Bioluminescent assay reagent and method

• US 4,665,022 A
• Filed: 02/17/1984
• Issued: 05/12/1987
• Est. Priority Date: 02/17/1984
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Schaeffer, James M., Hsueh, Aaron J.
• Primary Examiner(s): Marantz, Sidney
• Assistant Examiner(s): Spiegel, Jack
• Application Number:
  US06/581,242
• Time in Patent Office: 1,180 Days
• Field of Search: 436/536, 436/537, 436/501, 436/543, 436/544, 436/819, 436/547, 436/172, 435/7, 435/8, 260/112 R, 534/756, 534/764, 534/822, 534/824, 530/802
• US Class Current: 435/7.72
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 530/802 Chromogenic or luminescent peptides

Abstract

Reagent for immunoassay, ligand binding assay and ligand receptor assay in which luciferin is covalently bonded to a molecule having biological activity for bonding to a particular biologically active group of a material and method in which the luciferin and biologically active molecule conjugate is added to a material having a group for combination with the biological activity of the conjugated molecule, the mixture is incubated to bond the material to the reagent through the biological activity and the product of the bonding is reacted with luciferase to produce bioluminescence having intensity dependent on the concentration of the material being assayed.

Claims

1. A compound for use in bioluminescent assays comprising an amino terminated firefly luciferin derivative having bioluminescent activity, said amino terminated firefly luciferin derivative having the structure:

##STR4## wherein said amino terminated firefly luciferin derivative is covalently linked to a biologically active compound, said linkage formed by condensation between (1) a reaction product of a biological active compound and N-succinimidyl-3(2-pyridyldithio) propionate and (2) the product of reacting said amino terminated derivative of firefly luciferin with N-succinimidyl-3(2-pyridyldithio) propionate and reducing the reaction product.

2. A compound for use in the bioluminescent immuno assays, ligand binding assays or ligand receptor assays comprising an amino terminated firefly luciferin, said amino terminated firefly luciferin having the structure:

##STR5## and said amino terminated firefly luciferin being covalently joined to a protein retaining its bioluminescent properties wherein said joining is by peptide linkage resulting from carbodiimide condensation of carboxyl groups on said protein and the amino group of said amino terminated firefly luciferin.

3. Immunoassay, ligand binding assay or ligand receptor binding method determining the concentration of a material having a biologically active group comprising mixing with a sample of said material an assay reagent in which an amino terminated firefly luciferin having the structure:

##STR6## is covalently bonded by a peptide linkage to a compound having a group for biological combination with said material, said compound being selected from the group consisting of polypeptides and neurotransmitters;

said amino terminated firefly luciferin being covalently bonded by carbodiimide condensation of carboxyl groups carried by said biologically active group and amino group of said amino-terminated firefly luciferin, incubating said mixture under controlled conditions of time, temperature, pH and concentration to bond said material to said reagent to the active groups of said material and said reagent, separating the product resulting from the bonding of said material and said reagent, reacting luciferase with said product to coact with the luciferin moiety to produce bioluminescent.

4. Immunoassay, ligand binding assay or ligand receptor binding method for determining the concentration of a material having a biologically active group comprising mixing with a sample of said material an assay reagent in which an amino-terminated firefly lucifering having the structure:

##STR7## is covalently bonded by a disulfide linkage to a compound having a group for biological combination for said material;

said compounds being selected from the group consisting of polypeptides and neurotransmitters, said disulfide linkage formed by condensation between (1) a reaction product of a biologically active compound and N-succinimidyl-3-(2-pyridyldithio) propionate and (2) the product of reacting an amine terminated derivative of firefly luciferin with N-succinimidyl-3-(2-pyridyldithio) propionate and reducing the reaction product, incubating said mixture under controlled conditions of time, temperature, pH and concentration to bond said material to said reagent through the active groups of said material and said reagent, separating the product resulting from the bonding of said material and said reagent, reacting luciferase with said product to coact with the luciferin moitey to produce bioluminescence.