Fluorescence polarization immunoassay for heavy antigens

• US 4,681,859 A
• Filed: 09/21/1984
• Issued: 07/21/1987
• Est. Priority Date: 09/21/1984
• Status: Expired due to Term

• Current Assignee: Ortho Diagnostic Systems, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Original Assignee: Ortho Diagnostic Systems, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Inventors: Kramer, Peter B.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): DeSantis, Patricia L.
• Application Number:
  US06/653,382
• Time in Patent Office: 1,033 Days
• Field of Search: 436/546, 436/501, 436/512, 436/537, 436/536, 436/800, 935/76, 935/81, 935/88
• US Class Current: 436/501
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Methods for determining the presence of large molecular weight aqueous ligands. The sample ligands are made to compete with user supplied fluorescently labeled peptides for binding sites on a ligand binding partner. Presence of aqueous ligand and binding of ligand binding partner thereto leaves the fluorescently labeled peptides free to exhibit fluorescence depolorization effects.

Claims

1. A method for determining the presence of a ligand in an aqueous sample comprising the steps of:

providing a ligand binding partner capable of specifically combining with said sample ligand at a binding site on said ligand;

further providing fluorescently labeled epitope simulating peptide capable of specifically combining with said ligand binding partner whereby binding of (i) said sample ligand of (ii) said simulating peptide to said ligand binding partner blocks binding of (i) said simulating peptide or (ii) said sample ligand, respectively, to said ligand binding partner;

allowing said sample ligand and said simulating peptide to combine with said ligand binding partner;

illuminating said fluorescent label with polarized light; and

detecting fluorescence depolarization and relating said depolarization to the presence or absence of ligand in said sample.

2. In a method for detecting the presence of a ligand in an aqueous sample based upon its competition with a fluorescently labeled ligand for the binding site on an anti-ligand partner capable of specifically reacting with either sample ligand or labeled ligand and detecting the resultant differences in fluorescent depolarization and correlating said differences with the presence of ligand in said aqueous sample, the improvement comprising providing a fluorescently labeled epitope simulating peptide for simulating the region of the sample ligand which binds to the anti-ligand whereby the presence of ligands, with a molecular weight in excess of about 1000 daltons in an aqueous sample can be determined.