Method and apparatus for determination of glycosylated protein

• US 5,807,747 A
• Filed: 08/20/1996
• Issued: 09/15/1998
• Est. Priority Date: 06/13/1989
• Status: Expired due to Fees

• Current Assignee: Investime, Ltd.
• Original Assignee: Clinical Innovations LLC (Clinical Innovations)
• Inventors: Green, Bryan, Wallworth, Denise Maria
• Primary Examiner(s): Pyon, Harold Y.
• Assistant Examiner(s): CARRILLO, BIBI SHARIDAN
• Application Number:
  US08/697,176
• Time in Patent Office: 756 Days
• Field of Search: 422/56, 422/58, 436/15, 436/63, 436/86, 436/67, 436/164, 436/165, 436/169, 436/172, 436/66
• US Class Current: 436/67
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]

Abstract

A method of determining the glycosylated fraction of a protein in a sample includes the steps of examining the sample to determine the total quantity of protein in the sample (READ 1), removing the glycosylated fraction of the protein, and then reexamining the sample to measure the glycosylated fraction of the protein (READ 2). The glycosylated fraction of the protein may then be calculated by the formula (READ 1-READ 2)/READ ×100. The glycosylated fraction of the protein is removed by inserting a cartridge containing a binder into the sample, or by urging the sample through a cartridge containing a binder. Both of the cartridge assemblies preferably include a flow through body, a support material disposed in the body, and a binder material attached to the support wherein the binder has an affinity for the glycosylated fraction of the protein.

Claims

1. A method of determining a percentage of glycosylated fraction (% GP) of a protein in a sample consisting essentially of:

diluting said sample in a buffer solution thereby forming a buffered sample solution;

conducting a first examination (READ

     1) of said buffered sample solution using a measuring means to determine a total quantity of said protein including said glycosylated fraction in said sample;

inserting a cartridge assembly into said buffered sample solution, said cartridge assembly including a support material and a binder attached to said support material, said binder having an affinity for the glycosylated fraction of said protein, said binder binding to and removing the glycosylated fraction of said protein in said buffered sample solution from said buffered sample solution;

conducting a second examination (READ

     2) of the buffered sample solution using said measuring means to determine the total quantity of said protein remaining after removal of said glycosylated fraction of said protein;

wherein the glycosylated fraction of said protein is not measured during said second examination;

directly calculating from the total quantities of protein obtained by the first and second examinations the percentage of the glycosylated fraction of the protein in said sample by using the following equation;

##EQU2##

2. The method of claim 1 wherein said buffered sample solution is examined optically.

3. A method of determining a percentage of glycosylated fraction (% GP) of a protein in a sample consisting essentially of:

diluting said sample in a buffer solution thereby forming a buffered sample solution;

urging said buffered sample solution through an examination station;

conducting a first examination (READ

     1) of said buffered sample solution using a measuring means while said buffered sample solution is passing through said examination station to determine a total quantity of said protein including said glycosylated fraction in said buffered sample solution,

urging said buffered sample solution through a cartridge assembly, said cartridge assembly including a support material and a binder attached to said support material, said binder having an affinity for the glycosylated fraction of said protein, in said buffered sample solution, said binder binding to and removing the glycosylated fraction of said protein from said buffered sample solution when said buffered sample solution is urged therethrough;

urging said buffered sample solution through said examination station a second time;

conducting a second examination (READ

     2) of said buffered sample solution using said measuring means while said buffered sample solution is passing through said examination station to determine the total quantity of said protein remaining after removal of said glycosylated fraction wherein the glycosylated fraction of said protein is not measured during said second examination; and

directly calculating from the total quantities of protein obtained by the first and second examinations the percentage of the glycosylated fraction of the protein in said sample by using the following equation;

##EQU3##

4. The method of claim 3 wherein said buffered sample solution is examined optically.

5. A system for determining a percentage of glycosylated fraction (% GP) of a protein in a sample consisting essentially of:

a) a metering device for obtaining said sample;

b) a buffered solution for diluting said sample and forming a buffered sample solution;

c) an apparatus for holding said buffered sample solution, said apparatus comprising an entrance tube for receiving said buffered sample solution and a bypass tube, wherein said entrance tube is connected to said bypass tube;

d) a first valve means positioned in said entrance tube for transporting said buffered sample solution through said entrance tube;

e) a measuring means positioned adjacent said entrance tube for measuring a first value (READ

     1) representing a total quantity of said protein include said glycosylated fraction as said buffered sample solution is transported through said entrance tube by said first valve means;

f) a cartridge assembly positioned in said bypass tube, said cartridge assembly including a flow through body, a support material disposed in said flow through body, and a binder material imbedded in said support material, said binder material binding to and removing the glycosylated fraction of said protein from said buffered sample solution;

g) a second valve means positioned in said bypass tube for transporting said buffered sample solution from said entrance tube to said cartridge assembly to remove said glycosylated fraction of said protein, said buffered sample solution being measured a second time by said measuring means to determine a second value (READ

     2) representing the total quantity of protein remaining in said buffered sample solution after removal of said glycosylated fraction, wherein the percentage of glycosylated fraction of the protein in said buffered sample solution is determined from said first and said second values by the following equation;

##EQU4##

6. The apparatus of claim 5, wherein said measuring means comprises an optical examining device.

7. The apparatus of claim 5, wherein said binding material comprises phenylboronic acid.

8. The apparatus of claim 5, wherein said support material comprises a hydroxyethyl methacrylate co-polymer in particulate form.

9. The apparatus of claim 5, wherein said support material comprises a nylon membrane.

10. The apparatus of claim 5, wherein said metering device comprises cellulose acetate.