Patent EA-002710-B1 (EA)
Title: Assay for phosphatase-targeting toxins
Inventor: DOSKELAND STEIN OVE
IPC: G01N33/53
Filed: 1999-11-11
Abstract: 1. 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 binding to at least one of said toxins, to said non-immobilized ligand or to complexes of said toxin and said non-immobilized ligand, and said non-immobilized ligand is capable of binding to at least one of said immobilized ligand, to said toxin or to complexes of said toxin and said immobilized ligand whereby the proportion of said immobilized ligand bound by said toxin, said non-immobilized ligand or complexes of said toxin and said non-immobilized ligand is dependent on the toxin content of said sample and wherein the immobilized and/or non-immobilized toxin binding ligand is a protein phosphatase enzyme and 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) ; wherein the application of (i) and (ii) to the solid support may be performed separately, sequentially or simultaneously and if separately or sequentially, they can be performed in either order. 2. An assay method as claimed in claim 1 wherein said phosphatase-targeting toxin is produced by algae or by cyanobacteria, and, preferably, wherein the toxin to be determined is a heptatoxin or okadaic acid. 3. An assay method as claimed in any one of claims 1 to 2 wherein 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 sample is determined relative to the extent of non-immobilized ligand bound to or not bound to the binding sites of the immobilized ligand. 4. An assay method as claimed in any one of claims 1 to 3 wherein the presence or absence of a phosphatase-targeting toxin is determined. 5. An assay method as claimed in any one of claims 1 to 4 wherein the sample under investigation is surface or free moisture from shellfish, or water taken from the habitat in which such shellfish live, or water taken from domestic water supplies. 6. An assay method as claimed in any one of claims 1 to 5 wherein the immobilized or non-immobilized ligand is an antibody or antibody fragment. 7. An assay method as claimed in any one of claims 1 to 6 wherein the protein phosphatase enzyme is the binding ligand protein phosphatase 2A. 8. An assay method as claimed in any one of claims 1 to 7 wherein either the immobilised or non-immobilised ligands carries a reporter moiety and, preferably, wherein the non-immobilized ligand carries a reporter moiety. 9. An assay method as claimed in claim 8 wherein the non-immobilized ligand is a labelled peptide hepatotoxin or labelled okadaic acid and wherein the hepatoxin is preferably selected from nodularin, microcystin LC or microcystin YR. 10. An assay method as claimed in any one of claims 1 to 9 wherein the solid support is a dipstick or solid matrix and wherein the solid matrix is preferably polymeric or magnetic beads. 11. A kit for the detection of phosphatase-targeting toxins which inhibit protein phosphatases, said kit comprising: a solid phase upon which is immobilised a ligand; a non-immobilized ligand, preferably in aqueous solution or complexed to the immobilized ligand; and wherein the immobilized and/or non-immobilized ligand is a protein phosphatase enzyme; where neither of said immobilized and non-immobilized ligands includes a directly or indirectly detectable moiety, a reporter moiety capable of binding to one of said immobilized and non-immobilized ligands and generating a detectable signal, preferably said. detectable moiety or signal being directly readable without laboratory equipment. 12. A kit as claimed in claim 11 wherein said phosphatase-target ing toxin is produced by algae or by cyanobacteria. 13. A kit as claimed in either of claims 11 and 12 wherein said kit comprises: a solid phase upon which is immobilized a protein phosphatase enzyme as a toxin binding ligand; a reporter molecule capable of competitively inhibiting binding of phosphatase-targetfcing toxins to said toxin binding ligand and generating a signal readable without laboratory equipment. 14. A kit as claimed in any one of claims 11 to 13 wherein said kit comprises magnetically displaceable polymer micro spheres having immobilized thereon a protein phosphatase; gold sol labelled peptide hepatofcoxin molecules capable of competitively inhibiting cyanobacterial toxins binding to said protein phosphatase. 15. A kit as claimed in any one of claims 11 to 13 wherein said kit comprises magnetically displaceable polymer micro spheres having immobilized thereon a protein phosphatase; gold sol labelled okadaic acid molecules capable of competitively inhibiting algal toxins binding to said protein phosphatase. 16. Use of the kit as claimed in any one of claims 11 to 15 for the determination of phosphatase-targeting toxins.