Cleavable labels for use in binding assays

• US 4,780,421 A
• Filed: 04/03/1986
• Issued: 10/25/1988
• Est. Priority Date: 04/03/1986
• Status: Expired due to Fees

• Current Assignee: Sclavo SPA
• Original Assignee: Sclavo SPA
• Inventors: Rowley, Gerald L., Kameda, Naomi
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/847,505
• Time in Patent Office: 936 Days
• Field of Search: 436/518, 436/542, 436/545, 436/804, 436/536-538, 436/541, 436/800, 436/824, 435/7
• US Class Current: 436/518
• CPC Class Codes: C07D 493/10 Spiro-condensed systems
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/582 with fluorescent label
• 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 436/824 Immunological separation techniques

Abstract

An improvement in an assay method which relies on the detection of a labeled, solubilized specific binding complex is achieved by linking the label to a component of the complex through a bond cleavable under conditions compatible with the assay and with the complex in solution.

Claims

1. A method for determining the amount of an analyte in a sample suspected of containing the analyte, which method comprises:

reacting said sample with a reagent containing at least one substance specifically reactive with said analyte, obtaining an insoluble complex incorporating a cleavable detectable label, separating the complex from the liquid phase, redissolving said complex to obtain a solubilized complex, cleaving the detectable label from the solubilized complex, and detecting the cleaved label

said label being conjugated to a component of said complex, through at least one bond cleavable under conditions compatible with said assay, wherein the cleavable bond permits the label to be released from the solubilized complex prior to measuring the amount of said label.

2. The method of claim 1 wherein the analyte is an antigen and immune complexing is conducted by mixing the sample with a known amount of antigen conjugated to label, said conjugate containing the cleavable bond, and reacting the mixture with an antibody, or and immunospecific fragment thereof, against said antigen.

3. The method of claim 2 which further comprises reacting the mixture with an antibody (or immunospecific portion thereof) specific against the anti-antigen antibody, or and immunospecific fragment thereof.

4. The method of claim 1 wherein the analyte component is an antigen and immune complexing is conducted by mixing the sample containing antigen with anti-antigen antibody, or an immunospecific fragment thereof, conjugated to label, said conjugate containing the cleavable bond.

5. The method of claim 1 wherein the the analyte is an antigen and immune complexing is conducted by mixing the sample with anti-antigen antibody and with labeled anti-antibody antibody, or an immunospecific fragment thereof, conjugated to label, said conjugate containing the cleavable bond.

6. The method of claim 1 wherein the analyte is an antibody, or an immunospecific fragment thereof, and immune complexing is conducted by mixing the sample with a known amount of said antibody or an immunospecific fragment thereof, conjugated to label, said conjugate containing the cleavable bond, and reacting the mixture with an antigen against said antibody, or an immunospecific fragment thereof.

7. The method of claim 6 which further comprises reacting the mixture with an additional antibody, or an immunospecific fragment thereof, specific against the antigen.

8. The method of claim 1 wherein the analyte component is an antibody, or an immunospecific fragment thereof, and immune complexing is conducted by mixing the sample containing antibody, or an immunospecific fragment thereof, with an antigen immunoreactive with said antibody, or an immunospecific fragment thereof, said antigen being conjugated to label, and said conjugate containing the cleavable bond.

9. The method of claim 1 wherein the analyte component is an antibody, or an immunospecific fragment thereof, and immune complexing is conducted by mixing the sample with an antigen immunoreactive with said antibody, or an immunospecific fragment thereof, and with a different antibody, or an immunospecific fragment thereof, reactive with the antigen, said different antibody, or an immunospecific fragment thereof being conjugated to label and said conjugate containing the cleavable bond.

10. The method of claim 1 wherein the separation of complex from the liquid phase is conducted by precipitation followed by separating the precipitate from the liquid phase.

11. The method of claim 1 wherein the analyte component is a substance specifically reactive with the analyte which is a non-Ig-related protein.

12. The method of claim 1 wherein the separation of complex from the liquid phase is conducted by attaching the complex to a solid support and removing the support from contact with the liquid phase.

13. The method of claim 10 wherein the separation of the precipitate from the liquid phase is effected by filtration.

14. The method of claim 10 wherein the separation of the precipitate from the liquid phase is effected by centrifugation.