# US 6,719,975 B1 · Anti-hirudin polyclonal antibodies and their use for the identification, immunopurification and quantitative determination of hirudin

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- Number: US 6,719,975 B1
- Document ID: US6719975B1
- Status: Granted
- Kind: B1
- Application number: 08/603,182
- Filed: 1996-02-20
- Granted: 2004-04-13
- Earliest claimed: 1991-06-27
- Assignees: Farmitalia Carlo Erba S.p.A.
- Inventors: Antonio Molinari; Marco Gerna; Carla Giorgetti; Jacqueline Lansen; Romeo Roncucci
- Web page: https://patentgrep.com/patent/US6719975B1

## CPC

- C07K 16/38: against protease inhibitors of peptide structure
- C07K 14/815: from leeches, e.g. hirudin, eglin
- Y10S 530/855: Proteins from animals other than mammals or birds
- 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)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

A method of making antibodies to hirudin by immunizing with an immunogenic composition containing polymerized hirudin monomers in the absence of carrier protein is taught.

## Claims

### Claim 1

An immunogen consisting essentially of a set of hirudin polymers being devoid of any heterologous carrier protein and having sufficient immunogenicity, in the absence of conjugation to said carrier protein, to elicit an antibody which binds specifically to a natural or recombinant hirudin, and, the majority of said polymers having at least three monomer units, said monomers selected from the group consisting of HV1 (SEQ ID NO:1), HV2 (SEQ ID NO:2), and HV3 (SEQ ID NO:3).

### Claim 2 (depends on 1)

An immunogenic composition for administering to a host consisting essentially of the immunogen according to claim 1, and an adjuvant.

### Claim 3 (depends on 1)

A method of making the immunogen according to claim 1, consisting essentially of polymerizing said monomers of hirudin under conditions such that a majority of polymers formed have at least 3 monomer units, said conditions including a molar excess of cross-linking agent relative to said monomers, and an absence of carrier protein.

### Claim 4 (depends on 3)

The method according to claim 3, wherein said cross-linking agent is glutaraldehyde.

### Claim 5 (depends on 4)

The method according to claim 4, wherein said glutaraldehyde is present in 1.1 to 1.5 molar excess.