Signal enhancement in immunoassay by modulation of enzymatic catalysis
First Claim
1. A method for the detection of a ligand in a liquid comprising:
- (a) combining a first liquid suspected of containing a ligand with an antiligand affixed to a solid support and a tracer, said tracer comprising a first hydrolase, whereby said antiligand binds to said ligand and said tracer binds to one of said antiligand and said ligand to give a bound phase on said support;
(b) separating said support from said first liquid;
(c) contacting said support with a second liquid containing a second hydrolase and a blocked fluoroketone whereby said first hydrolase converts said blocked fluoroketone to a fluoroketone and releases said fluoroketone into said second liquid;
(d) adding to said second liquid a substrate for said second hydrolase whereby conversion of said substrate to a product by said second hydrolase is inhibited by said fluoroketone; and
(e) detecting said ligand by a signal associated with said conversion.
1 Assignment
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Accused Products
Abstract
A method for enzyme immunoassay for a ligand suspected to be present in a liquid sample includes signal amplification by use of at least two hydrolases and a blocked fluoroketone for one of the hydrolases. Ligand present in the liquid binds to an antiligand and a hydrolase-labeled tracer. The resulting bound fraction is separated and the hydrolase in the tracer removes the blocking group from the blocked fluoroketone. The fluoroketone activates or inhibits a second hydrolase which catalyzes the conversion of a substrate to a product. The presence or absence of the ligand in the liquid is indicated by a signal, such as a color change or a rate of color change, associated with the product. The invention includes a new class of hydrolase inhibitors and blocked fluoroketones and a kit of materials useful for performing the method of the invention.
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Citations
32 Claims
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1. A method for the detection of a ligand in a liquid comprising:
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(a) combining a first liquid suspected of containing a ligand with an antiligand affixed to a solid support and a tracer, said tracer comprising a first hydrolase, whereby said antiligand binds to said ligand and said tracer binds to one of said antiligand and said ligand to give a bound phase on said support; (b) separating said support from said first liquid; (c) contacting said support with a second liquid containing a second hydrolase and a blocked fluoroketone whereby said first hydrolase converts said blocked fluoroketone to a fluoroketone and releases said fluoroketone into said second liquid; (d) adding to said second liquid a substrate for said second hydrolase whereby conversion of said substrate to a product by said second hydrolase is inhibited by said fluoroketone; and (e) detecting said ligand by a signal associated with said conversion. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for the detection of an antigen in a liquid comprising
(a) combining a first liquid suspected of containing an antigen with first and second antibodies, said first antibody being affixed to a solid support and said second antibody having alkaline phosphatase conjugated thereto whereby said antigen binds to said first and second antibodies to give a bound phase on said support; -
(b) separating said support from said first liquid; (c) contacting said support with a second liquid containing an esterase and a blocked fluoroketone whereby said alkaline phosphatase converts said blocked fluoroketone to fluoroketone of said esterase, said fluoroketone being released into said second liquid; (d) adding to said second liquid a chromogen, the conversion of which to a colored product by said esterase is inhibited by said fluoroketone; and (e) detecting said antigen by detecting inhibition of formation of color in said second liquid. - View Dependent Claims (16, 17)
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18. A method for the detection of an antigen in a liquid comprising:
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(a) combining a first liquid suspected of containing an antigen with an antibody affixed to a solid support and with a tracer, said tracer comprising said antigen having alkaline phosphatase conjugated thereto, whereby said antigen and said tracer bind to said antibody to give a bound phase on said support; (b) separating said support from said first liquid; (c) contacting said support with a second liquid containing an esterase and a blocked fluoroketone whereby said alkaline phosphatase converts said blocked fluoroketone to fluoroketone of said esterase, said inhibitor being released into said second liquid; (d) adding to said second liquid a chromogen, the conversion of which to a colored product by said esterase is inhibited by said fluoroketone; and (e) detecting said antigen by detecting inhibition of formation of color in said second liquid. - View Dependent Claims (19, 20)
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21. A method for the determination of the concentration of a ligand in a liquid comprising:
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(a) combining a first liquid suspected of containing a ligand with an antiligand affixed to a solid support and with a tracer, said tracer comprising a first hydrolase, whereby said antiligand binds to said ligand and said tracer binds to one of said ligand and said antiligand, to give a bound phase on said support; (b) separating said support from said first liquid; (c) contacting said support with a second liquid containing a second hydrolase and a blocked fluoroketone whereby said first hydrolase converts said blocked fluoroketone to a fluoroketone, said fluoroketone being released into said second liquid; (d) adding to said second liquid a substrate for said second hydrolase whereby conversion of said substrate to a product having a detectable signal associated therewith by said second hydrolas is inhibited by said fluoroketone; (e) measuring the magnitude of said signal; and (f) determining the concentration of said ligand in said first liquid by comparing said magnitude with the magnitude of a signal associate with a product when steps (a) to (e) are repeated with liquid samples containing known quantities of said ligand. - View Dependent Claims (22, 23, 24)
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- 25. A kit for performing an assay for a liquid comprising an antiligand for a ligand, a first hydrolase and a blocked fluoroketone for a second hydrolase.
Specification