Analytical method

• US 6,984,494 B2
• Filed: 08/01/2001
• Issued: 01/10/2006
• Est. Priority Date: 08/15/2000
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Ralph, Peter
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US09/921,161
• Publication Number:
  US 20020090662A1
• Time in Patent Office: 1,623 Days
• Field of Search: 435/962, 435/973, 435/7.1, 435/7.9, 435/7.91
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/57415 of breast
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• 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 435/973 Simultaneous determination of more than one analyte

Abstract

The instant invention describes an analytical assay to accurately measure an analyte in the presence of an interfering substance.

Claims

1. A method for determining the total amount of an anti-HER2 antibody in a fluid sample in the presence of HER2 extracellular domain (ECD), comprising:

(a) providing a solid surface dual-coated with first antibody recognizing said anti-HER2 antibody in a free form and a second antibody recognizing said HER2 ECD when bound to said anti-HER2 antibody;

(b) contacting said dual-coated surface with said fluid sample, whereby said first antibody binds said free anti-HER2 antibody and said second antibody binds said HER2 ECD bound to said anti-HER2 antibody; and

(c) determining the total amount of said free anti-HER2 antibody and said anti-HER2 antibody bound to said HER2 ECD by contacting the product of step (b) with a detectably labeled secondary antibody recognizing said anti-HER2 antibody, wherein said detectably labeled secondary antibody recognizes an epitope which is different from the epitope recognized by said first antibody and is also different from the epitope recognized by said HER2 ECD, and

wherein the total amount of said free anti-HER2 antibody and said anti-HER2 antibody bound to said HER2 ECD is compared with a standard curve generated by using various concentrations of said purified anti-HER2 antibody in place of said fluid sample in step (b).

2. The method of claim 1, wherein said first and second antibodies are polyclonal antibodies.

3. The method of claim 1, wherein said first and second antibodies are monoclonal antibodies.

4. The method of claim 1, wherein said detectable label is an enzyme conjugated to said secondary antibody.

5. The method of claim 4, wherein said enzyme is peroxidase.

6. The method of claim 5, wherein said peroxidase is horseradish peroxidase.

7. The method of claim 4, wherein said enzyme is alkaline phosphatase.

8. The method of claim 4, wherein said enzyme is detected using a substrate for said enzyme.

9. The method of claim 8, wherein said substrate is ortho-phenylene diamine (OPD) for the detection of said peroxidase.

10. The method of claim 1, wherein sail solid surface is in a configuration of a test tube, a well, bead, a rod, or a trip.

11. The method of claim 10, wherein said configured solid surface is glass, plastic, or paper.

12. The method of claim 11, wherein said plastic is polystyrene or polyacrylate.

13. The method of claim 1, wherein said fluid sample comprises a biological fluid.

14. The method of claim 13, wherein sad biological fluid comprises serum or plasma.

15. The method of claim 1, wherein said anti-HER2 antibody is a murine monoclonal antibody.

16. The method of claim 15, wherein said murine anti-HER2 monoclonal antibody is 4D5.

17. The method of claim 16, wherein said anti-HER2 murine monoclonal antibody is a humanized version of recombinant 4D5 antibody.

18. The method of claim 17, wherein said humanized version of recombinant 4D5 anti-HER2 antibody is huMAb4D5-8 (rhuMAb HER2).

19. The method of claim 18, wherein said first antibody recognizes huMAb4D5-8 (rhuMAb HER2) and does not significantly cross-react with hum IgG, and said second antibody against HER2 ECD is a polyclonal or a monoclonal antibody.

20. The method of claim 19, wherein said first antibody recognizing huMAb4D5-8 (rhuMAb HER2 is a monoclonal antibody or a fragment derived th from, and said monoclonal antibody against HER2 ECD is 7C2 (ATCChB-12215).

21. The method according to any one of the claims 15-18, wherein said fluid sample is serum or plasma derived from a cancer patient undergoing anti-HER2 therapy.

22. The method of claim 21, wherein said cancer is breast cancer overexpressing HER2.