Bispecific antibody determinants

• US 4,444,878 A
• Filed: 12/21/1981
• Issued: 04/24/1984
• Est. Priority Date: 12/21/1981
• Status: Expired due to Term

• Current Assignee: Boston Biomedical Research Institute
• Original Assignee: Boston Biomedical Research Institute
• Inventors: Paulus, Henry P.
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/332,881
• Time in Patent Office: 855 Days
• Field of Search: 435/7, 435/18, 435/25, 435/28, 435/174, 435/175, 435/178, 435/179, 435/288, 435/291, 435/172, 435/188, 435/805, 435/810, 435/817, 435/177, 435/4, 436/548, 436/806, 436/528, 436/530, 436/512, 436/518, 436/808, 436/819, 204/195 B, 260/112 R, 422/55, 422/57
• US Class Current: 435/7.1
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C08L 2666/02 Organic macromolecular compounds, natural resins, waxes or and bituminous materials
• CPC Class Codes: C08L 2666/04 Macromolecular compounds according to groups C08L7/00 - C08L49/00, or C08L55/00 - C08L57/00; Derivatives thereof
• CPC Class Codes: C08L 35/04 Homopolymers or copolymers of nitriles C08L35/06, C08L35/08 take precedence
• CPC Class Codes: C08L 55/02 ABS [Acrylonitrile-Butadiene-Styrene] polymers
• CPC Class Codes: C08L 65/00 Compositions of macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain C08L7/00 - C08L57/00, C08L61/00 take precedence; Compositions of derivatives of such polymers
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: G01N 33/563 involving antibody fragments
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• 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 435/964 including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
• CPC Class Codes: Y10S 435/966 involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
• CPC Class Codes: Y10S 435/969 Multiple layering of reactants
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

A homogenous sample of identical bispecific antibody determinants, each determinant being composed of two L-H half-molecules linked by disulfide bonds, each L-H half-molecule being specific for a different antigenic determinant and including at least the F(ab'"'"') portion of a monoclonal IgG antibody. The bispecific antibody determinants are useful, e.g., in the formation of multilamellar assemblies and enzyme electrodes.

Claims

1. A homogenous sample of identical bispecific antibody determinants, each said determinant comprising two L-H half-molecules linked by disulfide bonds, each said L-H half-molecule being specific for a different antigenic determinant, and comprising at least the F(ab'"'"') portion of a monoclonal IgG antibody,

one said antigenic determinant comprising an antigenic site on a solid matrix, whereby said bispecific antibody determinant is capable of being immobilized on said solid matrix by binding to said matrix at said antigenic site,

said sample comprising a multilamellar assembly wherein

said antigenic site on said matrix is a site on a haptenic molecule attached to said matrix,

said bispecific antibody determinant is bonded to said haptenic molecule,

the other said antigenic determinant comprises a first antigenic site on a first protein molecule, said bispecific antibody determinant being bonded to said protein molecule, and

there is bonded to said first protein molecule, at a second antigenic site on said protein molecule, a second bispecific antibody determinant different from the determinant bonded to said haptenic molecule, each said second determinant comprising two L-H half molecules linked by disulfide bonds, each said L-H half molecule being specific for a different antigenic determinant, one said antigenic determinant being a second antigenic site on said first protein molecule, each said half-molecule comprising at least the F(ab'"'"') portion of a monoclonal IgG antibody.

2. The assembly of claim 1 wherein the other said antigenic determinant for which said second bispecific antibody determinant is specific is an antigenic site on a second protein molecule.

3. The assembly of claim 2 wherein each said first and second protein is an enzyme.

4. The assembly of claim 3 wherein

said assembly is useful for the measurement of a substance, and

said enzymes participate in a series of reactions which result in the production, from said substance, of a measurable effect which is a measure of said substance.

5. Electrode apparatus for the measurement in a sample of an unknown amount of a substance which is acted on by one or more enzymes to evolve a measurable ion or compound, said ion or compound evolved being a measure of said unknown substance, said electrode apparatus comprising

means for measuring said measurable ion or compound, and,

associated with said means for measuring said measurable ion or compound, a membrane having associated therewith a plurality of molecules of each said enzyme which acts on said substance to be measured and, bonded to the molecules of each said enzyme, a plurality of identical, bispecific antibody determinants, each said determinant comprising two L-H half-molecules linked by disulfide bonds, each said half-molecule being different from the other and comprising at least the F(ab'"'"') portion of a monoclonal IgG antibody, one said L-H half-molecule being specific for an antigenic site on the enzyme molecule to which it is bonded, the other half-molecule being specific for an antigenic determinant on said membrane, said bispecific antibody determinant being bonded thereto,

wherein said substance to be measured is acted on by more than one enzyme, and

the molecules of at least one of said enzymes are bonded to half-molecule of each of two different said bispecific antibody determinants at two different antigenic sites on said enzyme molecules.

6. Electrode apparatus for the measurement in a sample of an unknown amount of a substance which is acted on by one or more enzymes to evolve a measurable ion or compound, said ion or compound evolved being a measure of said unknown substance, said electrode apparatus comprising

means for measuring said measurable ion or compound, and,

associated with said means for measuring said measurable ion or compound, a membrane having associated therewith a plurality of molecules of each said enzyme which acts on said substance to be measured and, bonded to the molecules of each said enzyme, a plurality of identical, bispecific antibody determinants, each said determinant comprising two L-H half-molecules linked by disulfide bonds, each said half-molecule being different from the other and comprising at least the F(ab'"'"') portion of a monoclonal IgG antibody, one said L-H half-molecule being specific for an antigenic site on the enzyme molecule to which it is bonded, the other half-molecule being specific for an antigenic determinant on said membrane, said bispecific antibody determinant being bonded thereto,

wherein said substance to be measured is lactose,

said enzymes are β

-galactosidase and glucose oxidase, and

said measurable compound is oxygen,

wherein the molecules of said glucose oxidase are bonded to two different said bispecific antibody determinants at two different antigenic sites on said glucose oxidase molecules.