DETECTION OF HEMOGLOBIN A1C (HbA1c) IN BLOOD
An assay method for detecting and measuring the presence or amount of glycated hemoglobin in a sample using fluorescence resonance energy transfer (FRET).
1 . A method for measuring the amount of glycated hemoglobin (HbA1c) in a sample, the method comprising:
contacting the sample with an anti-hemoglobin (HbA 0 ) antibody labeled with a first fluorophore, wherein the anti-hemoglobin (HbA 0 ) antibody also binds glycated hemoglobin (HbA1c);
contacting the sample with an anti-glycated hemoglobin (HbA1c) antibody labeled with a second fluorophore;
incubating the sample for a time sufficient to obtain a dual labeled glycated hemoglobin (HbA1c); and
exciting the sample have dual labeled glycated hemoglobin (HbA1c) using a light source to detect fluorescence emission signal associated with fluorescence resonance energy transfer (FRET).
2 . The method according to claim 1 , wherein total hemoglobin is measured in the sample.
3 . The method according to claim 1 , wherein the sample includes red blood cells.
4 . The method according to claim 1 , wherein the red blood cells are from whole blood.
5 . The method according to claim 1 , wherein the red blood cells are lysed.
6 . The method according to claim 1 , wherein the sample does not include red blood cells.
7 . The method according to claim 1 , wherein the FRET emission signal is a time resolved FRET emission signal.
8 . The method according to claim 1 , wherein the first fluorophore is a FRET energy donor.
9 . The method according to claim 8 , wherein the FRET energy donor is a terbium cryptate.
10 . The method according to claim 1 , wherein the second fluorophore is a FRET acceptor.
11 . The method according to claim 10 , wherein the acceptor is a member selected from the group consisting of fluorescein-like (green zone), Cy5, DY-647, Alexa Fluor 488, Alexa Fluor 546, Allophycocyanin (APC), Phycoeruythrin (PE) and Alexa Fluor 647.
12 . The method according to claim 1 , wherein the acceptor compound is Alexa Fluor 647.
13 . The method according to claim 1 , wherein the excitation wavelength is between about 300 nm to about 400 nm.
14 . The method according to claim 1 , wherein the emission wavelength is about 450 nm to 700 nm.
15 . The method according to claim 1 , wherein the glycated hemoglobin (HbA1c) value is less than 5.7%.
16 . The method according to claim 1 , wherein the glycated hemoglobin (HbA1c) value is between than 5.7 to 6.4%.
17 . The method according to claim 1 , wherein the glycated hemoglobin (HbA1c) value is at least 6.4%.
18 . A method for measuring the amount of glycated hemoglobin (HbA1c) in vitro in a sample, the method comprising:
obtaining a sample from a subject;
contacting the sample with an anti-hemoglobin (HbA 0 ) antibody labeled with a first fluorophore, wherein the anti-hemoglobin (HbA 0 ) antibody also binds glycated hemoglobin (HbA1c);
contacting the sample with an anti-glycated hemoglobin (HbA1c) antibody labeled with a second fluorophore;
incubating the sample for a time sufficient to obtain a dual labeled glycated hemoglobin (HbA1c); and
exciting the sample have dual labeled glycated hemoglobin (HbA1c) using a light source to detect fluorescence emission signal associated with fluorescence resonance energy transfer (FRET), to determine the amount of glycated hemoglobin (HbA1c) in the sample.
19 . The method according to claim 18 , wherein the FRET energy donor is a terbium cryptate.
20 . The method according to claim 18 , wherein the second fluorophore is a FRET acceptor.