Combined glycated hemoglobin and immunoturbidometric glycated albumin assay from whole blood lysate

• US 5,284,777 A
• Filed: 03/04/1991
• Issued: 02/08/1994
• Est. Priority Date: 03/04/1991
• Status: Expired due to Fees

• Current Assignee: Wallac, Inc.
• Original Assignee: Isolab Inc.
• Inventors: Hocking, David R., Rosenthal, Murray A.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Scheiner, Toni R.
• Application Number:
  US07/664,401
• Time in Patent Office: 1,072 Days
• Field of Search: 436/518, 436/815, 436/548, 436/824, 436/825, 436/808, 436/536, 436/87, 436/88, 436/67, 436/17, 530/387
• US Class Current: 436/518
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• 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: Y10S 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]

Abstract

A method and kit for the isolation and quantitation of glycated hemoglobin and other glycated proteins, specifically albumin, from a single sample of whole blood. Glycated hemoglobin is calculated from an eluate resulting from the contact of the whole blood hemolysate with a boronated resin. Glycated albumin and other plasma proteins are calculated using immunoturbidimetry, resulting from reaction between a buffered antibody solution and the albumin or other protein in the previous eluate.

Claims

1. A method of quantitating a glycated non-hemoglobin protein in a whole blood lysate, said method comprising the steps of:

(a) separating glycated proteins from non-glycated proteins by contacting said lysate with a boronated affinity column, to obtain an eluate containing glycated hemoglobin and at least one glycated non-hemoglobin protein;

(b) contacting said eluate with an antibody which specifically binds said non-hemoglobin protein, said antibody being in solution buffered to a Ph of from about 7.0 to 9.0, whereby turbidity is produced in said solution; and

(c) determining the amount of said non-hemoglobin protein in said turbid solution by measuring said turbidity.

2. The method according to claim 1 wherein said glycated non-hemoglobin protein comprises albumin.

6. The method according to claim 1 wherein said antibody is a polyclonal antibody.

7. The method according to claim 1 wherein said antibody is a goat antihuman antibody.

8. The method according to claim 1 wherein said antibody specifically binds albumin in said solution.

3. The method according to claim 3 wherein said turbidity is determined by measuring the amount of light transmitted through said solution.

4. The method according to claim 3 wherein said transmitted light is measured at a wavelength of about 305 to about 405 nanometers.

5. The method according to claim 4 wherein said transmitted light is measured at a wavelength of about 340 to about 380 nanometers.

9. A method of determining amounts of glycated non-hemoglobin protein present in a sample of whole blood containing glycated hemoglobin, said method comprising the steps of:

(a) lysing said sample of whole blood;

(b) contacting said lysed sample of whole blood with a boronated resin to absorb glycated proteins, said glycated proteins comprising glycated hemoglobin and at least one glycated non-hemoglobin protein;

(c) eluting an eluate containing said glycated proteins from said boronated resin;

(d) contacting said eluate with an antibody which specifically binds said at least one non-hemoglobin glycated protein, said antibody being in a solution buffered to a pH of from about 7.0 to 9.0, whereby turbidity is produced in said solution; and

(e) determining the amount of said at least one glycated non-hemoglobin protein in said solution by measuring said turbidity and comparing said turbidity to that of a standard to quantitate the amount of said at least one glycated non-hemoglobin protein.

10. The method according to claim 9 further comprising the step of determining the amount of glycated hemoglobin in said eluate, said step occurring after step (c).

11. The method according to claim 9 wherein said at least one non-hemoglobin glycated protein is albumin.