Label modified immunoassays

• US 4,208,479 A
• Filed: 07/14/1977
• Issued: 06/17/1980
• Est. Priority Date: 07/14/1977
• Status: Expired due to Term

• Current Assignee: Dade Behring Marburg GmbH (Siemens AG)
• Original Assignee: Syva Co. (Siemens AG)
• Inventors: Maggio, Edward T., Zuk, Robert F.
• Primary Examiner(s): Wiseman, Thomas G.
• Application Number:
  US05/815,632
• Time in Patent Office: 1,069 Days
• Field of Search: 195/103.5 A, 195/103.5 R, 195/99, 195/127, 424/12, 424/8, 23/230 B
• US Class Current: 435/7.9
• CPC Class Codes: C07J 41/0016 Oximes
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives

Abstract

Methods and reagents are provided for immunoassays employing as reagents a labelled receptor, where the label is capable of providing a detectable signal, and modifying reagents, capable of modifying the signal obtained from the label. When ligand (ligand analog for monoepitopic ligands) is present in the assay medium, a complex is formed which inhibits interaction between the label modifying reagent and the label. By measuring the signal obtained in the presence of a known amount of ligand in the assay medium and comparing that signal with the signal obtained with an unknown sample suspected of containing ligand, one can qualitatively or quantitatively determine the amount of ligand in the unknown. For determinating anti(ligand) the assay is carried out in substantially the same way, except a source of ligand or ligand analog must be provided.

Claims

1. An assay method for determining a member of an immunological pair comprising a mono- or polyepitopic ligand and an anti(ligand) suspected of being present in a sample;

said method employing;

(1) labeled anti(ligand), wherein the label is capable of providing a detectable signal and said labeled anti(ligand) binds to said polyepitopic ligand to form a complex;

(2) a macromolecular modifier which interacts with said label to modify the signal produced from said label and is inhibited from interacting with said label by said complex;

(3) poly(ligand analog), wherein poly(ligand analog) has a plurality of epitopic sites capable of competing with said monoepitopic ligand for the binding sites of labeled anti(ligand), and capable of binding with anti(ligand) and labeled anti(ligand) to form a complex; and

(4) polyepitopic ligand;

with the proviso that poly(ligand analog) is only employed in an assay for monoepitopic ligand or anti(ligand) that binds monoepitopic ligand and polyepitopic ligand is only employed in an assay for anti(ligand) that binds polyepitopic ligand;

said method comprising;

(A) combining in an assay medium for a time sufficient to form said complexes which sterically inhibit the approach of the macromolecular modifier to said label;

(i) sample;

(ii) labeled anti(ligand);

(iii) modifier;

(iv) poly(ligand analog) in an assay for monoepitopic ligand or anti(ligand) that binds monoepitopic ligand; and

(v) polyepitopic ligand in an assay for anti(ligand) that binds polyepitopic ligand;

(B) exposing said assay medium to an agent which interacts with said label to provide said detectable signal; and

(C) measuring said detectable signal, wherein the measured detectable signal is directly related to the amount of said member in said medium.

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

to 50°

C.

3. A method according to claim 2, wherein said modifier is anti(label).

4. A method according to claim 3, wherein said label is a fluorescer.

5. A method according to claim 2, wherein said label is an enzyme.

6. A method according to claim 5, wherein said enzyme is an oxidoreductase.

7. A method according to claim 2, wherein said label is a member of a redox pair consisting of an oxidant and a reductant.

29. An assay composition for use in an assay method according to claim 1 which comprises the reagents labeled anti(ligand) and macromolecular modifier in relative amounts for substantially optimizing the response of the reagents in providing the detectable signal in relation to changes in the concentration of the member in said sample over the concentration range between 10-4 to 10-15 m.

30. An assay composition according to claim 29 wherein said modifier is anti(label).

31. An assay composition according to claim 29, including poly(ligand analog).

32. An assay composition according to claim 29, including polyepitopic ligand.

8. An assay method for determining a polyepitopic ligand suspected of being present in the sample;

said method employing;

(1) enzyme labeled anti(ligand), wherein the enzyme is capable of providing a detectable signal and said enzyme labeled anti(ligand) binds to said polyepitopic ligand to form a complex;

(2) anti(enzyme), which binds to said enzyme to substantially inhibit said enzyme;

said method comprising;

(A) combining in an assay medium

(i) sample;

(ii) enzyme labeled anti(ligand);

(B) incubating said assay medium for a time sufficient to form complexes which sterically inhibit the approach of said anti(enzyme) to said enzyme label;

(C) adding to said assay medium anti(enzyme), substrates and any cofactors for said enzyme to produce a detectable signal as a result of the chemical change in the substrates or cofactors; and

(D) measuring said detectable signal, wherein the measured detectable signal is proportional to the amount of said polyepitopic ligand in said assay medium.

9. A method according to claim 8, wherein said pH is in the range of about 6 to 9 and said temperature is in the range of about 15°

to 40°

C.

10. A method according to claim 9, wherein said enzyme is an oxidoreductase.

11. A method according to claim 10, wherein said oxidoreductase is a peroxidase and said substrates include hydrogen peroxide and a compound which forms a dye as a result of the enzymatic reaction.

12. A method according to claim 10, wherein said enzyme is glucose-6-phosphate dehydrogenase.

33. An assay composition for use in a method according to claim 9 which comprises the reagents enzyme labeled anti(ligand) and anti(enzyme) in relative amounts for substantially optimizing the response of the reagents in providing the detectable signal in relation to changes in the concentration of the member in said sample over the concentration range between 10-4 to 10-15 m.

34. An assay composition for use in a method according to claim 33, which comprises the reagents fluorescer labeled anti(ligand) and anti(fluorescer) in relative amounts for substantially optimizing the response of the reagents in providing the emitted light in relation to changes in the concentration of the ligand in said sample over the concentration range between 10-4 to 10-15 m.

13. An assay method for determining a monoepitopic ligand suspected of being present in the sample;

said method employing;

(1) poly(ligand analog)

(2) enzyme labeled anti(ligand), wherein the enzyme is capable of providing a detectable signal and said enzyme labeled anti(ligand) binds to said poly(ligand analog) to form a complex;

(3) anti(enzyme), which binds to said enzyme to substantially inhibit said enzyme;

said method comprising;

(A) combining in an assay medium

(i) sample;

(ii) enzyme labeled anti(ligand);

(iii) poly(ligand analog)

(B) incubating said assay medium for a sufficient time to form complexes which sterically inhibit the approach of said anti(enzyme) to said enzyme label;

(C) adding to said assay medium anti(enzyme), substrates and any cofactors for said enzyme to produce a detectable signal as a result of the chemical change in the substrates or cofactors; and

(D) measuring said detectable signal, wherein the measured detectable signal is directly related to the amount of said monoepitopic ligand in said assay medium.

14. A method according to claim 13, wherein said pH is in the range of about 6 to 9 and said temperature is in the range of about 15°

to 40°

C.

15. A method according to claim 14, wherein said enzyme is an oxidoreductase.

16. A method according to claim 14, wherein said oxidoreductase is a peroxidase and said substrates include hydrogen peroxide and a compound which forms a dye as a result of the enzymatic reaction.

17. A method according to claim 14, wherein said enzyme is glucose-6-phosphate dehydrogenase.

18. A method according to claim 13, wherein said monoepitopic ligand is of a molecular weight in the range of about 125 to 1,000.

19. An assay method for determining a polyepitopic ligand suspected of being present in a sample;

said method employing;

(1) fluorescer labeled anti(ligand), wherein said fluorescer labeled anti(ligand) binds to said polyepitopic ligand to form a complex; and

(2) anti(fluorescer);

said method comprising;

(A) combining in an assay medium at a pH of from about 6 to 9 and at a temperature in the range of about 15°

to 40°

C.

(i) sample;

(ii) fluorescer labeled anti(ligand);

(B) incubating said assay medium for a sufficient time to form complexes which sterically inhibit the approach of said anti(fluorescer) to the fluorescent label;

(C) adding anti(fluorescer) in an amount at least about stoichiometric with the amount of fluorescer and exciting fluorescer to an electronically excited state capable of emitting light; and

(D) measuring said emitted light, wherein the measured emitted light is directly related to the amount of said polyepitopic ligand present in said medium.

20. A method according to claim 19, wherein said anti(fluorescer) is conjugated with quencher.

21. A method according to claim 19, wherein said polyepitopic ligand is γ

-globulin.

22. An assay method for determining a monoepitopic ligand suspected of being present in a sample;

said method employing;

(1) poly(ligand analog), said poly(ligand analog) having a plurality of ligand analogs capable of competing with ligand for antiligand bonded to a water soluble hub nucleus

(2) fluorescer labeled anti(ligand), wherein said fluorescer labeled anti(ligand) binds to said poly(ligand analog) ligand to form a complex;

(3) anti(fluroescer);

said method comprising;

(A) combining in an assay medium at a pH of from about 6 to 9 and at a temperature in the range of about 15°

to 40°

C.

(i) sample;

(ii) fluorescer labeled anti(ligand);

(iii) poly(ligand analog)

(B) incubating said assay medium for a sufficient time to form complexes which sterically inhibit the approach of said anti(fluorescer) to the fluorescent label;

(C) adding anti(fluorescer) in an amount at least about stoichiometric with the amount of fluorescer and exciting fluorescer to an electronically excited state capable of emitting light; and

(D) measuring said emitted light, wherein the measured emitted light is directly related to the amount of said monoepitopic ligand present in said medium.

23. A method according to claim 22, wherein said anti(fluorescer) is conjugated with quencher.

24. An assay method for determining an anti(ligand) that binds to a polyepitopic ligand, said anti(ligand) suspected of being present in a sample, said method employing

(1) polyepitopic ligand;

(2) labeled anti(ligand), wherein the label is capable of providing a detectable signal and said labeled anti(ligand) and anti(ligand) bind to said ligand to form a complex;

(3) a macromolecular modifier which interacts with said label to modify the signal produced from said label and is inhibited from interacting with said label by said complex, said modifier being added in at least about an amount sufficient to interact with said label to modify said signal;

said method comprising;

(A) combining in an assay medium at a pH in the range of about 6 to 9 and at a temperature in the range of about 15°

to 40°

C. for a time sufficient to form complexes which sterically inhibit the approach of the macromolecular modified to said label

(i) sample;

(ii) labeled anti(ligand);

(iii) modifier;

(iv) polyepitopic ligand;

(B) exposing said assay medium to an agent which interacts with said label to provide said detectable signal; and

(C) measuring said detectable signal, wherein the measured detectable signal is directly related to the amount of said anti(ligand) in said medium.

25. A method according to claim 24, wherein said modifier is anti(label).

26. A method according to claim 25, wherein said label is an enzyme.

27. A method according to claim 25, wherein said label is a fluorescer and said modifier is antifluorescer in at least about stoichiometric amount based on the amount of said fluorescer in said assay medium.

28. An assay method for determining a member of an immunological pair comprising a mono- or polyepitopic ligand and an anti(ligand) suspected of being present in a sample;

said method employing;

(1) labeled anti(ligand), wherein the label is capable of providing a detectable signal and said labeled anti(ligand) binds to said polyepitopic ligand to form a complex;

(2) a Fab anti(label) which interacts with said label to modify the signal produced from said label and is inhibited from interacting with said label by said complex;

(3) poly(ligand analog), wherein poly(ligand analog) has a plurality of epitopic sites capable of competing with said monoepitopic ligand for the binding sites of labeled anti(ligand), and capable of binding with anti(ligand) and labeled anti(ligand) to form a complex; and

(4) polyepitopic ligand;

with the proviso that poly(ligand analog) is only employed in an assay for monoepitopic ligand or anti(ligand) that binds monoepitopic ligand and ligand is only employed in an assay for anti(ligand) that binds polyepitopic ligand;

said method comprising;

(A) combining in an assay medium for a time sufficient to form complexes which sterically inhibit the approach of the Fab anti(label) to said label;

(i) sample;

(ii) labeled anti(ligand);

(iii) Fab anti(label);

(iv) poly(ligand analog) in an assay for monoepitopic ligand or anti(ligand) that binds monoepitopic ligand; and

(v) ligand in an assay for anti(ligand) that binds poly(epitopic ligand);

(B) exposing said assay medium to an agent which interacts with said label to provide said detectable signal; and

(C) measuring said detectable signal, wherein the measured detectable liquid is directly related to the amount of said member in said medium.

35. An assay composition for use in a method according to claim 28 which comprises the combined reagents labeled anti(ligand) and Fab anti(label) in relative amounts for substantially optimizing the response of the reagents in providing the detectable signal in relation to changes in the concentration of the member in said sample over the concentration range between 10-4 to 10-15 m.

36. An assay composition according to claim 35, wherein said label is an enzyme.

37. An assay composition according to claim 35, wherein said label is a fluorescer.