# US 5,352,616 A · Analyte variant analysis

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- Number: US 5,352,616 A
- Document ID: US5352616A
- Status: Granted
- Kind: A
- Application number: 07/958,326
- Filed: 1993-02-19
- Granted: 1994-10-04
- Earliest claimed: 1990-06-21
- Assignees: Axis Biochemicals AS
- Inventors: Erling Sundrehagen
- Web page: https://patentgrep.com/patent/US5352616A

## CPC

- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G01N 33/538: by sorbent column, particles or resin strip {, i.e. sorbent materials}
- G01N 2333/79: Transferrins, e.g. lactoferrins, ovotransferrins
- Y10S 435/964: including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
- Y10S 435/975: Kit
- Y10S 436/808: Automated or kit
- 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)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The present invention provides a method of assessment of the concentration of a subset of variants in a population of proteinaceous analyte variants capable of separation by a fractionation system, wherein said population of variants are contacted with a population of labelled proteinaceous specific binding partners therefor to form labelled binding partner-analyte complexes therewith, which are then subjected to separation by the said fractionation system into one or more fractions containing said subset of analyte variants in complexed form and assessment of the amount of label in one or more fractions so obtained, the said population of specific binding partners having, prior to reaction, substantially uniform distribution or mobility in said fractionation system, compositions and test kits for use in such methods. The method of the invention is particularly suitable for the analysis of variants of the protein transferrin.

## Claims

### Claim 1

A method of assessment of the concentration of a subset of variants in a population of proteinaceous analyte variants capable of separation by a fractionation system, wherein said population of variants is contacted with a population of labelled proteinaceous specific binding partners therefor to form labelled binding partner-analyte complexes therewith, which are then subjected to separation by the said fractionation system into one or more fractions containing said subset of analyte variants in complexed form and assessment of the amount of label in one or more fractions so obtained, the said population of specific binding partners having, prior to reaction, substantially uniform distribution or mobility in said fractionation system, wherein said fractionation system is not SDS-PAGE.

### Claim 2 (depends on 1)

A method as claimed in claim 1 wherein the specific binding partner for the analyte variant is an antibody or a fragment thereof.

### Claim 3 (depends on 1)

A method as claimed in claim 1 wherein the specific binding partner is monovalent.

### Claim 4 (depends on 3)

A method as claimed in claim 3 wherein the specific binding partner is a F(ab) fragment of an antibody.

### Claim 5 (depends on 1)

A method as claimed in claim 1 wherein the fractionation system is based on charge or isoelectric point.

### Claim 6 (depends on 1)

A method as claimed in claim 1 for the assessment of variants of transferrin.

### Claim 7 (depends on 6)

A method as claimed in claim 6 wherein the fractionation system is selected from ion exchange, chromatofocusing or isoelectric focusing.

### Claim 8 (depends on 6)

A method as claimed in claim 6 additionally comprising saturating the population of transferrin variants with ferric ions prior to or simultaneously with the separation step.

### Claim 9 (depends on 1)

A method as claimed in claim 1 wherein substantially uniform distribution or mobility of the specific binding partner population in said fractionation system is obtained by modification and/or fractionation.

### Claim 10 (depends on 1)

A method as claimed in claim 1 wherein the proteinaceous analyte variants share a common biological function or property which is antigenicity or enzymatic activity.

### Claim 11 (depends on 10)

A method as claimed in claim 10, wherein the common biological function is common antigenicity.

### Claim 12 (depends on 1)

A method as claimed in claim 1, wherein the proteinaceous analyte variants are glycoprotein variants differing in the extent of glycosylation.

### Claim 13 (depends on 1)

A method as claimed in claim 1 for assessment of variants of transferrin, wherein the specific binding partner is monovalent and is an antibody or a fragment thereof and the fractionation system is based on charge or isoelectric point.

### Claim 14 (depends on 1)

A method as claimed in claim 1 wherein the population of analyte variants is present in excess of the binding partner population, whereby an assessment of the relative amounts of one or more different variants relative to the total concentration of all variants, is obtained.

### Claim 15 (depends on 7)

A method as described in claim 7 wherein the population of analyte variants is present in excess of the binding partner population, whereby an assessment of the relative amounts of one or more different variants relative to the total concentration of all variants, is obtained.