Enzyme-immunoassay for carcinoembryonic antigen

• US 4,467,031 A
• Filed: 08/16/1982
• Issued: 08/21/1984
• Est. Priority Date: 08/21/1981
• Status: Expired due to Fees

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Gallati, Harald, Brodbeck, Hans
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US06/408,388
• Time in Patent Office: 736 Days
• Field of Search: 436/813, 436/548, 436/826, 435/7, 435/810, 422/61
• US Class Current: 435/7.5
• CPC Class Codes: G01N 33/57473 involving carcinoembryonic antigen, i.e. CEA
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/963 Methods of stopping an enzyme reaction or stabilizing the test materials
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/813 Cancer
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives

Abstract

A solid phase-sandwich enzyme-immunoassay method for the rapid determination of carcinoembryonic antigen (CEA). The sample to be investigated is incubated with a first CEA antibody, which is bound to a water-insoluble carrier, and a second CEA antibody, to which peroxidase is bound directly or via a biotin/avidin bridge. The immunological reaction can be carried out in one or two steps. By the presence of 0.4-1.0 mol/l of phosphate ions, 0.3-0.4 mol/l of sulfate ions or 0.2-0.4 mol/l of tartrate ions in the immunological reaction or in the second step thereof (insofar as it is carried out in two steps) the incubation time period is shortened considerably. After the immunological reaction, the phases are separated, whereupon the peroxidase activity is measured either in the solid or in the liquid phase as the amount of CEA present in the sample.

Claims

1. In a solid phase-sandwich method for the rapid determination of carcino-embryonic antigen (CEA) which comprises incubating a sample to be investigated with a first CEA antibody, which is bound to or becomes bound to a water-insoluble carrier, and a second CEA antibody, to which peroxidase is bound directly or via a biotin/avidin bridge, separating the solid and liquid phase and measuring of the peroxidase activity either in the solid or in the liquid phase as the amount of CEA present, wherein the improvement comprises carrying out the incubation in the presence of 0.4-1.0 mol/l of phosphate ions, of 0.2-0.4 mol/l of sulfate ions or of 0.2-0.4 mol/l of tartrate ions.

2. The method according to claim 1, wherein the sample is incubated with the first CEA antibody, which is bound to or becomes bound to a water-insoluble carrier, and, after a washing step, is incubated with the second CEA antibody, to which peroxidase is bound directly or via a biotin/avidin bridge, whereby the second incubation is carried out in the presence of 0.4-1.0 mol/l of phosphate ions, of 0.2-0.4 mol/l of sulfate ions or of 0.2-0.4 mol/l of tartrate ions.

3. The method according to claim 1 or claim 2, wherein the peroxidase is bound directly to the second CEA antibody.

4. The method according to claim 3, wherein the two CEA antibodies are obtained from antiserum of different animal species and wherein peroxidase is bound directly to one of the antibodies.

5. The method according to claim 4, wherein CEA antibodies from goat and guinea pig are used and wherein in the peroxidase is bound directly to the goat antibody.

6. The method according to claim 1 or claim 2, wherein two different monoclonal mouse-CEA antibodies are used and wherein the peroxidase is bound to one of the monoclonal antibodies via a biotin/avidin bridge.

7. The method according to claim 3, wherein there is used a CEA antibody, which is obtained from an antiserum of an animal, as well as a monoclonal mouse-CEA antibody and wherein the peroxidase is bound directly to the antibody obtained from an antiserum.

8. The method according to claim 7, wherein as the CEA antibodies there are used goat-CEA antibody and monoclonal mouse-CEA antibody and wherein the peroxidase is bound directly to the goat-CEA antibody.

9. CEA antibody, to which peroxidase is coupled directly or via a biotin/avidin bridge, in an aqueous solution of pH 4-9, wherein the solution contains 0.4-1.0 mol/l of phosphate ions, 0.2-0.4 mol/l of sulfate ions or 0.2-0.4 mol/l of tartrate ions.

10. CEA antibody according to claim 9, wherein the peroxidase is coupled directly to the antibody.

11. A test kit for determining carcinoembryonic antigen (CEA) in a sample, comprising:

(a) a first CEA antibody which is bound to water-insoluble carrier;

(b) a second CEA antibody labeled with peroxidase wherein the peroxidase is bound directly or via a biotin/avidin bridge;

(c) an aqueous solution of pH 4-9 comprising 0.4-1.0 mol/l of phosphate ions, 0.2-0.4 mol/l of sulfate ions or 0.2-0.4 mol/l of tartrate ions.

12. The test kit according to claim 11, wherein the two CEA antibodies are obtained from antiserum of different animal species and wherein the peroxidase is bound directly to one of the antibodies.

13. A test kit according to claim 11, wherein CEA antibodies from goat and guinea pig are used and wherein the peroxidase is bound directly to the goat antibody.

14. The test kit according to claim 11, wherein two different monoclonal mouse-CEA antibodies are used and wherein the peroxidase is bound to one of the monoclonal antibodies via a biotin/avidin bridge.

15. The test kit according to claim 11, wherein there is used a CEA antibody, which is obtained from an antiserum of an animal, as well as a monoclonal mouse-CEA antibody and wherein the peroxidase is bound directly to the antibody obtained from an antiserum.

16. The test kit according to claim 11, wherein as the CEA antibodies there are used goat-CEA antibody and monoclonal mouse-CEA antibody and wherein the peroxidase is bound directly to the goat-CEA antibody.