Fluorescence quenching with immunological pairs in immunoassays

• US 3,996,345 A
• Filed: 06/30/1975
• Issued: 12/07/1976
• Est. Priority Date: 08/12/1974
• Status: Expired due to Term

• Current Assignee: Syva Co.
• Original Assignee: Syva Co.
• Inventors: Ullman, Edwin F., Schwarzberg, Moshe
• Primary Examiner(s): Wolk, Morris O.
• Assistant Examiner(s): Marantz, Sidney
• Application Number:
  US05/591,386
• Time in Patent Office: 526 Days
• Field of Search: 23/230 B, 424/12, 195/103.5 R, 260/112 B, 252/301.16
• US Class Current: 436/537
• CPC Class Codes: G01N 33/1833 Oil in water water in oil G01N33/2847
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/817 Steroids or hormones

Abstract

Immunoassays employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand present in the assay medium. In carrying out the assay, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antibodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The composition is irradiated with light at a wavelength, absorbed by the fluorescing molecule and the amount of fluorescence determined. By employing appropriate standards, the presence and amount of the ligand can be determined.

Claims

1. A method for determining in an assay solution, the presence of a ligand in an unknown suspected of containing said ligand, said ligand having at least one epitopic site, wherein two chromophores, Ch₁ and Ch₂, which form a fluorescer-quencher pair are employed as reagents, whereby in said assay solution the amount of fluorescer brought within quenching distance of said quencher is affected by the presence of ligand,

which comprises:

A. combining in an aqueous buffered medium to form an assay solution;

1. said unknown;

2. a source of Ch₁, as Ch₁ covalently bound to a first receptor composition capable of specific non-covalent binding to said ligand;

3. a source of Ch₂, as Ch₂ covalently bound to a second receptor composition capable of specific non-covalent binding to said ligand or as Ch₂ covalently or non-covalently bound to ligand analog, 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;

B. incubating said assay solution for a sufficient time for at least a portion of said receptor compositions 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.

2. A method according to claim 1, wherein said first and second receptor compositions are the same anti-ligand.

3. A method according to claim 1, wherein said first receptor composition is a combination of anti-ligand from a first species and anti(first anti-ligand) conjugated to Ch₁, and said second receptor composition is anti-ligand from a second species and anti(second anti-ligand) conjugated to Ch₂.

4. A method according to claim 1, wherein said ligand is of from about 110-2,000 molecular weight.

5. A method according to claim 1, wherein said ligand is a poly(amino acid) having a plurality of epitopic sites and of at least about 10,000 molecular weight.

6. A method according to claim 1, wherein said ligand is of greater than about 2,000 molecular weight and has a plurality of epitopic sites, and said sources of Ch₁ and Ch₂ are said first and second receptor compositions covalently bonded to Ch₁ and Ch₂ respectively.

7. A method according to claim 6, wherein said ligand is a serum protein.

8. A method according to claim 7, wherein said serum protein is a globulin.

9. A method according to claim 7, wherein said serum protein is an albumin.

10. A method according to claim 7, wherein said serum protein is a lipoprotein.

11. A method according to claim 7, wherein said serum protein is a glycoprotein.

12. A method according to claim 7, wherein said serum protein is a complement factor.

13. A method according to claim 7, wherein said serum protein is a blood clotting factor.

14. A method according to claim 6, wherein said ligand is at least a portion of a microorganism.

15. A method according to claim 14, wherein said ligand is a portion of the microorganism membrane.

16. A method according to claim 14, wherein said microorganism is a virus.

17. A method according to claim 6, wherein said ligand is a poly(amino acid) of greater than about 10,000 molecular weight.

18. A method according to claim 1, wherein said ligand is an assemblage of an antigen and antibody for said antigen.

19. A method according to claim 1, wherein said receptor compositions are antibodies, said aqueous medium has from 0 to 20 volume percent of an inert polar organic solvent, said aqueous medium is buffered in the range of from about 5 to 10, the amount of receptor added is sufficient to provide from about 20 to 80 percent quenching and the assay solution is incubated at a temperature in the range of about 0°

to 45°

C.

20. 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, Ch₁ and Ch₂, which form a fluorescer-quencher pair, one of the chromophores, Ch₁, is bound to receptor for ligand to form receptor-Ch₁, said receptor having binding sites capable of specifically binding to the epitopic sites of said ligand, the other of the chromophores, Ch₂ is bound to receptor for ligand to form receptor-Ch₂ or to ligand analog, either a single ligand analog being covalently bound to one or more chromophores to form ligand analog-(Ch₂)_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(Ch₂), 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;

22. A method according to claim 20, wherein said ligand is of from about 110 to 2,000 molecular weight, and said receptor is antibody.

23. A method according to claim 20, wherein said ligand is a poly(amino acid) having a plurality of epitopic sites and of at least about 10,000 molecular weight.

24. A method according to claim 20, wherein said ligand is of greater than about 2,000 molecular weight and has a plurality of epitopic sites, said receptor is antibody, and said source of Ch₂ is receptor-Ch₂.

26. A method according to claim 24, wherein said ligand is of from about 110 to 2,000 molecular weight and is a drug having a benzene ring separated from an amino group by from 2 to 3 aliphatic carbon atoms.

27. A method according to claim 24, wherein said ligand is a steroid.

29. A method according to claim 27, wherein said ligand is a poly(amino acid) of at least about 10,000 molecular weight.

31. A method according to claim 29, wherein said ligand has one epitopic site and is of a molecular weight in the range of about 110 to 2,000.

34. A method according to claim 29, wherein said polyfunctionalized nucleus molecule is a poly(amino acid) of at least 30,000 molecular weight.

21. unknown;

2. receptor-Ch₁ ; and

3. a source of Ch₂ capable of binding within quenching distance to receptor-Ch₂, as one of the following;

a. ligand analog-(Ch₂)_x ;

b. poly(ligand analog)-poly(Ch₂);

c. poly(ligand analog) and receptor-Ch₂ ;

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-Ch₂ ;

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.

25. 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, Ch₁ and Ch₂, which form a fluorescer-quencher pair, one of the chromophores, Ch₁ is bound to receptor for ligand to form receptor-Ch₁, 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-Ch₂, 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-Ch₁ ;

3. ligand analog-Ch₂ ;

the concentrations of receptor-Ch₁ and ligand analog-Ch₂ 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°

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.

28. 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, Ch₁ and Ch₂, which form a fluorescer-quencher pair, one of the chromophores, Ch₁, is bound to receptor for ligand to form receptor-Ch₁, 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-Ch₂ ;

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-Ch₁ ;

3. receptor-Ch₂ ;

B. incubating said assay solution at a temperature in the range of about 0°

-45°

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.

30. 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, Ch₁ and Ch₂, which form a fluorescer-quencher pair, one of the chromophores, Ch₁, is bound to receptor for ligand to form receptor-Ch₁, 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-Ch₂ ;

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-Ch₁ ;

3. poly(ligand analog) and receptor-Ch₂, 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°

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.

32. A method according to claim 30, wherein said ligand is a drug having a benzene ring separated by from 2 to 3 aliphatic carbon atoms from an amino group.

33. A method according to claim 30, wherein said ligand is a steroid.

35. 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 Ch₁ and a portion of which is bound to Ch₂, wherein Ch₁ and Ch₂ 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.

36. 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)-Ch₁ and anti(b-(anti-ligand)-Ch₂, wherein a and b designate a different species source for the anti-ligand and Ch₁ and Ch₂ 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)-Ch₁ ; and

5. anti(b-(anti-ligand)-Ch₂ ;

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.

37. 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)-Ch₁ and (anti-ligand)-Ch₂, wherein Ch₁ and Ch₂ 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)-Ch₁ ; and

4. (anti-ligand)-Ch₂ ;

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.

39. A combination according to claim 37, including a-(anti-ligand) and b-(anti-ligand).

38. In combination, anti(a-anti-ligand)-Ch₁ and anti(b-anti-ligand)-Ch₂ wherein a and b designate anti-ligand from two different species for the same ligand, said ligand having a plurality of epitopic sites, and Ch₁ and Ch₂ designate a fluorescer-quencher pair.