MXPA01004700A - Assay for phosphatase-targeting toxins - Google Patents
Assay for phosphatase-targeting toxinsInfo
- Publication number
- MXPA01004700A MXPA01004700A MXPA/A/2001/004700A MXPA01004700A MXPA01004700A MX PA01004700 A MXPA01004700 A MX PA01004700A MX PA01004700 A MXPA01004700 A MX PA01004700A MX PA01004700 A MXPA01004700 A MX PA01004700A
- Authority
- MX
- Mexico
- Prior art keywords
- immobilized
- toxin
- ligand
- phosphatase
- immobilized ligand
- Prior art date
Links
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Abstract
The invention provides an assay method for determining phosphatase targeting toxins which inhibit protein phosphatases comprising contacting a solid support having an immobilized ligand immobilized thereon with:(i) a sample suspected of being contaminated with toxin and (ii) a non-immobilized ligand, wherein said immobilized ligand is capable of generating directly or indirectly detectable signal when uncomplexed, when complexed by said toxin, when complexed by a complex of said toxin and said non-immobilized ligand or when complexed by said non-immobilized ligand or said non-immobilized ligand is capable of generating a directly or indirectly detectable signal when uncomplexed or when complexed, separating a bound fraction from a non-bound fraction;and directly or indirectly determining the non-immobilized ligand bound to the immobilized ligand (the bound fraction) or non-complexed in aqueous solution (the non-bound fraction).
Description
METHOD TO DETERMINE TOXINS THAT JOIN PHOSPHATASE
DESCRIPTION OF THE INVENTION The present invention relates to an assay method for the detection of phosphatase-binding toxins typically produced by microalgae such as, for example, cyanobacteria and dinoflagellates. Dinoflagellates are typically unicellular, photosynthetic, biflagellate algae. Some of the marine dinoflagellates (eg, Prorocentrum sp. And Dinophysis sp.) Produce toxins that bind to phosphatase such as okadaic acid and Dinophysis toxin, which causes gastrointestinal problems to be ingested by humans. Such algae can be problematic in this way if they contaminate the habitats of molluscs destined for consumption. Cyanobacteria, which are regularly referred to as blue-green algae, are also photosynthetic organisms that are mainly aquatic and inhabit coastal waters, the open sea and oceans, rivers, lakes and groundwater but can also be terrestrial and can be found in beds foliage and earth. Many species and strains of cyanobacteria, especially My crocys ti s sp., Aphani zonemon sp. , Anabena sp. , Nodularia sp. and Oscillatoria sp., produce toxins that if ingested by humans or other mammals, birds and even fish, can cause disease. The ingestion of such
Ref: 129429 toxins takes place through two main routes, either by drinking contaminated water or eating contaminated seafood. Cyanobacteria and dinoflagellates produce two particular types of toxins. The neurotoxins, for example, toxins and saxitoxins, cause paralysis in the victim and consequently the condition is regularly referred to as paralytic shellfish poisoning. Poisoning by such neurotoxins is rare but can be fatal. The other way toxins inactivate protein-phosphatase enzymes in body cells is by binding to enzymes and affecting their ability to dephosphorylate protein substrates. These toxins are relatively common, and some (such as dinoflagellate toxins, okadaic acid and dinophysis toxin) can cause nausea, vomiting and diarrhea and consequently the condition is regularly referred to as mollusc diarrhea poisoning. Some toxins that bind to the protein phosphatase are tumor promoters and exposure to these toxins can induce cancer. Others, such as microcystin and nodularin cyanobacterial toxins, are hepatotoxic and cause liver damage. The most prevalent toxins that bind to phosphatase are microcystin, nodularin and okadaic acid. The most common sources of dinoflagellate toxin poisoning are mollusks and fish liver, and the most common cause of poisoning by cyanobacterial toxins is drinking or bathing in contaminated water. However, both cyanobacterial and dinoflagellate toxins can be housed in mollusks and in water. A particularly common source of algae toxin poisoning is the mussel, since they accumulate toxins based on their diet in toxin-producing algae. Other molluscs, for example oysters, clams and shells, may also be affected. Additionally, domestic water suppliers, particularly if it originates from groundwater, can become contaminated with cyanobacteria and thus provide a direct route for the ingestion of the toxin. There is some interest regarding the consumption of algae and cyanobacteria as a food high in protein and dietary supplement. There are no official guidelines for the monitoring of contamination by toxins produced by the strains of algae or cyanobacteria collected and the commercialization of genera such as Anabena and Aphani zomenon is particularly worrisome since different strains that produce different strains can be found within these genera. toxins. In addition to the short-term discomfort, medical costs, commercial costs for the mollusc industry, losses of work hours, etc. resulting from exposure to algal toxins, the aforementioned microcystin and nodularin toxins that bind to phosphatase have been found to be tumor promoters and it is believed that repeated exposure to such toxins at clinical or subclinical level, particularly in combination with a high consumption of alcohol or smoking can result in cancer, especially of the liver. Currently, there are a number of different methods for the detection and quantification of algae toxins and cyanobacteria that bind to phosphatase. A standard method involves grinding mussels or other potential sources of toxins that bind to phosphatase and injection into mice of an extract from the ground mussel tissue. The presence and level of contamination by the toxin that bind to the phosphatase is then determined in relation to the survival of the mouse (Stabell et al (1992), Food Chem. Toxicol 30 (2): 139-44). Clearly, this is a time-consuming, unrefined and expensive method of assessing the safety and quality control of food. Another method for the determination of mollusc poisons that cause diarrhea (DSP) (EP-A-554458 of latron Laboratories Inc) involves the use of a first and second antibodies to the toxin in a conventional sandwich assay. Another method involves the measurement of the reduction in the enzymatic activity of phosphatase added exogenously thus detecting in the mollusks the presence of toxins that bind to the phosphatase. This again involves the grinding of mussels or other mollusk tissue, which releases endogenous phosphatases which interfere with the added phosphatase, compromising the sensitivity and precision of the test (Sim and Mudge (1994) in Detection Methods for Cyanobacterial Toxins Eds. Codd, Jeffries, Keevil and Potter, Royal Society of Chemistry, and Charles Holmes, US-A-5180665. There is therefore a great need for a rapid, sensitive, and non-expensive assay or method that allows the qualitative and / or quantitative determination of the presence of toxins which bind to the phosphatase, in particular toxins of algae and cyanobacteria which bind to the phosphatase, in water, products of molluscs and / or edible seaweed or cyanobacteria In particular, there is a need for an assay method that is simple enough to be carried out on site by relatively inexperienced or inexperienced personnel, for example fishmongers or water sanitation personnel and that does not require equipment of laboratory or special facilities for its realization. Thus, according to a first aspect, the present invention provides an assay method for the determination of phosphatase-binding toxins that inhibit protein phosphatases comprising contacting a solid support having a ligand immobilized therein. support with: (i) a sample suspected of being contaminated with the toxin; and (ii) a non-immobilized ligand, wherein the immobilized ligand is capable of binding with at least one of the toxins mentioned, to the non-immobilized ligand or to the complexes of the toxin and the non-immobilized ligand, and wherein the non-immobilized ligand is capable of binding to at least one of the immobilized ligands, the toxin or the toxin complexes and the immobilized ligand whereby the proportion of the ligand does not immobilized bound by the toxin, the non-immobilized ligand or the complexes of the toxin and the non-immobilized ligand is dependent on the toxin content of the sample and where the ligand or immobilized is capable of directly or indirectly generating a detectable signal when a complex is not formed, when it forms a complex by the toxin, when it forms a complex by the complex of the toxin and the non-immobilized ligand or when it forms a whole by the non-immobilized ligand or the non-immobilized ligand is capable of directly or indirectly generating a detectable signal when it does not form a complex or when it forms a complex, the separation of a bound fraction from an unbound fraction; and determining directly or indirectly the non-immobilized ligand bound to the immobilized ligand (the bound fraction) or does not form a complex in aqueous solution (the unbound fraction);
wherein the application of (i) and (ii) to the solid support can be carried out separately, sequentially or simultaneously and, separately or sequentially, it can be carried out in any order. Thus, in one embodiment the toxin determination may involve the determination of the non-immobilized ligand which has failed to bind directly or indirectly with the immobilized ligand. Where the non-immobilized ligand competes with the toxin for binding to the immobilized ligand, a high level of unbound ligand is indicative of a high toxin concentration. Where the non-immobilized ligand can form a complex with the toxin bound to the immobilized ligand, a high level of unbound ligand is then indicative of a low concentration of toxin. In another embodiment, the toxin determination involves the determination of the non-immobilized ligand which has been directly or indirectly bound to the immobilized ligand. Where the toxin and the non-immobilized ligand compete for binding to the immobilized ligand, a high level of binding to the ligand is indicative of a low level of toxin concentration. Where the non-immobilized ligand can form a complex with the toxin bound to the immobilized ligand, a high level of binding to the ligand is indicative of a high level of toxin concentration. Preferably, however, the method of the invention involves a competitive binding assay for the detection of toxins that bind to the phosphatase, in particular toxins of algae and cyanobacteria, wherein the toxin molecules present in a sample compete with the non-immobilized ligand. for a limited number of binding sites of the immobilized ligand and any toxin present in said mixture is determined relative to the degree of non-immobilized ligand bound or not to the binding sites of the immobilized ligand. As used herein, the terms "detection", "determination" or "assessment" include quantification in the sense of obtaining an absolute value for the amount or concentration of the toxins that bind to the phosphatase present in the sample, and also the estimation or semi-quantitative and qualitative determination. An index, ratio, percentage or molar indication of the level or amount of the toxin present can be determined or alternatively, an indication of the mere presence or absence of the toxin in the sample can be obtained. In a preferred aspect of the invention, the determination of a mere presence or absence or the semi-quantitative determination of the presence of the toxin is achieved. In this regard "absence" of toxin may mean that the concentration of the toxin is below the detection limit of the assay or is below a level considered safe or tolerable. The samples used in the test method of the invention can be any sample suspected of exposure to toxins that bind to the phosphatase, probably by exposure to microorganisms producing toxins that bind to the phosphatase, for example water that can be seawater, freshwater, groundwater, water taken from lakes, rivers, wells, streams, reservoirs, domestic water supplies or can be extracted from the moisture of molluscs for example by simple draining or extraction using a pipette or water in which the mollusc has been allowed to soak or can be a food product, food supplement, food supplement, alternative remedy or similar product which is produced by or with algae or cyanobacteria. Where the mollusk contains free water (for example in oysters), the assay may involve immersing an absorbent substrate
(the solid support) in the water. Alternatively, this may simply involve the pressure of an absorbent substrate against the wet pulp of the mollusk, for example after breaking or opening the shell. In a preferred aspect of the invention, the sample subject to investigation is the mollusc's moisture-free surface. All types of molluscs, for example the shells, shrimp, mussels, and oysters are susceptible to the test method of the invention but in a preferred aspect, the molluscs are mussels. In another preferred aspect, the sample under investigation is water taken from the habitat in which the molluscs live and in a further preferred aspect, the sample is water taken from domestic water suppliers. The sample used for the analysis can be used essentially in an untreated form but optionally can be filtered by any known method or diluted by the addition of water, buffer or any other aqueous medium prior to analysis and can be stored or preserved for example by cooling or freezing prior to analysis. As immobilized or non-immobilized ligand, any toxin-binding ligand can be used in the method of the invention, for example antibodies, which can be monoclonal or polyclonal, or fragments of antibodies, for example F (ab), F ( ab ') 2 or F (b). Such antibodies or antibody fragments can be monovalent or divalent and can be produced by hybridoma technology or be of synthetic origin, either as products of recombinant DNA technology or by chemical synthesis. For example, single-chain antibodies or other antibody derivatives or imitations thereof could be used. The antibodies or antibody fragments can be directed or stimulated against any epitope, component or structure of the toxins that bind to the phosphatase when appropriate. Alternatively, compounds with an affinity for the toxin could be used for example a small organic molecule or peptide, for example, an oligopeptide or polypeptide, capable of specifically binding to the toxin for example a specific linker selected from a library combinatorial or phage display chemistry or a binding sequence specifically of .DNA or RNA. Preferably, however, the toxin binding ligand of the present invention is a protein phosphatase enzyme, and even more preferably the protein-phosphatase binding ligand 2A (pp2A) is used in the assay method. Likewise, the second ligand used in the method of the invention can be any ligand that binds to the toxin competitively or non-competitively with the first ligand. Alternatively, the second ligand can be any ligand that competes with the toxin for binding to the first ligand. Preferably the first ligand is a toxin-binding ligand, more preferably a protein-phosphatase enzyme. One of the two ligands can be found immobilized and the other can be detectable directly or indirectly. In a preferred embodiment, the non-immobilized ligand must fulfill the functional requirements of competitively inhibiting the binding of the toxin to the immobilized ligand and can directly or indirectly produce a detectable signal, for example this can be a molecule which can be labeled using a direct or indirect signal constituting itself as part of any known form. Such ligands may also take the form of antibodies, which may be polyclonal or monoclonal, or fragments of antibodies for example the F (ab), F (ab ') 2 or F (b) fragments. Such antibodies or antibody fragments can be monovalent or divalent and can be produced by hybridoma technology or be of synthetic origin, either recombinant DNA technology or chemical synthesis. For example, single-chain antibodies or other derivatives of antibodies or imitations and small organic molecules, peptides, oligopeptides and polypeptides selected from combinatorial or phage display libraries could be used. Antibodies or antibody fragments can be directed or stimulated against any epitope, component or structure of the toxin molecule that binds to the phosphatase or the ligand that binds to the molecule that bind to the phosphatase when appropriate. Alternatively, compounds could be used with an affinity for the toxin or for the ligand which binds to the toxin, for example a small organic molecule or peptide, oligopeptide or polypeptide capable of specifically binding to the toxin or to the ligand that binds to the toxin. toxin, for example a specific linker selected from a combinatorial or phage display library, or a DNA or RNA binding sequence. The reporter part which can generally carry with it one of the ligands can be a binding site for a directly detectable part, for example a metal sol (for example gold sol), a chromosorph or fluorophore (for example cyanine, phthalocyanine, merocenintin, triphenylmethyl, equinancin, etc. see Topics in Applied Chemistry, Infrared. Absorbing Chromophores, edited by M. Matsuoka, Plenum Press, New York, NY, 1990, Topics in Applied Chemistry, The Chemistry and Application of Dyes, Waring et al., Plenum Press, New York, NY, 1990, and Handbook of Fluorescent Probes and Research Chemicals, Hautgland, Molecular Probes Inc. 1996, a radioactive label, an enzyme, a magnetic particle, a turbidity-inducing agent, etc., or this can already carry a directly detectable part.The immobilized ligand where the reporter part is carried would generally be a binding site for a directly detectable part whose binding site is It is either activated, or more generally deactivated, when the ligand is joined. Preferably, the reporter part is carried by the non-immobilized ligand. In a preferred embodiment of the invention, the non-immobilized ligand is a labeled one, for example peptide enzyme or peptide hepatotoxins labeled with chromophor or fluorophore, for example a hepatotoxin selected from nodularin, microcystin-LC or microcystin-YR or alternatively okadaic acid. While labeling with radioactive markers is possible, given that the test is primarily intended to be used on the site by non-professional users, the use of reporter parts that give a visual signal is preferred, for example chromophores, fluorophores, parts phosphorescent, turbidity-inducing agents, agents that induce the evolution of gas, etc. Where the signal-forming part is a material which binds to a binding site in one of the ligands, it will be conveniently contacted with the bound or unbound fraction, when appropriate, after separation of the ligand. the united and unbound fractions. In general, where the signal to be derived from the bound fraction is found, it will preferably be found to rinse the substrate, for example with water, to entrain the non-bound fraction before the ligand is detected or generated and detected. Any species or strain of algae or cyanobacterium which produces toxins that bind to the phosphatase can be subjected to the present invention but this is particularly applicable to the toxin produced by strains of cyanobacteria eg Microcystis aeroginosa, Anabena species , Nodularia spuragena and Anabena fl us-aquae or algae. For example, the microcystin-LR and microcyst-YR toxins are produced by My crocysti sp., The nodularin toxin is produced by Nodul aria sp. and the okadaic acid toxin is produced by Prorocentrum sp. The toxins subjected to the determination by means of the present method can also be any toxin whose target is the phosphatase produced by an alga or cyanobacterium, but in preferred aspects the peptide toxins are hepatotoxins (of which microcystin and nodularin are the most prevalent). ) or okadaic acid. Thus, in its most general sense, the method of the invention involves simply contacting a suspect sample of contamination with toxins that bind to the phosphatase, with a toxin-binding ligand and a reporter molecule capable of competing with said toxin by the binding sites of the ligands either simultaneously, sequential or separated in any order, the reporter molecule being optionally linked to the binding ligand, prior to exposure to the sample under investigation, and determination of the reporter molecule which is bound to the solid or free phase in the solution. The bound fraction can be separated from the unbound fraction, upon estimation of the reporter by any suitable means, for example, precipitation, centrifugation, filtration, chromatography means, capillary action or simply by draining. The solid phase can be, for example, in the form of a rod or a solid matrix in any known form, for example polymeric or magnetic beads, for example Dynabeads® (available from Dynal AS). In the preferred embodiments of the present invention, the solid phase in which the toxin-binding ligands are immobilized is in the form of Dynabeads®.
The reporter molecule can be estimated either in the bound or unbound fraction depending on the specific embodiment of the invention but preferably it is estimated in the bound fraction. The immobilized ligand can be immobilized by any known means, for example by binding or pairing the ligand to any of the well-known solid supports or matrices that are currently widely used or proposed for separation or immobilization, for example, the solid phases can take the shape of particles, sheets, gels, filters, membranes, fibers or capillaries or microtitre strips, tubes or well plates etc. and conveniently they can be made of glass, silica, latex, a polymeric material or magnetic beads. The techniques for binding the ligand to the solid support are well known in the art widely described in the literature. In the preferred embodiments of the present invention, the solid phase in which the binding ligands to the toxin that binds to the phosphatase are immobilized is in the form of Dynabeads®. The test method of the present invention is advantageous in that it can be carried out without the need for complex laboratory equipment and can be carried out by relatively inexperienced or inexperienced persons. Therefore, the test method is suitable for use at home, in stores or in the field and this can be done quickly and easily without the need for labor intensive and hazardous chemicals. The high degree of sensitivity which is of critical importance when analyzing samples where the toxin is present at very low levels, for example in the drinking water test or assessment of possible contamination with toxins that bind to the phosphatase is of particular advantage in the assay of the present invention. Typically the assay is capable of detecting toxins in picomolar concentrations, for example as low as 10 pM. Conveniently the assay can be used to detect toxins in the range of 15 to 560 pM. A further advantage of the present assay related to the existing techniques is that the present assay is not affected by the presence of endogenous phosphatases that may be present in the samples under analysis, particularly, for example, if the samples are taken from molluscs. In another embodiment of the present invention, a protein phosphatase is immobilized on a solid support, the immobilized phosphatase is contacted with the sample under investigation and any toxin whose target is the phosphatase that is present in the sample binds to the immobilized phosphatase. A source of reporter molecules that compete with the toxin for the phosphatase binding sites is added. The reporter molecules displace the toxin molecules from the binding sites to a degree that depends on the relative concentration of the toxin molecules and the reporter molecules. The degree of reporter molecules that join facilitates the determination of the toxin present in the sample under investigation. Reporters / preferred markers include radioactive labels, chromophores, (including fluorophores) and enzymes that give stimulation to chromogenic or fluorogenic products. Proximal titration markers and markers that give stimulation for a measurable change in light scattering are also considered. In an alternative embodiment, the phosphatase molecules blocked by the reporter immobilized on the solid support are contacted with the sample under investigation and any toxin whose target is the phosphatase present in the sample competes with reporter molecules bound to the phosphatase by displacing they are of the solid phase in the aqueous phase to a degree proportional to the amount of toxin present in the sample. The amount of reporter molecule that remains bound to the solid phase is then estimated to facilitate the determination of the presence of the toxin in the sample under investigation. Seen from a further aspect, the invention provides an equipment for the detection of toxins that bind to the cyanobacteria phosphatase or algae, according to the invention, the equipment comprises: a solid phase on which a ligand is immobilized;
a non-immobilized ligand, preferably in aqueous solution or bound to the immobilized ligand; where none of the immobilized and non-immobilized ligands include a directly or indirectly detectable part, a reporter part capable of binding to one of said immobilized and non-immobilized ligands and generating a detectable signal, said detectable part or signal being readable without laboratory equipment. In a preferred embodiment, the kit of the present invention comprises: a solid phase on which the binding ligands are immobilized to the toxin that binds to the phosphatase; a reporter molecule capable of competitively inhibiting the binding of the toxins that bind to the phosphatase to the toxin-binding ligand and that generates a readable signal without laboratory equipment. A particularly preferred embodiment of the equipment of the invention comprises magneticly displaceable polymer microspheres having immobilized therein a protein phosphatase; Gold-labeled peptide hepatotoxin molecules capable of competitively inhibiting the toxins of cyanobacteria that bind to protein-phosphatase. A further preferred embodiment of the equipment of the invention comprises magneticly displaceable polymer microspheres which have immobilized therein a protein phosphatase;
Okadaic acid molecules marked with sol de oro capable of competitively inhibiting the toxins of algae that bind to protein-phosphatase. In another preferred aspect, the use of the equipment involves immersing in a sample of water or mollusc fluid, a porous cellulosic substrate in which a toxin-binding ligand is immobilized and impregnated with a ligand labeled with chromophor (or fluorophore). etc.) competitively bound, allowing the saturated substrate to incubate for a predetermined period (removed from the sample or at a predetermined volume of the sample), removing unbound labeled ligand, for example by washing the substrate with water free of the toxin or leaving the substrate to soak for a predetermined period in a predetermined volume of toxin-free water, and inspecting the color of the substrate or the soaking water. Desirably, the substrate is mounted on a support, preferably one marked with calibration colors to facilitate comparing the color of the substrate or the soaking water, to determine the concentration of the toxin or to indicate whether the concentration of the toxin is present. above or below one or more of the minimum concentration. The invention will now be illustrated by the following non-limiting examples:
materials
The microcystin-YR, Microcystin-LR, okadaic acid, nodularin, caliculin A and tautomycin are purchased from Calbiochem (San Diego, CA). The Na125I free of Harvester and the [? -32P] ATP are obtained from .Amersham (Little Chalfont, UK). Albumin (RIA class), ammonium acetate, Chloramine T, dimethyl sulfoxide (DMSO), dithioerythritol (DTE), EDTA, EGTA, glycerol, Hepes, histone II-AS, sodium metabisulfite, and trypsin inhibitor (soybean) They are acquired from Sigma (St. Louis, MO). Acetonitrile and trifluoroacetic acid (TFA) are purchased from Rathburn (Walkerburn, Scotland). The partially purified type 2A protein phosphatase is purchased from Upstate Biotechnology (Lake Placid, NY) or purified in accordance with Resink et al. (Eur. J. Biochem. 133: 455-461 (1983)).
Iodinization of microcystin-YR Microcystin-YR (10 μg) was iodine with 1 mCi of Na ~ 25I (37 MBq) carrier-free using chloramine T as described by Ciechanover et al., (PNAS 77: 1365-1368 (1980 )). After the iodination reaction, iodine is separated from [1Z5I] | microcystin-YR using Sep-Pak® Plus cartridges (Waters, Milford, MA) in accordance with the method of Runnegar et al. (Toxicon 24: 506-509 (1986)). The [1 5I] microcystin-YR is applied to a column of Inertsil ODS-2 HPLC 3 x 250 mm Chrompack (Raritan, NJ) and eluted with a gradient of acetonitrile.
Competitive union test
The competitive binding assay is carried out in a volume of 0.5 ml buffered with 50 mM Hepes (pH 7.2), lmM EDTA, 0.3 mM EGTA, 1 mM DTE, 5 mM MnCl2, 0.5 mg ml "1 BSA and 0.2 mg ml "1 of trypsin inhibitor. Alga toxins are diluted in 100% DMSO and added to the assay at 0-100 nM for a final concentration of 10% DMSO. Add [125I] microcystin-YR (1 Ci / 13 ng) to 35 pM. Protein-phosphatase 2A (30 pM) is added to the end and the reaction mixture is incubated on ice overnight. [125I] microcystin-YR bound to protein-phosphatase 2A is separated from [125I] free microcystin-YR by gel filtration using Sephadex® G-50 fine from Pharmacia (Uppsala, Sweden) in 0.7 x 15 cm columns of Bio-Rad (Hercules, CA). A 50 mM Hepes buffer (pH 7.2) with lmM of EDTA and 0.3 mM of EGTA is used in the separation which is carried out at 4 ° C. The fraction containing [125I] microcystin-YR that binds to the protein-phosphatase 2A, is collected and the radioactivity is quantified by titration counting. The non-specific binding of [l25I] microcystin-YR is detected in a control reaction where the microcystin-YR is added to the excess (lM).
Example 1
The protein-phosphatase 2A is coupled to the magnetic beads (directly to the beads or biotinylation pathway of the phosphatase). The immobilized protein-phosphatase is then mixed with the sample and the radioactively labeled toxin (e.g. [12DI] microcystin-YR). The immobilized protein phosphatase is separated from the reaction mixture by magnetic force. The radioactivity associated with the protein phosphatase (magnetic beads) is detected by titration counting. The amount of the radioactive label associated with the protein phosphatase in the sample decreases as a function of the toxin that binds to the phosphatase.
Example 2
The protein-phosphatase 2A is coupled to the magnetic beads (directly to the beads or biotinylation pathway of the phosphatase). The immobilized protein phosphatase is then mixed with the sample and the toxin is coupled to the colored beads. The immobilized protein phosphatase is separated from the reaction mixture by magnetic force. The colored beads associated with the protein phosphatase (magnetic beads) are evaluated by sight or by a low magnification microscope (e.g., Nikon TMS). The amount of colored beads associated with the protein phosphatase (magnetic beads) decreases in the sample as a function of the toxin that binds to the phosphatase.
Example 3
The protein-phosphatase 2A is coupled to the magnetic beads (directly to the beads or biotinylation pathway of the phosphatase). The immobilized protein-phosphatase is then mixed with the sample and the immobilized toxin in beads carrying an immobilized enzyme. When properly incubated with a chromogenic or fluorogenic substrate, the enzyme is capable of producing a detectable product (colored or fluorescent). The immobilized protein phosphatase is separated from the reaction mixture by magnetic force. The color or fluorescence associated with the protein phosphatase (magnetic beads) is measured respectively by spectroscopy or fluorimetry. The amount of color / fluorescence associated with the magnetic beads decreases in the sample, as a function of the toxin that binds to the phosphatase.
Example 4
Proximal Titilation Assay: The protein phosphatase is biotinylated and immobilized to wells previously covered with streptavidin and a titilant (for example FlashPlate PLUS Streptavidin SMP103 from the NEN provider). The sample and the [125I] microcystin-YR are added to the wells. The amount of [125I] microcystin-YR bound to the immobilized protein-phosphatase is detected by titration counting.
Example 5
Inhibition of the binding of [125I] microcystin-YR to protein-phosphatase 2A in the presence of various toxins
Compound tested IC5cf (pM) nodularin 15 microcystin-LR 17 microstine-YR 75 okadaic acid 100 caliculin A 251 tautomycin 562 1 The tested compounds were incubated with [125I] microcyst ma-YR and protein-phosphatase 2A as described above. The IC50 value represents the concentration necessary to obtain a 50% inhibition of [125I] microcystin-YR bound to protein-phosphatase 2A. These values were determined according to Figure 3. The data represent an average of at least 3 separate experiments.
EXAMPLE 6 Effect of Exogenous Compounds in the Competitive Binding Assay in Comparison with the Protein-Phosphatase Assay Compounds Tested1 for Activity "" Binding Assay Competitive Protein-Phosphatase Assay
2 mM ATP 103.3 ± 0.2 9.8 ± 3.4
0. 5 mM ATP 101.6 ± 1.7 29.8 ± 5.6
0. 05 mM NaPPi 101.4 ± 4.1 14.2 ± 1.2
50 mM NaF 101.5 ± 1.9 7.7 ± 1.4
mM NaF 102.0 ± 3.3 62.6 ± 0.4
1 mg / ml casein 98.6 ± 4.5 3.4 ± 0.2
0. 02 mg / ml casein 98.9 ± 6.1 33.3 ± 4.9
mg / ml histone 2A 91.9 ± 1.8 1.4 ± 0.1
0. 002 mg / ml histone 95.2 ± 4.7 63.6 ± 4.0
0. 5 M NaCl 41.2 ± 0.7 44.4 ± 1.6
Seawater 34.8 ± 0.4 ND 10% seawater 87.3 ± 0.4 ND 10% DMSO 72.8 ± 2.3 97.9 ± 3.3
% MeOH 73.9 ± 0.5 87.4 ± 4.1
% acetonitrile 90.4 ± 5.4 88.2 ± 2.7
0. 4% Triton x-100 122.3 ± 1.0 60.2 ± 5.7
0. 4% of Nonidet P-40 106.0 ± 2.0 61.1 ± 1.3
0. 4% of CLAPS 90.0 ± 9.9 138.0 ± 34.4
The protein-phosphatase 2A was previously incubated with the compounds dissolved in 50mM Hepes (pH 7.2) or with the buffer alone (control) for 30 minutes on ice. The phosphatase activity was measured by dephosphorylation of the phosphohistone as described. The percentage of activity is related to the control reaction. 2 Activity in the competitive binding assay represents the ability of protein-phosphatase 2A to bind to [i2DI] microcyst-YR in the presence of the dissolved exogenous compound
in the shock absorber in relation to the shock absorber alone.
The data represent an average ± EMS of at least 3 separate experiments.
fifteen
twenty
¡¡H ^ Example 7 Sensitivity of the nodularin and microcystin-LR binding assay Percentage of inhibition of the binding of [125I] microcystin-YR1 Toxin (M) MiliQ water Drinking water Seawater Seawater, 1 / 102 Nodularine 1E-10 88.37 ± 0.31 88.75 ± O.le 72.18 ± 0.82 67.24 ± 0.66 00 5E-11 36.37 ± 2.28 36.12 ± l.?4 48.47 ± 0.79 52.98 ± 1.98 Microcysteine-LR 1E-10 84.91 ± 0.42 86.97 ± 1.12 73.31 ± 1.20 46.41 ± 5.97 5E-11 13.87 ± 3.16 12.85 ± 0.88 49.34 ± 3.82 38.61 ± 1.49 1 Nodularin and microcystin-LR were dissolved in water: milliQ, drink or sea in the concentration shown. Aliquots with 300 μl of these solutions were tested for their ability to compete for the binding of [125I] microcystin-YR with protein-phosphatase 2A as described above. 2 Dilution of sea water 1/10 was done with miliQ water.
The data represent the average ± EMS.
Example B
Equivalent of okadaic acid in mollusc extracts as determined by HPLC analysis and by the protein-phosphatase binding assay Extract 'AO equivalents by AO equivalents by HPLC analysis2 the Hepatopancreas binding assay μg / g (nM). nM)
1 0 0 85 2 0 0 45 3 0 0 70 4 4 2480 2100
1.2 748 755
6 0.8 496 805
1 The extracts were made from the hepatopancreas of mussels collected along the Norwegian coast. The extracts were analyzed by equivalents of okadaic acid by HPLC. 3 The extracts were diluted in 100% DMSO and tested for their ability to compete for the binding of [125I] microcystin-YR with protein-phosphatase 2A using the binding assay described above. The concentration of the okadaic acid equivalents was determined by comparing data with the standard curves of okadaic acid dissolved in 100% DMSC.
• ÉkiÉMiiiáiM Example 9
Annex diagrams Figure 1 of the attached diagram is a schematic diagram of the competitive binding assay for the detection of protein-phosphatase binding toxins. The protein-phosphatase 2A is incubated with [125I] microcystin-YR and another toxin directed towards the protein-phosphatase 2A. The toxin competes with the [125I] microcystin-YR to bind to the phosphatase. Adding large amounts of the toxin results in a reduction of the binding of [125I] microcystin-YR to the phosphatase and vice versa. After reaching equilibrium in the union, [125I] microcystin-YR bound to protein-phosphatase 2A is separated from [125I] free microcystin-YR by gel filtration chromatography. The fraction containing the [125I] microcystin-YR bound to the phosphatase is collected and the amount of radioactivity is determined by titration counting. Figure 2 of the accompanying diagrams demonstrate the effects of increasing amounts of toxins from different algae in the binding of [125I] microcystin-YR to protein-phosphatase 2A. The figure shows how the protein-phosphatase 2A (30 pM) was incubated in the presence of 35 pM of the [125I] microcystin-YR (1 Ci / 13 ng) and 0-100 nM of the toxins of the different algae. The [125I] microcystin-YR bound to the protein-phosphatase
2A was isolated by gel filtration chromatography and the radioactivity was determined by titration counting. Each curve represents the average of at least 3 separate experiments. Figure 3 of the attached diagrams shows the IC5: for the raicrocystin-LR that binds in the competitive binding assay. The binding of [125I] microcystin-YR to protein-phosphatase 2A was plotted as the ratio between [125I] microcystin-YR (Co-Cx) unbound and [~ 2ol] microcystin-YR (Cx) bound against the concentration of microcystin-LR. Co represents the amount of [125I] microcyst-YR bound in the absence of microcystin-YR and Cx represents the amount of [125I] microcystin-YR bound in the presence of various concentrations of microcystin-LR. Figure 4 of the accompanying diagrams illustrates the stability of [125I] microcystin-YR bound to protein-phosphatase 2A in the presence of excess microcystin-LR. Protein-phosphatase 2A (InM) was incubated in the presence of [125I] microcystin-YR (100 pM) for one hour. The microcystin-LR (2 μM) was added to the reaction mixture at time 0. The amount of [125I] microcystin-YR bound to the protein-phosphatase 2A was determined by the times indicated by gel filtration and titration counting as indicated. has described. The curve represents an average of 4 separate experiments. It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.
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Claims (20)
1. A test method for the determination of phosphatase binding toxins that inhibit protein phosphatases comprising contacting a solid support having a ligand immobilized on the support with: (i) a sample suspected of being contaminated with the toxin and (ii) a non-immobilized ligand, characterized in that the immobilized ligand is capable of binding with at least one of the toxins mentioned, to the non-immobilized ligand or to the complexes of the toxin and the non-immobilized ligand, and wherein the Non-immobilized ligand is capable of binding to at least one of the immobilized ligands, toxin or complexes of the toxin and immobilized ligand whereby the proportion of the immobilized ligand bound by the toxin, the non-immobilized ligand or the complexes of the toxin and non-immobilized ligand is dependent on the toxin content of the sample and wherein the ligand that binds to the immobilized or non-immobilized toxin is an enzyme rotein-phosphatase and wherein the immobilized ligand is capable of directly or indirectly generating a detectable signal when it is not complexed, when it forms a complex by the toxin, when it forms a complex by the toxin complex and the ligand does not immobilized or when it forms a set by the non-immobilized ligand or the non-immobilized ligand is able to directly or indirectly generate a detectable signal when it is not forming a complex or when it is forming a complex, the separation of a bound fraction from a fraction not united; and determining directly or indirectly the non-immobilized ligand bound to the immobilized ligand (the bound fraction) or non-bound in aqueous solution (the non-bound fraction); wherein the application of (i) and (ii) to the solid support can be carried out separately, sequentially or simultaneously and, separately or sequentially, it can be carried out in any order.
2. An assay method according to claim 1, characterized in that said toxin that binds to the phosphatase is produced by algae or cyanobacteria.
3. A test method according to any of claims 1 and 2, characterized in that the toxin to be determined is a hepatotoxin or okadaic acid.
4. A test method according to any of claims 1 to 3, characterized in that the toxin molecules present in the sample compete with the non-immobilized ligand for a limited number of binding sites of the immobilized ligand and any toxin present in said mixture is determines in relation to the degree of non-immobilized ligand bound or not to the binding sites of the immobilized ligand.
5. An assay method according to any of claims 1 to 4, characterized in that the absence or presence of toxin that binds to the phosphatase is determined.
6. An assay method according to any of claims 1 to 5, characterized in that the sample subject to investigation is either the moisture-free surface of the mollusc, or water taken from the habitat in which the mollusc lives, or water taken from domestic water suppliers.
7. A test method according to any of claims 1 to 6, characterized in that immobilized or non-immobilized ligand is an antibody or antibody fragment.
8. An assay method according to any of claims 1 to 7, characterized in that the protein-phosphatase enzyme is the protein-phosphatase binding ligand 2A.
9. A test method according to any of claims 1 to 8, characterized in that either the immobilized or non-immobilized ligand carries a reporter part.
10. A test method according to claim 9, characterized in that the non-immobilized ligand brings with it a reporter part.
11. An assay method according to claim 10, characterized in that the non-immobilized ligand is a labeled peptide hepatotoxin or labeled okadaic acid.
12. A test method according to claim 11, characterized in that the hepatotoxin is selected from nodularin, microcystin-LC or microcystin-YR.
13. A test method according to any of claims 1 to 12, characterized in that the solid support is a rod or solid matrix.
14. A test method according to claim 13, characterized in that the solid matrix is a polymeric material or magnetic beads.
15. A device for the detection of toxins that bind to the phosphatase that inhibits the protein phosphatases, the device characterized in that it comprises: a solid phase on which a ligand is immobilized; a non-immobilized ligand, preferably in aqueous solution or bound to the immobilized ligand; and wherein the immobilized and / or non-immobilized ligand is a protein-phosphatase enzyme. where none of the immobilized and non-immobilized ligands include a directly or indirectly detectable part, a reporter part capable of binding to one of said immobilized and non-immobilized ligands and generating a detectable signal, said detectable part or signal being readable without laboratory equipment
16. A kit according to claim 15, characterized in that the toxin that binds to the phosphatase is produced by algae or cyanobacteria.
17. Equipment according to any of claims 15 and 16, characterized in that the equipment comprises: a solid phase on which a protein-phosphatase enzyme is immobilized as a toxin-binding ligand; a reporter molecule capable of competitively inhibiting the binding of the toxins that bind to the phosphatase to the toxin-binding ligand and that generates a readable signal without laboratory equipment.
18. Equipment according to any of claims 15 to 17, characterized in that said equipment comprises magnetically displaceable polymeric microspheres having immobilized therein a protein phosphatase; Gold-labeled peptide hepatotoxin molecules capable of competitively inhibiting the toxins of cyanobacteria that bind to protein-phosphatase.
19. An equipment according to any of claims 15 to 17, characterized in that said equipment comprises magnetically displaceable polymeric microspheres having immobilized therein a protein phosphatase; Okadaic acid molecules marked with sol de oro capable of competitively inhibiting the toxins of algae that bind to protein-phosphatase.
20. The use of an equipment according to any of claims 15 to 19, for the determination of toxins that bind to the phosphatase.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9824772.9 | 1998-11-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA01004700A true MXPA01004700A (en) | 2002-07-25 |
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