Chemically induced fluorescence immunoassay

• US 4,220,450 A
• Filed: 04/05/1978
• Issued: 09/02/1980
• Est. Priority Date: 04/05/1978
• Status: Expired due to Term

• Current Assignee: Dade Behring Marburg GmbH (Siemens AG)
• Original Assignee: Syva Co. (Siemens AG)
• Inventors: Maggio, Edward T.
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US05/893,910
• Time in Patent Office: 881 Days
• Field of Search: 23/230 B, 424/8, 424/12, 195/103.5 A, 195/103.5 L, 435/7, 435/8
• US Class Current: 436/537
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/966 involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
• 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 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/817 Steroids or hormones

Abstract

A competitive protein binding method is provided for the determination of an analyte which is a member of an immunological pair consisting of ligand and receptor for the ligand. A chemiluminescent source is employed comprised of one or more individual members, one chemiluminescent source member being conjugated to one of the members of the immunological pair, so as to provide chemiluminescence adjacent to the site of conjugation. A quencher molecule is conjugated to a member of the immunological pair. When the members of the immunological pair bind, the quencher molecule is brought within quenching distance of the chemiluminescent source so as to inhibit the emission of light by the chemiluminescent source. The amount of analyte present in the assay medium affects the amount of binding between the members of the immunological pair which results in quenching of the chemiluminescence. By observing the light emitted from the assay medium, either from the chemiluminescent source of the quencher, the change in light emission in relation to the concentration of analyte present in the assay medium can be used to determine the amount of analyte present in the assay medium. By employing standards having known amounts of analyte, the amount of analyte in an unknown sample can be quantitatively determined.

Reagent kits can be provided having predetermined amounts of the reagents, so as to substantially optimize the sensitivity of the assay.

Claims

1. A method for determining in an assay solution, the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of an immunological pair consisting of ligand and antiligand, said ligand having at least one epitopic site and said antiligand being capable of specifically binding to said epitopic site of said ligand;

wherein a light emitting reciprocal pair are employed as labels, said light emitting reciprocal pair consisting of a chemiluminescence source having at least one component and a quencher capable of quenching the light emitted by said chemiluminescence source without collision, said labels being conjugated to members of said immunological pair to form a chemiluminescence label conjugate and a quencher label conjugate, and wherein in said assay solution the amount of quencher brought within quenching distance of said chemiluminescence source is related to the amount of analyte in said assay medium;

said method comprising;

A. combining in an aqueous medium to form an asssay solution;

1. said sample;

2. said chemiluminescence label conjugate;

3. said quencher label conjugate;

4. any additional components of said chemiluminescence source;

with the proviso that;

a. when said analyte is monoepitopic ligand and neither of said labels are conjugated to ligand, poly(ligand analog) is included in said assay solution;

b. when said analyte is polyvalent antiligand for monoepitopic ligand, poly(ligand analog)-label or poly(ligand analog) or the combination of quencher label ligand and chemiluminescer label ligand is included in said assay solution and when said analyte is monovalent antiligand, poly(ligand analog)-label is included in said assay solution;

c. when said analyte is antiligand for polyepitopic ligand and neither label is conjugated to ligand, ligand is included in said assay solution;

d. when said analyte is polyepitopic ligand and both the quencher label and the chemiluminescer label are bonded to ligand, antiligand is included in said assay solution;

wherein said ligand analog has at least one epitopic site common with said ligand and capable of competing with ligand for antiligand; and

B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.

2. A method according to claim 1, wherein the assay solution is at a pH in the range of about 5 to 11 and is at a temperature in the range of about 10°

to 50°

C.

3. A method according to claim 2, wherein said analyte is a haptenic ligand of from about 125 to 2,000 molecular weight.

4. A method according to claim 2, wherein said analyte is an antigen of at least about 2,000 molecular weight.

5. A method according to claim 2, wherein said analyte is antiligand.

6. A method for determining in an assay solution, the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of an immunological pair consisting of monoepitopic ligand of from about 125 to 2,000 molecular weight and antiligand, said antiligand being capable of binding to said epitopic site of said ligand, wherein a light emitting reciprocal pair are employed as labels, said light emitting reciprocal pair consisting of a chemiluminescence source having at least one component and a quencher dye capable of quenching the light emitted by said chemiluminescence source without collision, said labels being conjugated to members of said immunological pair to form a chemiluminescence conjugate and a quencher label conjugate, at least one of said labels being conjugated to other than ligand, and wherein in said assay solution the amount of quencher brought within quenching distance of said chemiluminescence source is related to the amount of analyte in said assay medium;

said method comprising;

A. combining in an aqueous medium at a pH in the range of 5 to 11 and a temperature in the range of 10°

to 50°

C. to form an assay solution;

1. said sample;

2. said chemiluminescence label conjugate;

3. said quencher label conjugate;

4. any additional components of said chemiluminescence source;

with the proviso that, when said analyte is antiligand for monoepitopic ligand, and neither label is conjugated to ligand, poly(ligand analog) is included in said assay solution, wherein said ligand analog of said poly(ligand analog) has an epitopic site common to said ligand and is capable of competing with said ligand for antiligand; and

B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.

7. A method according to claim 6, wherein said analyte is antiligand.

8. A method according to claim 6, wherein said analyte is monoepitopic ligand.

9. A method according to claim 8, wherein said chemiluminescence source has at least two components, and one of said label is conjugated to ligand and the other of said label is conjugated to antiligand.

10. A method according to claim 9, wherein said ligand is an alkaloid.

11. A method according to claim 9, wherein said ligand is a steroid.

12. A method according to claim 9, wherein said ligand is a lactam of from 5 to 6 annular members.

13. A method according to claim 9, wherein said ligand is an aminoalkylbenzene, and said alkyl is of from 2 to 3 carbon atoms.

14. A method according to claim 9, wherein said ligand is a benzheterocycle.

15. A method according to claim 9, wherein said ligand is a purine.

16. A method according to claim 9, wherein said ligand is an amino acid.

17. A method according to claim 16, wherein said amino acid is a polyiodothyronine.

18. A method according to claim 9, wherein one of said components of said chemiluminescence source is an enzyme and said chemiluminescence label conjugate is ligand conjugated to said enzyme.

19. A method according to claim 18, wherein said enzyme is a peroxidase and another component of said chemiluminescence source is luminol.

20. A method for determining in an assay solution, the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of an immunological pair consisting of polyepitopic ligand of at least about 2,000 molecular weight and antiligand, said antiligand being capable of binding to the epitopic sites of said ligand, wherein a light emitting reciprocal pair is employed as labels, said light emitting reciprocal pair consisting of a chemiluminescence source having at least one component and a quencher dye capable of quenching the light emitted by said chemiluminescence source, said labels being conjugated to members of said immunological pair to form a chemiluminescence label conjugate and a quencher label conjugate, at least one of said labels being conjugated to other than ligand, and wherein in said assay solution the amount of quencher brought within quenching distance of said chemiluminescence source is related to the amount of analyte in said assay medium;

said method comprising;

A. combining in an aqueous medium at a pH in the range of about 5 to 11 and at a temperature in the range of about 10°

to 50°

C.;

1. said sample;

2. said chemiluminescence label conjugate;

3. said quencher label conjugate;

4. any additional components of said chemiluminescence source;

with the proviso that when said analyte is antiligand and neither of said labels is conjugated to ligand, ligand is included in said assay solution; and

B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.

21. A method according to claim 20, wherein said analyte is antiligand.

22. A method according to claim 20, wherein said analyte is polyepitopic ligand.

23. A method according to claim 22, wherein said chemiluminescence source has at least two components, and one of said labels is conjugated to ligand and the other of said labels is conjugated to antiligand.

24. A method according to claim 23, wherein said ligand is a polypeptide.

25. A method according to claim 24, wherein said polypeptide is a globulin.

26. A method according to claim 25, wherein said globulin is an immunoglobulin.

27. A method according to claim 24, wherein said polypeptide is a hormone.

28. A method according to claim 23, wherein said component of said chemiluminescence source conjugated to a member of said immunological pair is an enzyme.

29. A method according to claim 28, wherein said enzyme is a peroxidase and luminol is another component of said chemiluminescence source.

30. A method for determining in an assay solution, the presence of human globulin in a sample suspected of containing said human globulin, wherein said assay employs as reagents anti(human globulin) and a light emitting reciprocal pair employed as labels, said light emitting reciprocal pair consisting of a chemiluminescence source comprised of peroxidase and luminol and a quencher dye capable of quenching the light emitted by said chemiluminescence source, said peroxidase being conjugated to human globulin to form a human globulin-peroxidase conjugate and said quencher dye being conjugated to anti(human globulin) to form quencher label anti(human globulin), and wherein in said assay solution the amount of quencher brought within quenching distance of said chemiluminescence source is related to the amount of human globulin;

said method comprising;

A. combining in an aqueous medium at a pH in the range of about 7 to 10 and at a temperature in the range of about 10°

to 50°

C. to form an assay solution;

1. said sample;

2. said peroxidase-human globulin conjugate;

3. said quencher-anti(human globulin) conjugate;

4. any additional components of said chemiluminesence source; and

B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of human globulin.

31. A method according to claim 30, wherein said quencher dye is fluorescein.

32. A method for determining in an assay solution, the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of an immunological pair consisting of monoepitopic ligand of from about 125 to 2000 molecular weight and antiligand, said antiligand being capable of binding to said epitopic site of said ligand, wherein a label is employed which is one component of a chemiluminescence source having at least one component, said label being conjugated to said ligand to form a chemiluminescence label conjugate;

said method comprising;

A. combining in an aqueous medium at a pH in the range of 5 to 11 and at a temperature in the range of 10°

to 50°

C. to form an assay solution;

1. said sample;

2. said chemiluminescence conjugate;

3. any additional components of said chemiluminescence source;

with the proviso that when said analyte is ligand, antiligand is added; and

B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.