Saxitoxin detection and assay method
AssigneeYale University
InventorsEdward Moczydlowski, Gomathi Krishnan, Maria A. Morabito
Life of this patent
Estimating the term…
Abstract
Tagged saxiphilin and tagged fragments thereof that bind saxitoxin and saxitoxin derivatives, methods of producing tagged saxiphilin and tagged fragments and methods of detecting and measuring saxitoxin and saxitoxin derivatives in samples.
Claims54
A method of detecting saxitoxin (STX) or STX derivatives in a sample, comprising:
- (a) contacting a tagged saxiphilin or a tagged fragment thereof which binds STX or a STX derivative with a sample to be assessed under conditions appropriate for binding of tagged saxiphilin or a tagged fragment thereof with STX or a STX derivative, and
- (b) determining whether binding of tagged saxiphilin or a tagged fragment thereof to STX or a STX derivative occurs in (a),
- wherein if binding occurs, then STX or a STX derivative is detected in the sample.
A method of detecting STX in a sample, comprising:
- (a) combining a saturating concentration of tagged saxiphilin or a tagged fragment thereof which binds STX or a STX derivative bound to a solid support with labeled STX and increasing amounts of a sample containing unlabelled STX;
- (b) measuring the amount of labeled STX bound to the solid support;
- and
- (c) determining the amount of unlabelled STX in the sample,
- wherein STX is detected in the sample by comparing the amount of labeled STX bound to the solid support with the amount of labeled STX bound to the solid support in an appropriate control.
A method of detecting STX in a sample, comprising:
- (a) combining a saturating concentration of tagged saxiphilin or a tagged fragment thereof which binds STX or a STX derivative with labeled STX and increasing amounts of a sample containing unlabelled STX within the wells of a metal-chelate microtitre plate;
- (b) measuring the amount of labeled STX bound to each well;
- and
- (c) determining the amount of unlabelled STX in the sample,
- wherein STX is detected in the sample by comparing the amount of labeled STX bound to each well with the amount of labeled STX bound to each well in an appropriate control.
Tagged saxiphilin or a tagged fragment thereof which binds STX or a STX derivative, comprising saxiphilin or a fragment thereof, and a tag selected from the group consisting of:
- (a) a Flag epitope;
- (b) a hexahistidine sequence;
- (c) a streptavidin sequence (Strep-tag);
- (d) calmodulin binding peptide (CBP);
- (e) maltose binding protein (MBP);
- (f) cellulose binding domain (CBD) tag;
- (g) myc epitope;
- (h) hemmaglutinin (HA) tag;
- (i) glutathione-S-transferase (GST) fusion;
- and (j) thioredoxin fusion, and combinations of any of (a)-(j).
Tagged saxiphilin comprising the amino acid sequence of SEQ ID NO:
- 12 or SEQ ID NO:
- 14.
Isolated nucleic acid comprising a nucleic acid sequence that encodes a tagged saxiphilin or a tagged fragment thereof which binds STX or a STX derivative, comprising saxiphilin or a fragment thereof, and a tag selected from the group consisting of:
- (a) a Flag epitope;
- (b) a hexahistidine sequence;
- (c) a streptavidin sequence (Strep-tag);
- (d) calmodulin binding peptide (CBP);
- (e) maltose binding protein (MBP);
- (f) cellulose binding domain (CBD) tag;
- (g) myc epitope;
- (h) hemmaglutinin (HA) tag;
- (i) glutathione-S-transferase (GST) fusion;
- and (j) thioredoxin fusion, and combinations of any of (a)-(j).
Isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO:
- 11 or SEQ ID NO:
- 13.
A vector comprising a nucleic acid encoding tagged saxiphilin or a tagged fragment thereof which binds STX or a STX derivative, wherein the tagged saxiphilin or tagged fragment thereof comprises a tag selected from the group consisting of:
- (a) a Flag epitope;
- (b) a hexahistidine sequence;
- (c) a streptavidin sequence (Strep-tag);
- (d) calmodulin binding peptide (CBP);
- (e) maltose binding protein (MBP);
- (f) cellulose binding domain (CBD) tag;
- (g) myc epitope;
- (h) hemmaglutinin (HA) tag;
- (i) glutathione-S-transferase (GST) fusion;
- and (j) thioredoxin fusion, and combinations of any of (a)-(j).
Host cells comprising an exogenous nucleic acid encoding tagged saxiphilin or a tagged fragment thereof which binds STX or a STX derivative, wherein the tagged saxiphilin or tagged fragment thereof comprises a tag selected from the group consisting of:
- (a) a Flag epitope;
- (b) a hexahistidine sequence;
- and (c) a streptavidin sequence (Strep-tag);
- (d) calmodulin binding peptide (CBP);
- (e) maltose binding protein (MBP);
- (f) cellulose binding domain (CBD) tag;
- (g) myc epitope;
- (h) hemmaglutinin (HA) tag;
- (i) glutathione-S-transferase (GST) fusion;
- and (j) thioredoxin fusion, and combinations of any of (a)-(j).
Method of producing tagged saxiphilin or a tagged fragment thereof comprising:
- culturing cells that contain nucleic acid encoding tagged saxiphilin or a tagged fragment thereof under conditions appropriate for expression of the nucleic acid, thereby producing tagged saxiphilin or a tagged saxiphilin fragment.
A kit for detecting the amount of saxitoxin or saxitoxin derivatives in a sample comprising:
- (a) tagged saxiphilin or tagged saxiphilin fragment;
- and
- (b) labeled STX or labeled STX derivative.
A kit for detecting the amount of saxitoxin or saxitoxin derivatives in a sample comprising:
- (a) tagged saxiphilin or tagged saxiphilin fragment immobilized on a solid support;
- and
- (b) labeled STX or labeled STX derivative.
A kit for measuring the amount of saxitoxin or saxitoxin derivatives in a sample comprising:
- (a) tagged saxiphilin or tagged saxiphilin fragment;
- and
- (b) labeled STX or labeled STX derivative.
A kit for measuring the amount of saxitoxin or saxitoxin derivatives in a sample comprising:
- (a) tagged saxiphilin or tagged saxiphilin fragment immobilized on a solid support;
- and
- (b) labeled STX or labeled STX derivative.
Dependencies are inferred from the claim text.