Immunoassay element and process and immunoassay
First Claim
1. An immunoassay element for quantitatively analyzing a ligand by determining a change in enzymatic activity of a labelling enzyme caused by a competitive reaction betweenthe ligand anda linked product comprising(i) the ligand or a derivative thereof having a common antigenic determinant with the ligand and(ii) a high molecular weight compound having a molecular weight of not less than 50,000 daltons for specific binding toan enzyme labelled antibody which can react and bind specifically with the ligand and the linked product, wherein the high molecular weight compound sterically hinders enzymatic activity in specific binding complexes comprising the enzyme labelled antibody and the linked product;
- said element comprising;
(a) a substrate layer comprising a non-diffusible substrate composed of a polymer which is capable of being digested by the labelling enzyme into a diffusible oligomer, which is capable of migrating out of the substrate layer; and
(b) a reagent layer comprising a fragmenting enzyme for further fragmenting the diffusible oligomer into a detectable monomer, the reagent layer also comprising a reagent composition which react with the detectable monomer to form a dye having an absorption peak in the visible wavelength range;
wherein the labelling enzyme is an endo-active hydrolase to digest the polymer into the diffusible oligomer and the fragmenting enzyme is an exo-active hydrolase which is capable of digesting the diffusible oligomer into detectable monomer.
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Abstract
An immunoassay element for quantitatively analyzing a ligand by determining the change in enzymatic activity caused by a reaction between the ligand, a linked product of the ligand and a high molecular weight compound and an enzyme-labelled antibody. The immunoassay element comprises a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of the enzyme, and a reagent layer containing a fragmenting enzyme for further fragmenting the non-difussible material. Also provided is a process for quantitatively analyzing a ligand contained in a sample by the use of the immunoassay element.
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Citations
21 Claims
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1. An immunoassay element for quantitatively analyzing a ligand by determining a change in enzymatic activity of a labelling enzyme caused by a competitive reaction betweenthe ligand anda linked product comprising
(i) the ligand or a derivative thereof having a common antigenic determinant with the ligand and (ii) a high molecular weight compound having a molecular weight of not less than 50,000 daltons for specific binding to an enzyme labelled antibody which can react and bind specifically with the ligand and the linked product, wherein the high molecular weight compound sterically hinders enzymatic activity in specific binding complexes comprising the enzyme labelled antibody and the linked product;said element comprising; (a) a substrate layer comprising a non-diffusible substrate composed of a polymer which is capable of being digested by the labelling enzyme into a diffusible oligomer, which is capable of migrating out of the substrate layer; and (b) a reagent layer comprising a fragmenting enzyme for further fragmenting the diffusible oligomer into a detectable monomer, the reagent layer also comprising a reagent composition which react with the detectable monomer to form a dye having an absorption peak in the visible wavelength range; wherein the labelling enzyme is an endo-active hydrolase to digest the polymer into the diffusible oligomer and the fragmenting enzyme is an exo-active hydrolase which is capable of digesting the diffusible oligomer into detectable monomer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. An immunoassay element for quantitatively analyzing a ligand by determining a change in enzymatic activity of a labelling enzyme caused by a competitive reaction betweenthe ligand anda linked product comprising
(i) the ligand or a derivative thereof having a common antigenic determinant with the ligand and (ii) a high molecular weight compound having a molecular weight of not less than 50,000 daltons for specific binding to an enzyme labelled antibody which can react and bind specifically with the ligand and the linked product, wherein the high molecular weight compound sterically hinders enzymatic activity in specific binding complexes comprising the enzyme labelled antibody and the linked product;said element comprising; (a) a substrate layer comprising a non-diffusible substrate composed of a polymer which is capable of being digested by the labelling enzyme into a diffusible oligomer, which is capable of migrating out of the substrate layer; and (b) a reagent layer comprising a fragmenting enzyme for further fragmenting the diffusible oligomer into a detectable monomer; and (c) a water-permeable layer which contains a reagent composition which reacts with the detectable monomer to form a dye having an absorption peak in the visible wavelength range; wherein the labelling enzyme is an endo-active hydrolase to digest the polymer into the diffusible oligomer and the fragmenting enzyme is an exo-active hydrolase which is capable of digesting the diffusible oligomer into the detectable monomer. - View Dependent Claims (17, 18, 19, 20, 21)
Specification