Non-competitive threshold ligand-receptor assays

A method for determining the presence or amount of at least one target ligand above a threshold concentration in a fluid sample suspected of containing the target ligand, the target ligand capable of specifically binding to both a ligand receptor, and to a ligand receptor conjugate, said ligand receptor conjugate comprising a ligand receptor coupled to a signal development element, the method comprising:

• a. creating a reaction mixture by combining an amount of the ligand receptor with a volume of the fluid sample to achieve a concentration of the ligand receptor such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the ligand receptor is capable of specifically binding the threshold concentration of the target ligand in the sample to form a ligand receptor:target ligand complex;
• b. creating a composition by combining an amount of the ligand receptor conjugate with the reaction mixture to achieve a concentration of the ligand receptor conjugate greater than the threshold concentration to form a ligand receptor conjugate:target ligand complex when the target ligand concentration is greater than the threshold concentration;
• and,
• c. detecting the ligand receptor conjugate:target ligand complex formed when the target ligand concentration is greater than the threshold concentration by contacting the composition with a solid phase comprising a second ligand receptor immobilized thereto to specifically bind the ligand receptor conjugate:target ligand complex, and generating a signal from the signal development element of the bound ligand receptor conjugate:target ligand complex, whereby the bound target ligand is determined to be at a concentration greater than the threshold concentration.

A method for determining the presence or amount of at least one target ligand above a threshold concentration in a fluid sample suspected of containing the target ligand, the target ligand capable of specifically binding to both a ligand receptor, and to a ligand receptor conjugate, said ligand receptor conjugate comprising a second ligand receptor coupled to a signal development element, the method comprising:

• a. forming a reaction mixture by contacting the fluid sample:
• (i) with an amount of the ligand receptor to achieve a concentration of the ligand receptor such that subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the ligand receptor is capable of specifically binding the threshold concentration of the target ligand present in the sample;
• and,
• (ii) and with an amount of the ligand receptor conjugate to achieve a concentration of the ligand receptor conjugate capable of specifically binding more than the threshold concentration of the sample to form a ligand receptor conjugate:target ligand complex when target ligand concentration is greater than the threshold concentration;
• and,
• b. detecting the ligand receptor conjugate:target ligand complex formed when the target ligand concentration is greater than the threshold concentration by contacting the composition with a solid phase comprising ligand receptor immobilized thereto to specifically bind the ligand receptor conjugate:target ligand complex and generating a signal from the signal development element of the bound ligand receptor conjugate:target ligand complex, whereby the bound target ligand is determined to be at a concentration greater than the threshold concentration.

A method for determining the presence or amount of at least one target ligand above a threshold concentration in a fluid sample suspected of containing the target ligand, the target ligand capable of specifically binding to both a ligand receptor, and to a ligand receptor conjugate, said ligand receptor conjugate comprising a second ligand receptor coupled to a signal development element, the method comprising:

• a. contacting the fluid sample with an amount of the ligand receptor to form a reaction mixture, such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, below the threshold concentration of the target ligand in the sample, substantially all of the target ligand is specifically bound to the ligand receptor as target ligand:receptor complexes;
• b. contacting the reaction mixture with an amount of the ligand receptor conjugate to form a target ligand:ligand receptor conjugate complex when the target ligand is present at greater than the threshold concentration;
• and,
• c. detecting the ligand receptor conjugate:target ligand complex formed when the target ligand concentration is greater than the threshold concentration by providing a solid phase comprising one or more zones, a zone comprising one or more ligand receptors which are non-diffusibly immobilized to the zone to specifically bind the ligand receptor conjugate:target ligand complex, and generating a signal from the signal development element of the bound ligand receptor conjugate:target ligand complex.

A method for determining the presence or concentration of at least one target ligand which specifically binds to both to a ligand receptor conjugate and a ligand receptor, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of providing a detectable signal, in a fluid sample suspected of containing said target ligand, said method comprising:

• a. contacting said fluid sample with an amount of said ligand receptor to form a reaction mixture, the amount of said ligand receptor selected such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, substantially all of the target ligand in the sample is bound by the ligand receptor when the target ligand is present at a concentration less than a threshold concentration;
• b. contacting said reaction mixture with an amount of said ligand receptor conjugate, such that when the concentration of the target ligand in the sample is present at a concentration greater than the threshold concentration, complexes of ligand receptor conjugate:target ligand are formed;
• and,
• c. detecting the ligand receptor conjugate:target ligand complexes formed when the target ligand concentration is greater than the threshold concentration by providing a terminal solid phase comprising one or more ligand receptors which specifically bind each ligand receptor conjugate:target ligand complex, said ligand receptors being non-diffusibly immobilized to the terminal solid phase in at least one distinct spatial locus, and generating a signal from the signal development element of the bound ligand receptor conjugate:target ligand complex.

A method for determining the presence or amount of at least one target ligand above a threshold concentration in a fluid sample suspected of containing the target ligand, the target ligand capable of specifically binding to both a ligand receptor, and to a ligand receptor conjugate, said ligand receptor conjugate comprising a second ligand receptor coupled to a signal development element, the method comprising:

• a. forming a reaction mixture by contacting the fluid sample:
• (i) with an amount of the ligand receptor to achieve a concentration of the ligand receptor such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the ligand receptor is capable of specifically binding the threshold concentration of the target ligand present in the sample;
• and,
• (ii) and with an amount of the ligand receptor conjugate to achieve a concentration of the ligand receptor conjugate capable of specifically binding more than the threshold concentration of the sample to form a ligand receptor conjugate:target ligand complex when target ligand concentration is greater than the threshold concentration;
• and,
• b. detecting the ligand receptor conjugate:target ligand complex formed when the target ligand concentration is greater than the threshold concentration by providing a solid phase comprising one or more zones, a zone comprising one or more ligand receptors which are non-diffusibly immobilized to the zone to specifically bind the ligand receptor conjugate:target ligand complex, and generating a signal from the signal development element of the bound ligand receptor conjugate:target ligand complex.