a stainless steel sample plate. Once the solvent had evaporated off (namely 2 min after having deposited the samples), the sample plate was introduced into the MALDI-TOF instrument for the mass data acquisition.
(147) TABLE-US-00004 TABLE 4 Physicochemical characterization of the eluates obtained from the wells in which the Torpedo nAChRs were incubated with cyclized imines MALDI-TOF Mass Intensity UPLC/MS/MS Samples ([M + H].sup.+) (counts) ([M + H].sup.+) Gymnodimine A 508.35 22000 508.4 (standard) Gymnodimine A 508.34 2000 (eluate from 6 wells) 13-SPX C 692.54 16000 692.4 (10 M standard) 13-SPX C 692.53 4906.4 (eluate from 6 wells) 13,19-SPX C 678.62 20000 678.4 (10 M standard) 13,19-SPX C 678.52 1800 (eluate from 6 wells)
(148) As demonstrated in this example, the use of the device obtained by means of the method of the invention makes it possible, for example, to capture cyclized imines which can then be eluted, for example with methanol. The use of the device obtained by means of the method of the invention therefore advantageously makes it possible to facilitate the isolation/characterization of nAChR ligands. In particular, it advantageously makes it possible to facilitate the isolation/physicochemical characterization of an nAChR agonist/antagonist, which is for example competitive. Thus, this device can also advantageously be used in programs for searching for new toxins.
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