# US 2015/0147784 A1 · Reagents and Methods for Detecting Influenza Virus Proteins

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- Number: US 2015/0147784 A1
- Document ID: US20150147784A1
- Status: Published application
- Kind: A1
- Application number: 14/556,415
- Filed: 2014-12-01
- Published: 2015-05-28
- Earliest claimed: 2009-06-01
- Assignees: Her Majesty the Queen in right of Canada, as represented by the Minister of Health
- Applicants: Her Majesty the Queen in Right of Canada as Represented by the Minister of Health
- Inventors: Xuguang Li; Runtao He; Gary Van Domselaar
- Web page: https://patentgrep.com/patent/US20150147784A1

## CPC

- G01N 33/56983: {Viruses}
- A61K 39/12: Viral antigens
- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61P 31/16: for influenza or rhinoviruses
- A61P 37/04: Immunostimulants
- C07K 16/108: Orthomyxoviridae (F), e.g. influenza virus
- C07K 16/40: against enzymes
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12Y 302/01018: Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
- G01N 33/573: for enzymes or isoenzymes
- A61K 2039/505: {comprising antibodies}
- A61K 2039/55566: {Emulsions, e.g. Freund's adjuvant, MF59}
- A61K 2039/6081: {Albumin; Keyhole limpet haemocyanin [KLH]}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- G01N 2333/924: acting on glycosyl compounds (3.2)
- G01N 2333/9506: derived from viruses
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Two universally conserved sequences from influenza type A neuraminidases were identified by large scale sequence analysis then chemically modified and conjugated to carrier proteins to generate mono-specific and monoclonal antibodies. The two antibodies, one targeting the N-terminus of the type A neuraminidase and the other sequence close to enzymatic active site, were capable of binding to all 9 subtypes of neuraminidase while demonstrating remarkable specificity against the viral neuraminidase sequences since no cross-reactivity against allantoic proteins was observed. Quantitative analyses of NA using slot blot suggest that the antibodies can be used for NA antigen quantitation in vaccines. These represent the first time the antibody-based immunoassay can be used for NA quantitative determination.

## Claims

### Claim 1

A method of preparing antibodies against influenza virus neuraminidase comprising inoculating an animal with an effective amount of a composition comprising an influenza neuraminidase peptide consisting of the amino acid sequence as set forth in SEQ ID No. 1 attached to a first end of a spacer, and a carrier protein attached to a second end of the spacer; and, after the animal has produced antibodies against said composition, recovering said antibodies from said animal.

### Claim 2 (depends on 1)

The method according to claim 1 wherein the spacer is selected from the group consisting of amino acids, peptides, phosphoramidite, ε-aminohexanoic acid and 6-aminocaproic acid.

### Claim 3 (depends on 1)

The method according to claim 1 wherein the carrier protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), thyroglobulin (THY) and human gamma globulin (HOG).

### Claim 4 (depends on 1)

The method according to claim 1 wherein the spacer is 6-aminocaproic acid.

### Claim 5 (depends on 1)

The method according to claim 1 wherein the carrier protein is attached to the spacer by a linker.

### Claim 6 (depends on 5)

The method according to claim 5 wherein the linker is selected from the group consisting of an amino acid, a peptide of 2-10 amino acids and KKC.

### Claim 7 (depends on 5)

The method according to claim 5 wherein the linker is KKC and the spacer is 6-aminocaproic acid.

### Claim 8

A method of preparing monoclonal antibodies against influenza virus neuraminidase comprising inoculating an animal with an effective amount of a composition comprising an influenza neuraminidase peptide consisting of the amino acid sequence as set forth in SEQ ID No. 1 attached to a first end of a spacer, and a carrier protein attached to a second end of the spacer; and, after the animal has produced antibodies against said composition, removing antibody-producing cells from said animal, fusing a respective one of said antibody-producing cells with a respective one of an immortal cell line, thereby producing a respective one hybridoma cell, and selecting for hybridoma cells producing antibodies against said composition.

### Claim 9 (depends on 8)

The method according to claim 8 wherein the spacer is selected from the group consisting of amino acids, peptides, phosphoramidite, ε-aminohexanoic acid and 6-aminocaproic acid.

### Claim 10 (depends on 8)

The method according to claim 8 wherein the carrier protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), thyroglobulin (THY) and human gamma globulin (HGG).

### Claim 11 (depends on 8)

The method according to claim 8 wherein the spacer is 6-aminocaproic acid.

### Claim 12 (depends on 8)

The method according to claim 8 wherein the carrier protein is attached to the spacer by a linker.

### Claim 13 (depends on 8)

The method according to claim 8 wherein the linker is selected from the group consisting of an amino acid, a peptide of 2-10 amino acids and KKC.

### Claim 14 (depends on 8)

The method according to claim 8 wherein the linker is KKC and the spacer is 6-aminocaproic acid.