Analysis and assay of glycated haemoglobins by capillary electrophoresis, buffer compositions and kits for capillary electrophoresis
The invention relates to a method for analysis by capillary electrophoresis of glycated haemoglobins comprising at least one globin chain comprising a glucose residue bound to the amino acid in the N-terminal position, contained in a biological sample, said method comprising using a buffer composition comprising at least one compound which is capable of specifically complexing glucose residues of one or several glycated haemoglobin(s) and of providing said glycated haemoglobin(s)with several negative electric charges at an alkaline pH. By way of example this compound may be 3,4- or 3,5-dicarboxyphenylboronic acid, preferably 3,5-dicarboxyphenylboronic acid. Said method may in particular be used to separate and assay haemoglobin HbA 1c present in a biological sample optionally comprising other haemoglobins, in particular other minor fractions. The invention also concerns buffer compositions for use in said analysis, as well as kits for the analysis and for the assay of glycated haemoglobins by capillary electrophoresis.
1. A method for analysis by capillary electrophoresis of one or several glycated haemoglobin(s) comprising at least one globin chain comprising a glucose residue bound to the amino acid in the N-terminal position of the beta globin chain, contained in a biological sample comprising haemoglobins, said method comprising the following steps
a. introducing a buffer composition and a biological sample into an electrophoresis capillary, said buffer composition comprising at least one compound which specifically complexes the glucose residue(s) bound to an amino acid in the N-terminal position in glycated haemoglobins of the biological sample and provides said glycated haemoglobin(s) with several negative electric charges at an alkaline pH,wherein said compound which specifically complexes the glucose residue(s) of glycated haemoglobins of the biological sample and provides negatice charges at an alkaline pH comprises two or more than two functional groups, at least one of said functional groups specifically complexing one or more glucose residue(s), thereby providing one negative electric charge per complexed glucose residue,and at least one off said functional groups does not complex the glucose residue(s) thereby providing each glucose residue with one or more additional negative charges at an alkaline pH and is:
(i) a boronate compound with general formulal RB(OH) 2 or RB(OH) 3 , in which the group R comprises at least one arvl and/or alkyl (linear,branched or cyclic) and/or an aralkyl and/or other functional groups or heteroatoms, and/or a combination thereof, and said group R provides glycated hamoglobins with one or several negative electric charge(s) at an alkaline pH for each glucose residue complexed with the boronate group; wherein said boronate compound is a dicaroxyphenylboronic acid or
(ii) a salt of a dicarboxyphenylboronic acid;
b. performing capillary electrophoresis on the buffered biological sample to separate constituents of the biological sample; and
c. detecting one or several haemoglobin(s) present in the biological sample.
2. A method according to claim 1 , in which the concentration in the buffer composition of the compound which specifically complexes glucose residues of glycated haemoglobin(s) of the biological sample and provides negative charges at an alkaline pH is in stoechiometric excess with respect to the total quantity of proteins, compared with the total quantity of all of the haemoglobins present in the biological sample or compared with the total quantity of all of the haemoglobins comprising glucose present in the biological sample.
3. A method according to claim 1 , in which the concentration in the buffer composition of the compound which specifically complexes glucose residues of glycated haemoglobins of the biological sample and provides negative charges at an alkaline pH is in the range 0.10 to 100 mM.
4. A method according to claim 1 , in which the buffer composition further comprises a flow retardant.
5. A method according to claim 4 , in which the concentration of flow retardant in the buffer composition is in the range 0.10 to 40 mM.
6. A method according to claim 1 , in which the buffer composition further comprises:
a buffer compound having a pKa in the range 8.0 to 11.0; and/or
a base; and/or
a salt,; and/or
a diluting solution.
7. A method according to claim 6 , in Which the buffer compound is a zwitterionic compound.
8. A method according to claim 6 , in which the concentration of buffer compound in the buffer composition is in the range 20 to 500 mM.
9. A method according to claim 1 , in which the buffer composition has a pH of 9 or more.
10. A method according to claim l, further comprising a step for generating an electropherogram from a detection signal which is proportional to the quantity of haemoglobin(s) detected.
11. A method according to claim 1 , further comprising a step for determining the quantity of one or several haemoglobin(s) present in the biological sample and/or of the proportion of one or several haemoglobin(s) present in the biological sample with respect to the total quantity of proteins, the total quantity of haemoglobin or the quantity of certain haemoglobins present in the biological sample.
12. A method according to claim 1 , further comprising a step for quantification of one or several haemoglobin(s) present in the biological sample compared with one or several standardized calibrator(s).
13. A method according to claim 1 , in which the biological sample is a blood sample.
14. A method according to claim 1 , in which the biological sample is diluted in a haemolyzing solution.
15. The method according to claim 1 , wherein the boronate compound is a dicarboxphenylboronic acid selected from 3,4-dicarboxyphenylboronic acid and 3,5-dicarboxyphenylboronic acid.