Fluorescence quenching with immunological pairs in immunoassays

A method for determining in an assay solution, the presence of a ligand in an unknown suspected of containing said ligand, said ligand having one or more epitopic sites;

• in which is employed two chromophores, Ch.sub.1 and Ch.sub.2, which form a fluorescer-quencher pair, one of the chromophores, Ch.sub.1, is bound to receptor for ligand to form receptor-Ch.sub.1, said receptor having binding sites capable of specifically binding to the epitopic sites of said ligand, the other of the chromophores, Ch.sub.2 is bound to receptor for ligand to form receptor-Ch.sub.2 or to ligand analog, either a single ligand analog being covalently bound to one or more chromophores to form ligand analog-(Ch.sub.2).sub.x, where x is on the average at least one, or a plurality of ligand analogs and chromophores being covalently bound to a polyfunctionalized nucleus molecule to form poly(ligand analog)-poly(Ch.sub.2), wherein ligand analog is a mono- or polyvalent radical, a substantial proportion of which defines one or more epitopic sites capable of competing with ligand for the binding sites of said receptor;
• and
• wherein the amount of fluorescer within quenching distance of quencher is related to the amount of ligand present in said assay solution;
• which comprises:
• A. combining in an aqueous buffered medium to form an assay solution:
• 1. unknown;
• 2. receptor-Ch.sub.1 ;
• and
• 3. a source of Ch.sub.2 capable of binding within quenching distance to receptor-Ch.sub.2, as one of the following:
• a. ligand analog-(Ch.sub.2).sub.x ;
• b. poly(ligand analog)-poly(Ch.sub.2);
• c. poly(ligand analog) and receptor-Ch.sub.2 ;
• wherein poly(ligand analog) has a plurality of ligand analogs covalently bound to a polyfunctionalized nucleus molecule;
• or
• when said ligand has a plurality of epitopic sites,
• d. receptor-Ch.sub.2 ;
• B. incubating said assay solution for a sufficient time for at least a portion of the receptor present to combine with at least a portion of any ligand present;
• C. irradiating said incubated assay solution with light at a wavelength within the absorption spectrum of said fluorescer;
• and
• D. measuring the amount of fluorescence from said assay solution as compared to an assay solution having a known amount of ligand.

A method for determining in an assay solution, the presence of a ligand in an unknown suspected of containing said ligand, said ligand having one epitopic site,

• in which is employed two chromophores, Ch.sub.1 and Ch.sub.2, which form a fluorescer-quencher pair, one of the chromophores, Ch.sub.1 is bound to receptor for ligand to form receptor-Ch.sub.1, said receptor having a binding site capable of specifically binding to the epitopic site of said ligand, the other of the chromophores is covalently bound to ligand analog to form ligand analog-Ch.sub.2, wherein ligand analog is a monovalent radical, a substantial proportion of which defines an epitopic site capable of competing with ligand for a binding site of said receptor;
• and
• wherein the amount of fluorescer within quenching distance of quencher is related to the amount of ligand present in said assay solution;
• which comprises:
• A. combining in an aqueous buffered medium at a pH in the range of about 5 to 10 to form an assay solution;
• 1. unknown:
• 2. receptor-Ch.sub.1 ;
• 3. ligand analog-Ch.sub.2 ;
• the concentrations of receptor-Ch.sub.1 and ligand analog-Ch.sub.2 providing from about 20 to 80% quenching of the fluorescence in the absence of ligand;
• B. incubating said assay solution at a temperature in the range of about 0-45.degree. C for a sufficient time for at least a portion of the receptor present to combine with at least a portion of any ligand present;
• C. irradiating said incubated assay solution with light at a wavelength within the absorption spectrum of said fluorescer;
• and
• D. measuring the amount of fluorescence from said assay solution as compared to an assay solution having a known amount of ligand.

A method for determining in an assay solution, the presence of a ligand in an unknown suspected of containing said ligand, said ligand having a plurality of epitopic sites,

• in which is employed two chromophores, Ch.sub.1 and Ch.sub.2, which form a fluorescer-quencher pair, one of the chromophores, Ch.sub.1, is bound to receptor for ligand to form receptor-Ch.sub.1, said receptor having binding sites capable of specifically binding to the epitopic sites of said ligand, the other of the chromophores is bound to receptor for ligand to form receptor-Ch.sub.2 ;
• wherein the amount of fluorescer within quenching distance of quencher is related to the amount of ligand present in said assay solution;
• which comprises:
• A. combining in an aqueous buffered medium at a pH in the range of about 5 to 10, to form an assay solution;
• 1. unknown;
• 2. receptor-Ch.sub.1 ;
• 3. receptor-Ch.sub.2 ;
• B. incubating said assay solution at a temperature in the range of about 0.degree.-45.degree. C for a sufficient time for at least a portion of the receptor present to combine with at least a portion of any ligand present;
• C. irradiating said incubated assay solution with light at a wavelength within the absorption spectrum of said fluorescer;
• and
• D. measuring the amount of fluorescence from said assay solution as compared to an assay solution having a known amount of ligand.

A method for determining in an assay solution, the presence of a ligand in an unknown suspected of containing said ligand, said ligand having one or more epitopic sites,

• in which is employed two chromophores, Ch.sub.1 and Ch.sub.2, which form a fluorescer-quencher pair, one of the chromophores, Ch.sub.1, is bound to receptor for ligand to form receptor-Ch.sub.1, said receptor having binding sites capable of specifically binding to the epitopic sites of said ligand, the other of the chromophores is bound to receptor for ligand to form receptor-Ch.sub.2 ;
• wherein the amount of fluorescer within quenching distance of quencher is related to the amount of ligand present in said assay solution;
• which comprises:
• A. combining in an aqueous buffered medium at a pH in the range of 5 to 10 to form an assay solution;
• 1. unknown;
• 2. receptor-Ch.sub.1 ;
• 3. poly(ligand analog) and receptor-Ch.sub.2, wherein ligand analog is a mono- or polyvalent radical, a substantial proportion of which defines one or more epitopic sites capable of competing with ligand for the binding sites of said receptor and poly(ligand analog) has a plurality of ligand analogs covalently bound to a polyfunctionalized nucleus molecule;
• B. incubating said assay solution at a temperature in the range of about 0-45.degree. C for a sufficient time for at least a portion of the receptor present to combine with at least a portion of any ligand present;
• C. irradiating said incubated assay solution with light at a wavelength within the absorption spectrum of said fluorescer;
• and
• D. measuring the amount of fluorescence from said assay solution as compared to an assay solution having a known amount of ligand.

A method for determining in an assay solution the presence of a ligand or anti-ligand in an unknown suspected of containing said ligand or anti-ligand, said ligand and anti-ligand having a plurality of epitopic sites;

• in which is employed anti(anti-ligand), a portion of which is bound to Ch.sub.1 and a portion of which is bound to Ch.sub.2, wherein Ch.sub.1 and Ch.sub.2 form a fluorescer-quencher pair;
• combining with said unknown (1) anti-ligand for the determination of the presence of ligand, or (2) ligand for the determination of the presence of anti-ligand;
• separating anti-ligand bound to ligand from unbound anti-ligand;
• combining in an aqueous buffered assay solution said bound anti-ligand with said anti(anti-ligand)s;
• irradiating said assay solution with light at a wavelength within the absorption spectrum of said fluorescer;
• and
• measuring the amount of fluorescence from said assay solution as compared to an assay solution having a known amount of ligand or anti-ligand.

A method for determining in an assay solution the presence of a ligand in an unknown suspected of containing said ligand, said ligand having a plurality of epitopic sites, in which method is employed a-(anti-ligand), b-(anti-ligand), anti(a-(anti-ligand)-Ch.sub.1 and anti(b-(anti-ligand)-Ch.sub.2, wherein a and b designate a different species source for the anti-ligand and Ch.sub.1 and Ch.sub.2 form a fluorescer-quencher pair, wherein the amount of fluorescer within quenching distance of quencher is related to the amount of ligand present in said assay solution,

• which comprises:
• A. combining in an aqueous buffered medium to form an assay solution:
• 1. unknown;
• 2. a-(anti-ligand);
• 3. b-(anti-ligand);
• 4. anti(a-(anti-ligand)-Ch.sub.1 ;
• and
• 5. anti(b-(anti-ligand)-Ch.sub.2 ;
• B. incubating said assay solution for a sufficient time for at least a portion of the anti-ligands and anti(anti-ligand)s to combine with ligand and anti-ligand respectively;
• C. irradiating said incubated assay solution with light at a wavelength within the absorption spectrum of said fluorescer;
• and
• D. measuring the amount of fluorescence from said assay solution as compared to an assay solution having a known amount of ligand.

A method for determining in an assay solution, the presence of anti-ligand for a ligand in an unknown suspected of containing said anti-ligand,

• in said method is employed (anti-ligand)-Ch.sub.1 and (anti-ligand)-Ch.sub.2, wherein Ch.sub.1 and Ch.sub.2 form a fluorescer-quencher pair;
• wherein anti-ligand present in the unknown diminishes the amount of quenching of said fluorescer by said quencher;
• and
• which comprises:
• combining in an aqueous buffered assay medium:
• 1. said unknown;
• 2. said ligand, when said ligand is polyepitopic, or poly(ligand analog) when said ligand is monoepitopic;
• 3. (anti-ligand)-Ch.sub.1 ;
• and
• 4. (anti-ligand)-Ch.sub.2 ;
• irradiating said assay solution with light at a wavelength within the absorption spectrum of said fluorescer;
• and
• measuring the amount of fluorescence from said assay solution as compared to an assay solution having a known amount of anti-ligand.