Cascade immunoassay by multiple binding reactions
First Claim
1. A method for determining an analyte in a liquid comprising:
- (a) combining a first liquid suspected of containing an analyte with a tracer comprising an unmasking enzyme and with a solid support having an antianalyte and a first immunologically active component randomly affixed thereto whereby said analyte binds to said antianalyte and said tracer binds to one of said analyte and said antianalyte to give a first bound fraction on said support;
(b) replacing said first liquid with a second liquid containing a second immunologically active compound conjugated to a masking group, said unmasking enzyme removing said masking group to give free second component which binds to said first component, and a signal enzyme conjugated to one of said second component and a third immunologically active component specific for said second component, said signal enzyme becoming affixed to said support;
(c) replacing said second liquid with a third liquid containing a substrate which converted by said signal enzyme to a product; and
(d) determining said analyte by a signal associated with said product.
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Accused Products
Abstract
A method for enzyme immunoassay includes contacting under binding conditions a liquid suspected of containing an analyte, an antianalyte affixed to a solid support and a tracer having an enzyme conjugated thereto. A bound fraction is separated from the liquid and incubated in a second liquid with a masked ligand. The masked ligand is converted by the enzyme on the bound fraction to give free lignad which binds to an antiligand. A signal system, such as a signal enzyme and substrate therefor, or a label-loaded vesicle and vesicle lysing agent, is added to generate a signal used to detect or measure the analyte in the liquid. The invention includes a kit of materials useful in performing the assay of the invention.
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Citations
31 Claims
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1. A method for determining an analyte in a liquid comprising:
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(a) combining a first liquid suspected of containing an analyte with a tracer comprising an unmasking enzyme and with a solid support having an antianalyte and a first immunologically active component randomly affixed thereto whereby said analyte binds to said antianalyte and said tracer binds to one of said analyte and said antianalyte to give a first bound fraction on said support; (b) replacing said first liquid with a second liquid containing a second immunologically active compound conjugated to a masking group, said unmasking enzyme removing said masking group to give free second component which binds to said first component, and a signal enzyme conjugated to one of said second component and a third immunologically active component specific for said second component, said signal enzyme becoming affixed to said support; (c) replacing said second liquid with a third liquid containing a substrate which converted by said signal enzyme to a product; and (d) determining said analyte by a signal associated with said product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for determining an analyte in a liquid comprising:
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(a) combining a first liquid suspected of containing an analyte with a tracer comprising an unmasking enzyme and with a solid support having an antianalyte and an antiligand randomly affixed thereto whereby said analyte binds to said antianalyte and said tracer binds to said analyte to give a bound fraction on said support; (b) separating said support from said first liquid; (c) suspending said support in a second liquid; (d) adding to said second liquid a ligand conjugated to a masking group and a liposome having ligand conjugated thereto and having a signal enzyme encapsulated therein, said unmasking enzyme removing said masking group to give free ligand, said antiligand binding competitively with said ligand on said liposome and said free ligand; (e) adding complement and a substrate for signal enzyme to said second liquid, said complement lysing a liposome having conjugated thereto ligand bound to antiligand, said lysing causing release of said signal enzyme, the released signal enzyme converting said substrate to a product; and (f) determining said analyte by a signal associated with said product. - View Dependent Claims (23, 24, 25)
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26. A method for determining an antigen in a fluid comprising:
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(a) combining a first liquid suspected of containing a antigen with a first antibody conjugated to an unmasking enzyme and a solid support having randomly affixed thereto an antiligand and a second antibody whereby said first and second antibodies bind to said antigen to give a first bound fraction on said support; (b) separating said support from said first liquid; (c) suspending said support in a second liquid; (d) adding to said second liquid a first increment of a ligand conjugated to a signal enzyme and a second increment of said ligand conjugated to a masking group, said unmasking enzyme converting the masked ligand to free ligand, said free ligand and said first increment of ligand binding competitively with said antiligand on said support; (e) separating said support from said second liquid; (f) suspending said support in a third liquid; (g) adding to said third liquid a substrate for said signal enzyme whereby said signal enzyme converts said substrate to a product; and (h) determining said antigen by a signal associated with said product.
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27. A method for determining an antigen in a fluid comprising:
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(a) combining a first liquid suspected of containing an antigen with a first antibody conjugated to an unmasking enzyme and a solid support having randomly affixed thereto a first antiligand and a second antibody whereby said first and second antibodies bind to said antigen to give a first bound fraction on said support; (b) separating said support from said first liquid; (c) suspending said support in a second liquid; (d) adding to said second liquid a masked ligand and a second antiligand conjugated to a signal enzyme, said unmasking enzyme converting said masked ligand to a free ligand, said free ligand binding to said first and second antiligands whereby said signal enzyme becomes affixed to said support; (e) separating said support from said second liquid; (f) suspending said support in a third liquid; (g) adding to said third liquid a substrate for said signal enzyme whereby said signal enzyme converts said substrate to a product; and (h) determining said antigen by a signal associated with said product.
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- 28. A kit of materials for performing an assay for an analyte comprising a solid support having randomly affixed thereto an antianalyte and an immunologically active compound, a tracer comprising an unmasking enzyme conjugated to an analyte, a masked ligand and a signal enzyme.
Specification