Antibody steric hindrance immunoassay with two antibodies

• US 3,935,074 A
• Filed: 12/17/1973
• Issued: 01/27/1976
• Est. Priority Date: 12/17/1973
• Status: Expired due to Term

• Current Assignee: Syva Co. (Siemens AG)
• Original Assignee: Syva Co. (Siemens AG)
• Inventors: Leute, Richard K., Rubenstein, Kenneth Edward
• Primary Examiner(s): Monacell, A. Louis
• Assistant Examiner(s): Fan, C. A.
• Application Number:
  US05/425,376
• Time in Patent Office: 771 Days
• Field of Search: 195/103.5 R, 23/230 B, 424/12
• US Class Current: 435/7.9
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 436/803 Stabe free radicals, e.g. spin immunoassay
• 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

A novel immunoassay is provided, as well as particular reagents, for determining the presence of a ligand. A Reagent is employed having at least two epitopes, one of the epitopes being common with the ligand, and the other epitopes being foreign to the ligand. The two epitopes are positioned in the Reagent so that antibody bound to one of the epitopes sterically inhibits the binding of antibody to the second epitope.

In carrying out the assay, the sample, the Reagent, and antibodies to the two epitopes are combined. Because of the steric inhibition to the simultaneous binding of the two antibodies to the Reagent, the amount of antibody bound to the epitope of the Reagent foreign to the ligand will be related to the amount of ligand present in the assay medium. By analyzing directly or indirectly for the antibody bound to the epitope foreign to the ligand, and comparing the results to known standards, qualitative or quantitative determinations of the amount of ligand may be made. Various detector systems may be employed for determining the amount of antibody to the foreign epitope which is unbound or bound. These systems include stable free radicals, enzymes, radioactive labels, fluorescers, and the like.

Claims

1. A method for determining the presence of a ligand in an unknown suspected of containing said ligand which comprises:

combining in an aqueous medium said unknown, antiligand, anti(detector ligand) and Reagent, wherein said Reagent has a pair of epitopes in close proximity so that simultaneous binding of receptors to said pair of epitopes is sterically inhibited, wherein one of said pair of epitopes is recognized by said antiligand and the other of said pair of epitopes is recognized by said anti(detector ligand);

determining by a detectant the amount of remaining unbound anti(detector ligand) or anti(detector ligand) bound to Reagent; and

determining the presence of ligand by comparing said amount determined with said amount determined with a medium having a known amount of ligand.

2. A method according to claim 1, wherein said aqueous medium is buffered at a pH in the range of 6 to 9 and said determining is carried out at a temperature in the range of about 15°

to 40°

C.

3. A method according to claim 2, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and a stable free radical group.

4. A method according to claim 3, wherein said stable free radical group is a cyclic nitroxide.

5. A method according to claim 2, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and a radioactive atom.

6. A method according to claim 2, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and an enzyme, wherein said activity of said enzyme is substantially reduced when said epitope recognized by said anti(detector ligand) is bound to antibody.

7. A method according to claim 6 wherein said enzyme is lysozyme, glucose-6-phosphate dehydrogenase, malate dehydrogenase, or amylase.

8. A method according to claim 2, wherein said ligand is a drug of molecular weight in the range of 125 to 2,000.

9. A method according to claim 8, wherein said ligand is an alkaloid.

10. A method according to claim 8, wherein said ligand is a catecholamine.

11. A method according to claim 8, wherein said ligand is an opiate.

12. A method according to claim 8, wherein said ligand is a cardiac glycoside.

13. A method according to claim 8, wherein said ligand is a vitamin.

14. A method according to claim 8, wherein said ligand is an iodo substituted thyronine.

15. A method according to claim 8, wherein said ligand is a steroid.

16. A method according to claim 2, wherein said ligand is an amino acid or poly(amino acid) of from 2 to 100 amino acid units.

17. A method for determining the presence of a ligand in an unknown suspected of containing said ligand which comprises:

combining in an aqueous medium buffered at a pH in the range of 6 to 9, said unknown, Reagent and antiligand, wherein said Reagent has a pair of epitopes in close proximity, so that simultaneous binding of antibodies to said pair of epitopes is sterically inhibited, wherein one of said pair of epitopes is recognized by said antiligand and the other of said pair of epitopes is recognized by an anti(detector ligand);

incubating for sufficient time for a steady state to be obtained;

adding anti(detector ligand); and

determining by a detectant at a temperature in the range of 15°

to 40°

C, the amount of remaining unbound anti(detector ligand) or anti(detector ligand) bound to Reagent; and

determining the presence of ligand by comparing said amount determined with said amount determined with a medium having a known amount of ligand.

18. A method according to claim 17, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and a stable free radical group.

19. A method according to claim 17, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and a radioactive atom.

20. A method according to claim 17, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and an enzyme whose activity is substantially reduced when said epitope is bound to said anti(detector ligand).

21. A method for determining the presence of a ligand in an unknown suspected of containing said ligand which comprises:

combining in an aqueous medium buffered at a pH in the range of 6 to 9, said unknown and antiligand;

incubating said aqueous medium for sufficient time to obtain a steady state;

adding Reagent to said aqueous medium, wherein said Reagent has a pair of epitopes in close proximity, so that simultaneous binding of antibodies to said pair of epitopes is sterically inhibited, wherein one of said pair of epitopes is recognized by said antiligand and the other of said pair of epitopes is recognized by an anti(detector ligand);

incubating for a sufficient time to establish a steady state;

adding anti(detector ligand);

determining by means of a detectant the amount of remaining unbound anti(detector ligand) or anti(detector ligand) bound to Reagent; and

determining the presence of ligand by comparing said amount determined with a medium having a known amount of ligand.

22. A method according to claim 21, wherein said detectant includes a detector molecule having an antibody recognized by said anti(detector ligand) and a stable free radical group.

23. A method according to claim 21, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and a radioactive atom.

24. A method according to claim 21, wherein said detectant includes a detector molecule having an epitope recognized by said anti(detector ligand) and an enzyme whose activity is substantially reduced when said epitope is bound to said anti(detector ligand).