# US 5,210,017 A · Ligand-mediated immunofunctional hormone binding protein assay method

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 5,210,017 A
- Document ID: US5210017A
- Status: Granted
- Kind: A
- Application number: 07/615,538
- Filed: 1990-11-19
- Granted: 1993-05-11
- Earliest claimed: 1990-11-19
- Assignees: Genentech, Inc.
- Inventors: Lena M. S. Carlsson; Ross G. Clark; Wai L. T. Wong
- Web page: https://patentgrep.com/patent/US5210017A

## CPC

- C07K 16/26: against hormones {; against hormone releasing or inhibiting factors}
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/72: for hormones {(for neuromediators C07K14/70571)}
- C07K 16/2869: {against hormone receptors (for antibodies against neuromediator receptors C07K16/286)}
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- G01N 2333/61: Growth hormones [GH] (Somatotropin)
- G01N 2333/65: Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
- Y10S 435/975: Kit
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

A ligand-mediated immunofunctional assay (LIFA) method for detecting the presence and the concentration of polypeptide hormone binding proteins which comprises capturing the binding protein with a solid phase bound first antibody, saturating the bound hormone binding protein with the ligand polypeptide hormone, and detecting the bound ligand polypeptide hormone with a detectably labeled second antibody specific for the ligand polypeptide hormone. In the absence of added saturating polypeptide hormone, the LIFA measures the amount of hormone binding protein bound to the endogenous ligand polypeptide hormone. A growth hormone binding protein assay illustrates the method of the present invention.

## Claims

### Claim 1

A ligand-mediated immunofunctional method for determining the amount of polypeptide hormone binding protein in a liquid sample comprising:
(a) contacting said liquid sample with: 1) a first antibody attached to a solid phase carrier, wherein said first antibody is specific for epitopes on said polypeptide hormone binding protein such that in the presence of antibody the hormone binding sites remain available on the binding protein for binding to the ligand polypeptide hormone, thereby forming a complex between said first antibody and said polypeptide hormone binding protein; and 2) said ligand polypeptide hormone for a period of time sufficient to saturate all available ligand hormone binding sites on said hormone binding protein thereby forming a saturated complex;
(b) contacting said saturated complex with a detectably labeled second antibody which is specific for epitopes on said ligand polypeptide hormone which are available for binding when said ligand polypeptide hormone is bound to said hormone binding protein; and
(c) quantitatively analyzing the amount of said second labeled antibody bound as a measure of said polypeptide hormone binding protein in said liquid sample.

### Claim 2 (depends on 1)

The method of claim 1 wherein steps (a) 1) and (a) 2) occur sequentially.

### Claim 3 (depends on 1)

The method of claim 1 wherein said polypeptide hormone binding protein is selected from the group consisting of: a growth hormone binding protein, an insulin-like growth factor binding protein, a platelet derived growth factor (PDGF) binding protein, a nerve growth factor (NGF) binding protein, an insulin binding protein, a corticotropin releasing factor (CRF) binding protein, a transforming growth factor beta (TGF-) binding protein and an activin binding protein.

### Claim 4 (depends on 3)

The method of claim 3 wherein said polypeptide hormone binding protein is human growth hormone binding protein and the ligand polypeptide hormone is human growth hormone.

### Claim 5 (depends on 1)

The method of claim 1 wherein the ligand polypeptide hormone in step (a) 2) is selected from the group consisting of: a growth hormone, an epidermal growth factor, an insulin-like growth factor-1, an insulin-like growth factor-2, a platelet derived growth factor, a nerve growth factor, and activin, or a fragment of such polypeptide hormone having binding affinity for said hormone binding protein.

### Claim 6 (depends on 1)

The method of claim 1 wherein said liquid sample is selected from the group consisting of: serum, plasma, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, spinal fluid, saliva, sputum, tears, perspiration, mucus, tissue culture medium, tissue extracts and cellular extracts.

### Claim 7 (depends on 1)

The method of claim 1 wherein an assayable marker is attached to said ligand polypeptide hormone of step (a), wherein quantitative analysis of the amount of the assayable marker bound and of the amount of the second labeled antibody bound is a measure of saturation of available ligand binding sites.

### Claim 8 (depends on 1)

The method of claim 1 wherein said hormone binding protein is glycosylated.

### Claim 9 (depends on 1)

The method of claim 1 wherein step (a)1) and (a)2) occur simultaneously.

### Claim 10

A method for determining the amount of endogenous ligand polypeptide hormone bound to a polypeptide hormone binding protein in a liquid sample comprising:
(a) contacting said liquid sample with a first antibody attached to a solid phase carrier, wherein said first antibody is specific for epitopes on said polypeptide hormone binding protein such that in the presence of antibody the hormone binding sites remain available on the binding protein for binding to a ligand polypeptide hormone, thereby forming a complex between said first antibody and said polypeptide hormone binding protein;
(b) contacting said complex with a detectably labeled second antibody which is specific for epitopes on said ligand polypeptide hormone which are available for binding when said ligand polypeptide hormone is bound to said hormone binding protein; and
(c) determining the amount of said second labeled antibody bound as a measure of endogenous polypeptide hormone bound to said polypeptide hormone binding protein in said liquid sample.

### Claim 11 (depends on 10)

The method of claim 10 wherein said polypeptide hormone binding protein is selected from the group consisting of: a growth hormone binding protein, an epidermal growth factor binding protein, an insulin-like growth factor binding protein, a platelet derived growth factor (PDGF) binding protein, a nerve growth factor (NGF) binding protein, insulin binding protein, a corticotropin releasing factor (CRF) binding protein, a transforming growth factor beta (TGF-.beta.) binding protein, and an activin binding protein.

### Claim 12 (depends on 10)

The method of claim 10 wherein said polypeptide hormone binding protein is human growth hormone binding protein; said ligand hormone is human growth hormone; said first antibody is specific for human growth hormone binding protein; and said detectable labeled second antibody is specific for human growth hormone.

### Claim 13 (depends on 12)

The method of claim 12 wherein said antibody specific for human growth hormone is a monoclonal antibody and said detectable label is an enzyme.

### Claim 14 (depends on 13)

The method of claim 13 wherein said enzyme is selected from the group consisting of: horseradish peroxidase, glucoamylase, alkaline phosphatase, glucose oxidase, and beta-D-galactosidase.

### Claim 15

A kit useful in the assaying of polypeptide hormone binding proteins in fluids comprising:
(a) an assay device containing a solid phase carrying a first antibody, wherein said first antibody is specific for epitopes on a polypeptide hormone binding protein such that the hormone binding sites remain available for binding to the ligand polypeptide hormone;
(b) a ligand polypeptide hormone with affinity for the binding protein in the conjugate of item (a); and
(c) a detectably labeled second antibody which is specific for epitopes on said ligand polypeptide hormone, wherein said epitopes are available for binding when said ligand polypeptide hormone is bound to said hormone binding protein.

### Claim 16 (depends on 15)

The kit of claim 15 further comprising:
(d) an indicator means to provide a visual indication of the presence of said second antibody and thereby indicate the presence of said polypeptide hormone binding protein.

### Claim 17 (depends on 16)

The kit of claim 16 wherein said first antibody is specific for human growth hormone binding protein, said ligand polypeptide hormone is human growth hormone; and said detectably labeled second antibody is specific for human growth hormone.

### Claim 18 (depends on 17)

The kit of claim 17 wherein said second antibody is anti-hGH produced by hybridoma HGH-B, and said detectable label is an enzyme selected from the group consisting of: horseradish peroxidase, glucoamylase, alkaline phosphatase, glucose oxidase, and beta-D-galactosidase.

### Claim 19

In a method of assaying for the presence and amount of a polypeptide hormone binding protein in a liquid sample using a modified immunometric assay utilizing a first and second monoclonal antibody wherein the second antibody is labeled and is quantitated as a measure of the presence and amount of said polypeptide hormone binding protein, the improvement comprising:
(a) attaching the polypeptide hormone binding protein in the liquid sample to a solid phase using a first monoclonal antibody specific for epitopes on said polypeptide hormone binding protein such that the hormone binding sites remain available for binding to the ligand polypeptide hormone, thereby forming a complex between said antibody and said polypeptide hormone binding protein;
(b) saturating the complex of step (a) with said ligand polypeptide hormone for a period of time sufficient to saturate all available ligand hormone binding sites on said hormone binding protein thereby forming a saturated complex;
(c) contacting said saturated complex with a detectably labeled second monoclonal antibody which is specific for epitopes on said ligand polypeptide hormone which are available for binding when said ligand polypeptide hormone is bound to said hormone binding protein.

### Claim 20 (depends on 19)

The method of claim 19 wherein said polypeptide hormone binding protein is growth hormone binding protein and said ligand polypeptide hormone is growth hormone.

### Claim 21 (depends on 20)

The method of claim 20 wherein said growth hormone binding protein is human growth hormone binding protein and said growth hormone is human growth hormone.

### Claim 22 (depends on 21)

The method of claim 21 wherein said detectable label is an enzyme.

### Claim 23 (depends on 22)

The method of claim 22 wherein said enzyme is selected from the group consisting of: horseradish peroxidase, glucoamylase, alkaline phosphatase, glucose oxidase, and beta-D-galactosidase.

### Claim 24 (depends on 20)

The method of claim 20 wherein said growth hormone is a fragment of human growth hormone having binding affinity for human growth hormone binding protein.