Use of calcium in immunoassay for measurement of C-reactive protein

• US 5,358,852 A
• Filed: 12/21/1992
• Issued: 10/25/1994
• Est. Priority Date: 12/21/1992
• Status: Expired due to Term

• Current Assignee: Clinical Diagnostic Systems Inc.
• Original Assignee: Eastman Kodak Company
• Inventors: Wu, Annie L.
• Primary Examiner(s): Scheiner, Toni R.
• Assistant Examiner(s): Green, Lora M.
• Application Number:
  US07/993,569
• Time in Patent Office: 673 Days
• Field of Search: 422/68.1, 422/56, 435/7.9, 435/7.92, 435/7.94, 435/970, 436/518, 436/528, 436/531, 436/532, 436/815
• US Class Current: 435/7.94
• CPC Class Codes: G01N 2333/4737 C-reactive protein
• 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 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Abstract

A specific binding immunoassay method for reducing the "hook" effect for the measurement of C-Reactive Protein has been discovered for both solution and dry analytical elements comprising contacting a liquid sample containing C-reactive protein in the presence of calcium ions with (a) a first antibody Ab1 specific for C-reactive protein, Ab1 being immobilized on a water-insoluble substrate and (b) a labeled, unbound second antibody Ab2 specific for C-reactive protein to obtain a water-insoluble complex of Ab1, ligand, and Ab2; (2) separating the water-insoluble complex from the liquid sample and unreacted Ab2; and (3) measuring either the amount of Ab2 associated with said water-insoluble complex or the amount of unreacted Ab2 as an indication of the amount of C-reactive protein in the sample.

Claims

1. A specific binding assay method comprising:

(1) contacting a liquid sample containing C-reactive protein (CRP) in the presence of from 0.01M to 0.5M calcium ions with (a) a first polyclonal antibody Ab1 specific for CRP, Ab1 being immobilized on a water-insoluble substrate and (b) a labeled, unbound second monoclonal antibody Ab2 specific for CRP to obtain a water-insoluble complex of Ab1, ligand, and Ab2;

(2) separating the water-insoluble complex from the liquid sample and unreacted Ab2; and

(3) measuring either the amount of Ab2 associated with said water-insoluble complex or the amount of unreacted Ab2 as an indication of the amount of C-reactive protein in the sample.

2. A method according to claim 1 wherein said calcium is present in an amount ranging from 0.02M to 0.2M.

3. A method according to claim 1 wherein said calcium is present in an amount of about 0.1M.

4. A method according to claim 1 wherein said calcium is incorporate into said fluid sample prior to said contacting in step (1).

5. A method according to claim 1 wherein said label on Ab2 is an enzyme.

6. An analytical element comprising a porous spreading zone and in the porous spreading zone, or a different zone, a first polyclonal antibody Ab1 specific to C-Reactive Protein (CRP), Ab1 being immobilized on a water-insoluble particulate substrate and a labeled, unbound second monoclonal antibody Ab2 specific for CRP, wherein calcium ions are incorporated into said porous spreading zone in an amount ranging from 0.01M to 0.5M.

7. A method according to claim 6 wherein said calcium is present in an amount ranging from 0.02M to 0.2M.

8. A method according to claim 7 wherein said calcium is present in an amount of about 0.1M.