Analysis and assay of glycated haemoglobins by capillary electrophoresis, buffer compositions and kits for capillary electrophoresis

Abstract

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.sub.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.

Claims

1. A method for analysis by capillary electrophoresis of glycated hemoglobin A.sub.1c comprising at least one beta globin chain and a glucose residue bound to the amino acid in the N-terminal position of the beta globin chain, contained in a biological sample, said method comprising the following steps: a. introducing a buffer composition and 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 hemoglobin A.sub.1c of the biological sample, and provides said glycated hemoglobin A.sub.1c with several negative electric charges at an alkaline pH, and wherein said compound which specifically complexes the glucose residue(s) of hemoglobin A.sub.1c of the biological sample and provides negative charges at an alkaline pH comprises two or more than two functional groups, at least one of said functional group(s) specifically complexing one or several glucose residue(s), thereby providing one negative electric charge per complexed glucose residue, the other, one of the others or all of the other functional group(s), which do not complex said glucose residue(s), providing each of said glucose residue with one or several additional negative electric charge(s) at an alkaline pH and; b. performing capillary electrophoresis on the buffered biological sample for separating the constituents of said biological sample, thereby producing an electrophoretic profile comprising a series of fractions, wherein the glycated hemoglobin A.sub.1c fraction is separated from other hemogloblins including hemoglobin A.sub.1a and hemoglobin A.sub.1b found in the biological sample, and; c. performing a step for detection of hemoglobin A.sub.1c present in the biological sample.

2. The method according to claim 1, wherein the functional group(s) specifically complexing one or several glucose residue(s) interacts with two vicinal hydroxyl groups of a glucose residue.

3. The method according to claim 1, in which the compound which specifically complexes glucose residues of hemoglobin A.sub.1c of the biological sample and provides negative charges at an alkaline pH comprises one or several groups which are anionisables at an alkaline pH, that are one or several carboxylate(s), carboxyl(s), sulphonate(s) and/or sulphonyl(s).

4. The method according to claim 1, in which the compound which specifically complexes glucose residues of hemoglobin A.sub.1c of the biological sample and provides negative charges at an alkaline pH comprises one (or more) boronyl and/or boronate group(s), and is: (i) a boronate compound with general formula RB(OH).sub.2 or RB(OH).sub.3.sup., in which the group R comprises at least one aryl 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 hemoglobins with one or several negative electric charge(s) at an alkaline pH for each glucose residue complexed with the boronate group; or (ii) a salt of said boronate compound.

5. The method according to claim 4, in which the boronate compound is a polysubstituted phenylboronate that is a phenylboronate which is disubstituted with carboxyl and/or sulphonyl groups.

6. The method according to claim 4, in which the boronate compound is a dicarboxyphenylboronic acid.

7. The method according to claim 6, in which the boronate compound is a dicarboxyphenylboronic acid selected from 3,4-dicarboxyphenylboronic acid and 3,5-dicarboxyphenylboronic acid.

8. The method according to claim 1, in which the concentration in the buffer composition of the compound which specifically complexes glucose residues of glycated hemoglobin A.sub.1c 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 hemoglobins present in the biological sample or compared with the total quantity of all of the hemoglobins comprising glucose present in the biological sample.

9. The method according to claim 1, in which the concentration in the buffer composition of the compound which specifically complexes glucose residues of glycated hemoglobin A.sub.1c of the biological sample and provides negative charges at an alkaline pH is in the range 0.10 to 100 mM.

10. The method according to claim 1, in which the buffer composition further comprises a flow retardant.

11. The method according to claim 10, in which the concentration of flow retardant in the buffer composition is in the range 0.10 to 40 mM.

12. The 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, for example water.

13. The method according to claim 12, in which the buffer compound is a zwitterionic compound.

14. The method according to claim 12, in which the concentration of buffer compound in the buffer composition is in the range 20 to 500 mM.

15. The method according to claim 1, in which the buffer composition has a pH of 9 or more.

16. The method according to claim 1, further comprising a step for generating an electropherogram from a detection signal which is proportional to the quantity of hemoglobin A.sub.1c detected.

17. The method according to claim 1, further comprising a step for determining the quantity of one or several hemoglobin (s) present in the biological sample and/or of the proportion of one or several hemoglobin (s) present in the biological sample with respect to the total quantity of proteins, the total quantity of hemoglobin or the quantity of hemoglobin A.sub.1c present in the biological sample.

18. The method according to claim 1, further comprising a step for quantification of one or several hemoglobin (s) present in the biological sample compared with one or several standardized calibrator(s).

19. The method according to claim 1, in which the biological sample is a blood sample.

20. The method according to claim 1, in which the biological sample is diluted in a haemolyzing solution.

21. A method of diagnosing diabetes in a biological sample of human or non-human mammal and/or monitoring the glycaemic balance in a biological sample of human or non-human mammal, which comprise the following steps: a. performing the method of claim 1 on a biological sample of human or non-human mammal, wherein the at least one compound which specifically complexes glucose residue(s) comprises one boronyl and/or boronate group(s), and b. performing a step of diagnosing diabetes and/or monitoring the glycaemic balance based on results from step a.

22. The method for diagnosing diabetes in a human or non-human mammal and/or monitoring the glycaemic balance in a human or non-human mammal according to claim 21, wherein the compound comprising one (or more) boronyl and/or boronate group(s), is (i) a boronate compound with general formula RB(OH).sub.2 or RB(OH).sub.3.sup., in which the group R comprises at least one aryl 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 hemoglobins with one or several negative electric charge(s) at an alkaline pH for each glucose residue complexed with the boronate group; or (ii) a salt of said boronate compound.

23. The process for separating, by capillary electrophoresis glycated hemoglobin A.sub.1c comprising at least one beta globin chain comprising a glucose residue bound to the amino acid in the N-terminal position of the beta globin chain, from other hemoglobins including hemoglobin A.sub.1a and hemoglobin A.sub.1b present in a biological sample, which comprises: a. contacting a biological sample with a compound which specifically complexes the glucose residue(s) of glycated hemoglobin A.sub.1c in said biological sample, and provides said glycated hemoglobin A.sub.1c with several negative charges at an alkaline pH, said compound comprising two or more than two functional groups, at least one of said functional group(s) specifically complexing one or several glucose residue(s), the other, one of the others or all of the other functional group(s), which do not complex said glucose residue(s), providing each of said glucose residue with one or several additional negative electric charge(s) at an alkaline pH, and said compound comprising one (or more) boronyl and/or boronate group(s), and; b. a step of performing capillary electrophoresis on the biological sample obtained from step a., thereby displacing the electrophoretic migration peak corresponding to said glycated hemoglobin A.sub.1c from the other hemoglobins including hemoglobin A.sub.1a and hemoglobin A.sub.1b, and detecting said glycated hemoglobin A.sub.1c.

24. The process according to claim 23, wherein the compound comprising one (or more) boronyl and/or boronate group(s), is (i) a boronate compound with general formula RB(OH).sub.2 or RB(OH).sub.3.sup., in which the group R comprises at least one aryl 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 hemoglobins with one or several negative electric charge(s) at an alkaline pH for each glucose residue complexed with the boronate group; or (ii) a salt of said boronate compound.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: electropherogram obtained on CE Agilent from a normal human blood sample using the analysis buffer described in U.S. Pat. No. 5,599,433, which contains 100 mM of CAPS and 300 mM of boric acid (pH: 11.00; temperature: 24 C.; voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

(2) FIG. 2: standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing different purified fractions of glycated haemoglobin using the analysis buffer described in U.S. Pat. No. 5,599,433, which contains 100 mM of CAPS and 300 mM of boric acid (pH: 11; temperature: 24 C.; voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

(3) FIG. 3: standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CAPSO and 10 mM of putrescine but no borate compound, nor boronate compound (pH: 10.20; temperature: 24 C.; voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

(4) FIG. 4: standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CAPSO, 10 mM of putrescine and 50 mM de borate (pH: 10.20; temperature: 24 C.; voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

(5) FIG. 5: standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CAPSO, 10 mM of putrescine and 50 mM of 3-carboxyphenylboronic acid (pH: 10.20; temperature: 24 C.; voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

(6) FIG. 6: standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CAPSO, 10 mM of putrescine and 50 mM of 3,5-dicarboxyphenylboronic acid (pH: 10.20; temperature: 24 C.; voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

(7) FIG. 7: standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CAPSO, 15 mM of putrescine and 100 mM of 3,5-dicarboxyphenylboronic acid (pH: 10.20; temperature: 30 C.; voltage: 10 kV, i.e. 310 V/cm; injection: 50 mbars 20 s).

(8) FIG. 8: standard electrophoretic profiles A.sub.0, A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing HbA.sub.0 and/or different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CHES, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid at a pH of 9.40. Fused silica capillary, uncoated (temperature: 34 C.; voltage: 17.3 kV i.e. 520 V/cm; injection 50 mbars 20 s).

(9) FIG. 9: standard electrophoretic profiles A.sub.0, A.sub.1a,b and A.sub.1c obtained on CE Agilent from reference samples containing HbA.sub.0 and/or different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CHES, 20 mM of putrescine and 30 mM of 3,4-dicarboxyphenylboronic acid at a pH of 9.40. Fused silica capillary, uncoated (temperature: 34 C.; voltage: 17.3 kV i.e. 520 V/cm; injection 50 mbars 20 s).

(10) FIG. 10: standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained on Capillaries (Sebia) from reference samples containing different purified fractions of glycated haemoglobin, using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. Fused silica capillary, uncoated (temperature: 34 C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 20 mbars 6 s).

(11) FIG. 11: electropherogram obtained on Capillaries (Sebia) from a normal human blood sample diluted to th in the haemolyzing solution (water+1 g/L de Triton X100), using a buffer composition containing (A) 200 mM of CAPSO buffer, 10 mM of putrescine and 50 mM of 3,5-dicarboxyphenylboronic acid at a pH of 10.2, or (B) 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. Fused silica capillary, uncoated (temperature: 34 C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars 6 s).

(12) FIG. 12: study of the influence of the concentration of 3,5-dicarboxyphenylboronic acid in the buffer composition on the separation between A.sub.1c and A.sub.0 haemoglobins (temperature: 30 C.; voltage: 10 kV, i.e. 310 V/cm).

(13) FIG. 13: electropherogram obtained on Capillaries (Sebia) from a normal human blood sample comprising a variant HbE, diluted to .sup.th in the haemolyzing solution (water+1 g/L de Triton X100), using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused silica capillary, uncoated (temperature: 34 C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars 6 s).

(14) FIG. 14: electropherogram obtained on Capillaries (Sebia) from a control AFSC diluted to .sup.th in the haemolyzing solution (water+1 g/L de Triton X100), using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused silica capillary, uncoated (temperature: 34 C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars 6 s).

(15) FIG. 15: electropherogram obtained on Capillaries (Sebia) from a pool of normal blood comprising F and Bart's variants, diluted to .sup.th in the haemolyzing solution (water+1 g/L de Triton X100), using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused silica capillary, uncoated (temperature: 34 C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars 6 s).

(16) FIG. 16: electropherogram obtained on Capillaries (Sebia) from a normal human blood sample comprising a HbD variant, diluted to .sup.th in the haemolyzing solution (water+1 g/L de Triton X100), using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused silica capillary, uncoated (temperature: 34 C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars 6 s).

(17) FIG. 17: comparison of the results obtained by the inventors by capillary electrophoresis using the Capillaries (Sebia) analyzer with the results obtained by HPLC with the Variant II Turbo (Bio-Rad) analyzer.

(18) FIG. 18A: agarose gel of HbA.sub.1c carried out on Hydrasys (Sebia) automated machine. Tracks 1 and 2: weak (5.0%) A.sub.1c calibrator and strong (10.8%) A.sub.1c calibrator. Tracks 3 to 9: normal whole blood incubated for 3 h at 37 C. with glucose at a concentration of 0 g/L (reference; track 3), 1 g/L (track 4), 5 g/L (track 5), 10 g/L (track 6), 20 g/L (track 7), 30 g/L (track 8) and 50 g/L (track 9).

(19) FIG. 18B: capillary electrophoresis profiles obtained with the Capillaries (Sebia) analyzer with the samples of FIG. 18A. The analysis was not, however, carried out for the sample containing 1 g/L of glucose as there was no visible difference in the gel (FIG. 18A). The samples were diluted to .sup.th in the haemolyzing solution (water+1 g/L de Triton X100), using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused silica capillary, uncoated (temperature: 34 C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars 6 s).

(20) FIG. 18C: table summarizing values for HbA.sub.1c obtained on agarose gel and with the Capillaries (Sebia) analyzer for the analyses of the blood presented in FIGS. 18A and 18B.

EXAMPLES

(21) A. Apparatus and Methods

(22) Capillary Electrophoresis

(23) The principle of separation is free solution capillary electrophoresis at an alkaline pH (pH>9), in order to obtain negatively charged haemoglobin fractions (the isoelectric point of haemoglobins is in the range 6.05 to 7.63).

(24) The capillary electrophoresis of biological samples was carried out on a capillary electrophoresis apparatus provided with 8 fused silica capillaries with an internal diameter of 25 microns, a useful length of 16 cm and a total length of 18 cm (Capillaries (Sebia) capillary electrophoresis system) or on capillary electrophoresis apparatus provided with one fused silica capillary with an internal diameter of 25 microns, with a useful length of 24 cm and a total length of 32 cm (.sup.3DCE capillary electrophoresis system from Agilent Technologies).

(25) Detection was carried out at a wavelength of 425 nm. The blood samples were diluted in a haemolyzing solution (Triton X100 1 g/L in water) and injected by hydrodynamic injection. The capillary was washed before each analysis with 0.25 M sodium hydroxide, then with the buffer composition.

(26) Buffer Composition

(27) The buffer compositions in which the capillary electrophoresis was carried out comprised water, a buffer compound with a pKa in the range 8 to 11 (CAPS, CAPSO or CHES depending on the case), a base allowing the pH to be adjusted to the desired value, an optional flow retardant (putrescine), and an optional borate compound (boric acid) or boronate.

(28) 3,5-dicarboxyphenylboronic acid (3,5-dCPBA) was obtained from the firms Combi-blocks Inc. (San Diego, USA) and Apollo Scientific Ltd (Cheshire, United Kingdom).

(29) 3,4-dicarboxyphenylboronic acid (3,4-dCPBA) was synthesized by the firm BoroChem SAS (Caen, France).

(30) B. Results

Example 1

(31) Capillary electrophoresis was carried out from normal human blood (comprising haemoglobins HbA.sub.0, HbA.sub.1 and HbA.sub.2) diluted to th in a haemolyzing solution (1 g/L de Triton X100 dissolved in demineralized water), using the analysis buffer described in U.S. Pat. No. 5,599,433, which contained 100 mM of CAPS and 300 mM of boric acid, pH 11. The electropherogram obtained is presented in FIG. 1. The separation between the peaks of haemoglobins is poor.

Example 2

(32) Capillary electrophoresis was carried out from reference samples (Exocell, USA), containing different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using the analysis buffer described in U.S. Pat. No. 5,599,433, which contained 100 mM of CAPS and 300 mM of boric acid, pH 10.20. The standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained are presented in FIG. 2. The separation between the HbA.sub.1c and HbA.sub.1a,b haemoglobins is clearly insufficient; the HbA.sub.1c electrophoretic peak overlaps the HbA1.sub.a/HbA1.sub.b peaks.

Example 3

(33) Capillary electrophoresis was carried out from reference samples (Exocell, USA), containing different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CAPSO and 10 mM of putrescine (pH10.20) but no borate compound, and also no boronate compound. The standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained are presented in FIG. 3. The peak corresponding to HbA.sub.1c co-migrated with that of HbA.sub.o.

Example 4

(34) Capillary electrophoresis was carried out from reference samples (Exocell, USA), containing different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CAPSO, 10 mM of putrescine and 50 mM de borate (pH 10.20). The standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained are presented in FIG. 4. The peak corresponding to HbA.sub.1c lies between the peaks corresponding to HbA.sub.o and HbA.sub.1a/HbA.sub.1b and is too close to the peak corresponding to other HbA.sub.1s to allow a reliable assay of HbA.sub.1c.

Example 5

(35) Capillary electrophoresis was carried out from reference samples (Exocell, USA), containing different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CAPSO, 10 mM of putrescine and 50 mM of 3-carboxyphenylboronic acid (pH 10.20). The standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained are presented in FIG. 5. The peak corresponding to HbA.sub.1c lies between the peaks corresponding to HbA.sub.1b and HbA.sub.1a.

Example 6

(36) Capillary electrophoresis was carried out from reference samples (Exocell, USA), containing different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CAPSO, 10 mM de DAB and 50 mM of 3,5-dicarboxyphenylboronic acid (pH 10.20). The standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained are presented in FIG. 6. The peak corresponding to HbA.sub.1c lies after the peaks corresponding to HbA.sub.1b and HbA.sub.1a.

Example 7

(37) Capillary electrophoresis was carried out from reference samples (Exocell, USA) containing different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CAPSO, 15 mM of flow retardant (putrescine) and 100 mM of 3,5-dicarboxyphenylboronic acid (pH 10.20). The standard electrophoretic profiles for A.sub.1a,b and A.sub.1c obtained are presented in FIG. 7. The peak corresponding to HbA.sub.1c lies after the peaks corresponding to HbA.sub.1b and HbA.sub.1a and is distinct from these peaks; the separation between the haemoglobin HbA.sub.1c and the haemoglobins HbA.sub.1a and HbA.sub.1b is excellent.

Example 8

(38) Capillary electrophoresis was carried out from reference samples containing HbA.sub.0 and/or different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CHES, 20 mM of flow retardant (putrescine) and 30 mM of 3,5-dicarboxyphenylboronic acid (at a pH of 9.40). The standard electrophoretic profiles A.sub.0, A.sub.1a,b and A.sub.1c obtained are presented in FIG. 8. The peak corresponding to HbA.sub.1c lies after the peaks corresponding to HbA.sub.0, HbA.sub.1b and HbA.sub.1a and is clearly distinct from these peaks; the separation between the haemoglobin HbA.sub.1c and the HbA.sub.1a and HbA.sub.1b haemoglobins is excellent.

Example 9

(39) Capillary electrophoresis was carried out from reference samples containing HbA.sub.0 and/or different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c, or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CHES, 20 mM of flow retardant (putrescine) and 30 mM of acid 3,4-dicarboxyphenylboronic (at a pH of 9.40). The standard electrophoretic profiles A.sub.0, A.sub.1a,b and A.sub.1c obtained are presented in FIG. 9. It will be seen that the peak corresponding to HbA.sub.1c lies after the peaks corresponding to HbA.sub.0, HbA.sub.1b and HbA.sub.1a and is clearly distinct from these peaks; the separation between the haemoglobin HbA.sub.1c and the HbA.sub.1a and HbA.sub.1b haemoglobins is excellent.

(40) By comparing the electrophoretic profiles of examples 8 and 9, it will be seen that 3,5-dicarboxyphenylboronic acid can produce a slightly better result in terms of separation of HbA.sub.1c compared with the other fractions, while 3,4-dicarboxyphenylboronic acid allowed a slightly better result to be obtained in terms of focussing.

Example 10

(41) Capillary electrophoresis was carried out on Capillaries (Sebia) from reference samples (Exocell, USA) containing different purified fractions of glycated haemoglobin (fractions A.sub.0 and A.sub.1c or fractions A.sub.0, A.sub.1b and A.sub.1a), using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. The standard electrophoretic profiles for A1.sub.a,b and A.sub.1c obtained are presented in FIG. 10. It will be seen that the peak corresponding to HbA.sub.1c lies after the peaks corresponding to HbA.sub.1b and HbA.sub.1a and is clearly distinct from these peaks; the separation between the haemoglobin HbA.sub.1c and the HbA.sub.1a and HbA.sub.1b haemoglobins is excellent.

Example 11

(42) Analyses by capillary electrophoresis were carried out on normal human blood diluted to th in haemolyzing solution (water+1 g/L de Triton X100), using a buffer composition containing either 200 mM of CAPSO buffer, 10 mM of putrescine and 50 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 10.20, or 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. The electropherograms obtained on Capillaries (Sebia) using a fused silica capillary, uncoated, are presented in FIGS. 11A and 11B respectively. In both cases, complete separation of the haemoglobin HbA.sub.1c from the other haemoglobins forms is observed.

Example 12

(43) The influence of concentration of 3,5-dicarboxyphenylboronic acid in the buffer composition on the separation between the A.sub.1c and A.sub.o haemoglobins was studied. The buffer composition used contained 200 mM of CAPSO, 15 mM of putrescine and 0 to 120 mM of 3,5-dicarboxyphenylboronic acid. The results are presented in FIG. 12. The A.sub.1c/A.sub.o separation increased with the concentration of 3,5-dicarboxyphenylboronic acid.

Example 13

(44) Capillary electrophoreses were carried out on Capillaries (Sebia) using four different samples diluted by th in the haemolyzing solution (water+1 g/L de Triton X100): normal human blood comprising a HbE variant (FIG. 13), a AFSC control (FIG. 14), a pool of normal blood comprising F and Bart's variants (FIG. 15) and normal human blood comprising a HbD variant (FIG. 16). Capillary electrophoresis was carried out in a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, using a fused silica capillary, uncoated.

(45) FIGS. 13-16 show the absence of interference from the principal variants of haemoglobin (E, F, S, C, D and Bart) with the HbA.sub.1c fraction. Note, however, that in the case of Bart's haemoglobin, the resolution is not complete between the Hb Bart and HbA.sub.1c fractions. As a consequence, in order to be able to assay HbA.sub.1c in the presence of Hb Bart, these two fractions should be capable of being quantified using a suitable integration method. If this is not possible, it will be necessary to alert the user to this, in case he observed this type of profile with a shoulder on the expected peak.

Example 14

(46) The results obtained by capillary electrophoresis by the method of invention using the Capillaries (Sebia) analyzer were compared with the results obtained with one of the reference techniques: HPLC with the Variant II Turbo (Bio-Rad).analyzer.

(47) Capillary electrophoresis was carried out from whole blood diluted to th in the haemolyzing solution (water+1 g/L de Triton X100), in a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40.

(48) FIG. 17 shows the very good correlation of this novel technique for analysis by capillary electrophoresis with the analysis of HbA.sub.1c by HPLC with the Variant II Turbo from Bio-Rad. After calibration of the EC data using 2 calibrators (weak A1.sub.c and strong A1.sub.c), the values obtained by the method of invention were very close to those obtained by the reference method.

Example 15: Study Demonstrating the Absence of Interference of the Labile Fraction of HbA1c With the Assay of HbA1c Using the Method of the Invention

(49) Hypothesizing that the compound complexing glucose and providing negative charges at an alkaline pH used in the context of the analysis method of the invention would be capable of interacting with blood glucose (this interaction is hypothetical and has not been demonstrated), a study of any interference of free glucose on the result of the blood of HbA.sub.1c was carried out as follows: normal blood was incubated for 3 hours at 37 C. with different concentrations of glucose (0 to 50 g/L) in order to create the labile form of HbA.sub.1c (form obtained before rearrangement of the molecule (Amadori rearrangement)). Once incubation had been carried out, the blood samples were centrifuged and the pellets obtained were reconstituted in physiological water and the haemolyzing solution (15 L of pellet+25 L physiological water+160 L of haemolyzing solution (water+1 g/L Triton X100)) was then analyzed in parallel using a Hydrasys automated machine from Sebia (HbA.sub.1c gel) and using the Capillaries (Sebia) technique, using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40.

(50) The HbA1c gel obtained by the analysis on the Hydrasys automated machine (see FIG. 18A) confirmed the formation of labile fraction of the HbA1c migrating to the same level as the HbA1c and in an increasing concentration as the concentration of glucose increased during incubation with the blood. It should be noted that in gel, under the normal conditions of use defined by Sebia, the labile fraction did not appear, in particular because of the acid pH of the haemolyzing solution.

(51) In contrast, the analyses carried out on Capillaries (Sebia) with a buffer composition of the invention (200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40), on the same blood samples showed that the assay was not perturbed by the presence of free glucose, regardless of the concentration of incubated glucose, in the range studied (0 to 50 g/L): the profiles and the values of HbA1c were unchanged (see FIGS. 18B and 18C).

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