Signal enhancement in immunoassay by modulation of enzymatic catalysis

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.

A method for the detection of an antigen in a liquid comprising:

• (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.

A method for the determination of the concentration 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 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.