AU661207B2 - Simultaneous determination of HbA1c and haemoglobin variants with a glycation analogus to HbA1c - Google Patents

Abstract

The invention relates to a method for the immunological determination of the content of glycosylated haemoglobin in a blood sample, in which an antibody which recognises HbA1c, HbS1c and HbC1c is used, to the antibody used in this method, and to a method for producing an antibody of this type.

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Boehrlnger Mannhelm GmbH ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

INVENTION TITLE: Simultaneous determination of HbAlc and haemoglobin variants with a glycation analogus to HbAlc The following statement is a full description of this invention, including the best method of performing it known to me/us:oooo *t e oooe o roooe ooo I e la The invention concerns a method for the immunological determination of the content of glycated haemoglobin in a blood sample in which antibodies are used which recognize HbAlc, HbSIc and HbC1c as well as a process for the production of such antibodies.

Haemoglobin which transports oxygen and CO 2 and is located in the erythrocytes is composed of four protein chains of which two in each case have the same structure. It is mainly composed of two non-glycosylated a and B chains in each case. Usually more than 90 of the haemoglobin in blood is in this form denoted HbA 0 Variants of the a chain as well as of the B chain of haemoglobin with an altered amino acid sequence are known which impair the transport function of the haemoglobin molecule and lead to so-called haemoglobinopathies. In the most well-known of these haemoglobinopathies, sickle-cell anaemia, the hydrophilic glutamic acid is replaced by the hydrophobic valine at position 6 of the B chain. This results in a hydrophobic cyclisation between this valine and the valine at position 1 of the B chain. This structure causes an aggregation of HbS molecules containing such an altered B chain which influences the transport function of this altered haemoglobin. HbC, another pathological haemoglobin, also differs from HbA by an amino acid substitution at position 6 of tie B chain. In -I 2 this case glutamic acid is substituted by lysine. Apart from these, numerous further haemoglobin variants are known with a defined amino acid substitution HbA2, HbE).

Glycated haemoglobin derivatives are formed from these haemoglobin variants in vivo by a non-enzymatic reaction with glucose. This non-enzymatic glycation is a slow continuous reaction which proceeds irreversibly and is essentially dependent on the blood glucose concentration. In the glycation process a Schiff's base is formed between the aldehyde group of glucose and the free amino acid of the haemoglobin. The aldimine formed in this process rearranges by means of an Amadori rearrangement to form a N-(l-deoxy-D-fructose-l-yl) residue. The glycated haemoglobin is stable in this rearranged form.

The glycated haemoglobin which usually forms is denoted HbAlc. The aforementioned glycated haemoglobin variants are denoted HbSIc or HbC1c. These are formed by glycation of the free amino group of the valine or lysine residue which is located at the N-terminal end of the 8 chain of haemoglobin. The various haemoglobin variants are glycated in this process to the same extent as normal HbA 0 Sosenko et al., Diabetes Care 3 (1980), 590 593).

The concentration of g'lycated haemoglobins in blood depends on the blood glucose concentration. The proportion of glycated haemoglobins in relation to total haemoglobin is normally in the range of 3 6 in adults, When the blood sugar level is increased this proportion increases up to 15 The determination of ~BBIP~R~9~8~eL~ L r I _r 'P r I, the proportion of N-terminally glycated haemoglobin is therefore a reliable parameter for monitoring the blood glucose level. Since the erythrocytes have an average half life of 120 days, the dete.imination of glycated haemoglobin in blood provides a parameter for the blood glucose level which is independent of a short-term increase e.g. after a carbohydrate-rich meal.

The determination of glycated haemoglobin in blood is thus of great importance for the diagnosis and monitoring of diabetes mellitus. A number of chromatographic and electrophoretic methods for the detection of HbAlc have therefore been developed.

However, the glycated haemoglobin variants cannot be determined simultaneously with HbAlc using these methods Sosenko et al., Diabetes Care 3 (1980), 590 593, D. Goldstein et al., CRC Critical Reviews in Clinical Laboratory Sciences 21 (1984), 187 225 and Allen et al., Annual Clinical Biochemistry 29 (1992), 426 429).

SAlthough, in addition to HbAlc glycated haemoglobin variants such as HbSlc or HbC1c, are also determined simultaneously using affinity chromatographic methods, *this method is not specific for glycation at the N-terminus of the B chain since all glycations of the haemoglobin molecule on lysine residues or on the a chain) are measured equally Goldstein et al., CRC SCritical Reviews in Clinical Laboratory Sciences 21 (1984), 187 225). As a result the values of the chromatographic methods for the proportion of N-terminally glycated haemoglobin are too low in a patient with one of the aforementioned haemoglobinopathies whereas the affinity chromatographic methods in general measure higher values. These priorart methods can therefore not be used for the diagnosis ~LLP~-~PCLP DLslLI I 4 and monitoring of diabetes mellitus in blood samples of patients with haemoglobinopathies.

It has now surprisingly turned out that by using antibodies which can be obtained by immunization with an immunogen containing the glycated oligopeptide fructose- Val-His-Leu-Thr-Pro or a part thereof as the hapten component, antibodies are obtained which recognize HbAlc, HbSIc and HbCl c and are suitable for use in immunological metnods of determination for HbAlc, HbSlc and HbClc.

Accordingly the invention concerns the use of antibodies which recognize HbAlc, HbSlc and HbClc and are obtainable by immunization with at least one immunogen which contains the glycated oligopeptide fructose-Val- His, fructose-Val-His-Leu, fructose-Val-His-Leu-Thr and/or fructose-Val-His-Leu-Thr-Pro as the hapten component for the immunological, simultaneous determination of HbAlc, HbS1c and HbClc.

2 The fact that antibodies produced in this way recognize HbAlc, HbSlc and HbClc is also particularly surprising because the amino acid substitution at position 6 of the B chain of HbS and HbC also leads to a change in the tertiary structure in the region of the epitope recognized by the antibody and consequently it would have been expected that antibodies which are obtained with the aforementioned immunogen would differentiate between HbAlc, HbS1c and HbClc.

The simultaneous immunological determination of N-terminally glycated haemoglobin HbAlc, HbSlc and HbClc can be achieved with these antibodies by means of all 5 current immunoassays such as e.g. ELISA, fluorescence immunoassay, radioimmunoassay, fluorescence-polarisation immunoassay (FPIA), cloned enzyme donor immunoassay (CEDIA) or enzyme multiplied immunoassay technique (EMIT). The test can be carried out in this process as a homogeneous test, e.g. as a competitive turbidimetric immunoassay and also as a heterogeneous test.

The test is preferably carried out according to the principle of the agglutination test such as e.g. TINIA or latex particle-enhanced immunoassay (LPIA). These are competitive tests in which the antigen from the sample competes with a polyhapten, in which several haptens are present bound to a high molecular carrier protein, for binding to a specific antibody. The hapten used for the immunization (preferably fructose-Val-His-Leu-Thr-Pro) is preferably used as the hapten which is present bound to a high molecular carrier. In the absence of antigen from the sample this immunogen is cross-linked by the antibodies used in the test to form large aggregates which cause a certain turbidity of this test solution.

High molecular polyhapten is displaced from these aggregates by the antigen to an extent corresponding to the amount of antigen in the sample. As a result the turbidity decreases in a manner proportional to the amount of antigen. By comparison with the turbidity decrease which is observed on addition of known amounts of glycated haemoglobin, it is possible to determine of the amount of glycated haemoglobin (HbAlc, HbSlc and HbClc) in the sample solution. For this HbAlc, HbSlc or HbC1c can be used alone or as mixtures for the standard.

In addition it is preferred that the method according to the invention is carried out according to the CEDIA, EMIT, FPIA or ELISA technique.

6 In the CEDIA technique, the antigen alone from the sample to be analysed causes an association of inactive enzyme acceptor and enzyme donor to form an active enzyme whose activity is thus proportional to the amount of antigen in the sample to be analysed. (Henderson et al., Clinical Chemistry 32 (1986), 1637 1641). Certain enzymes such as e.g. B-galactosidase are used for this test which are present as two components each of which is enzymatically inactive, namely a large polypeptide (enzyme acceptor) and a small polypeptide (enzyme donor), and these components associate spontaneously to form an enzymatically active protein. The hapten which is to be detected as the analyte is bound to the enzyme donor in such a way that the association of the enzyme donor with the enzyme acceptor to form the active enzyme is not impeded by this binding. This association is, however, inhibited when an antibody against the antigen binds to the antigen-enzyme donor complex. Therefore in a reagent solution in which enzyme acceptor, antigenenzyme 2nor complex and the corresponding antibody are present, no active enzyme is formed and no enzymatic activity is measured. After addition of the sample solution the antigen from this sample solution now displaces the antibody from the binding to the antigenenzyme donor complex and thus enables formation of the *'oo active enzyme.

In the enzyme multiplied immunoassay technique (EMIT), the hapten to be detected is coupled covalently to the S"marker enzyme in such a way that the enzymatic activity is retained. However, after an antibody binds to the hapten cozmpsnent, the substrate binding to the enzyme is S"sterically hindered so that enzymatic conversion of the substrate cannot take place. As in the CEDIA technique, the antigen from the sample solution to be determined 7 then also in this case displaces the antibody from the enzyme-bound hapten and thus enables an enzymatic activity which is proportional to the concentration of the antigen to be analysed in the sample solution (Gunzer et al., "Kontakte III, 1980, 3 11 and K.

Rubenstein, Biochemical and Biophysical Research Communications 47 (1972), 846 851).

In the fluorescence polarisation immunoassay (FPIA), the hapten to be determined is labelled with a fluorescent substance. These molecules absorb light energy and release it as light of a longer wavelength in a period of about 10- 8 sec. If the fluorophore is excited by polarized light, then the degree of polarisation of the emitted light is dependent on the speed of rotation of the tracer (analyte-fluorophore conjugate). Binding of the tracer to an antibody impedes the rotation of the fluorophore. The free tracer rotates more rapidly and depolarizes the excitatory light more than the larger, more inert antibody-tracer complex. The more analyte is present in the sample, the less antibody-tracer Scomplexes are formed and the less fluorescence polarisation can be measured Dandliker et al., Journal of Exp. Med. 122 (1965), 1029).

In immunoassays based on the ELISA principle, binding of an enzyme-labelled antibody to an antigen from the sample solution to be determined, immobilized before or during the detection reaction, is determined by measuring the enzyme marker in the solid phase.

The invention in addition concerns monoclonal and polyclonal antibodies which recognize HbAlc, HbSl c and HbC1c and are obtainable by immunizat.'on with an 8 immunogen which contains the glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu or fructose-Val- His-Leu-Thr-Pro as the hapten, and isolating the antibodies from the serum of the immunized animals by known methods.

A further subject matter are monoclonal and polyclonal antibodies obtainable by immunising a mammal with a mixture of an immunogen which contains fructose-Val-His- Leu-Thr as the hapten and with at least one further immunogen which contains fructose-Val-His, fructose-Val- His-Leu and/or fructose-Val-His-Leu-Thr-Pro as the hapten component, and isolation of the antibodies from the serum of the immunized animals.

A preferred subject matter of the invention are monoclonal antibodies which recognize HbAlc, HbSlc and HbClc and are obtainable by immunization with the said immunogen, immortalization of the spleen cells of the immunized animals, cloning those immortalized cells .which produce the desired antibody and isolation of the •o antibody from the cells producing the antibodies according to known methods.

The immunogen can be produced analogously to the method described in EP-A 0 329 994 (which is a subject matter of the disclosure in the present patent application). In this process the hapten is bound to a carrier protein such as e.g. KLH (keyhole limpet hemocyanin), B-galactosidase or edALtin. KLH is preferably used as the carrier protein. It is expedient to couple the hapten and carrier protein by means of coupling groups such as e.g. lysine, cysteine and the maleimidohexyl group.

9 It is particularly preferable to use an immunogen which has a high coating density (number of bound hapten groups corresponds to 5 25 of the weight of the carrier protein). An antiserum of high serum titre is obtained by this means. A high serum titre is understood to mean that the polyclonal antiserum obtained can be used in high dilutions preferably 1:10 and more) for the determination of the glycated haemoglobins.

The animals which are usually used to obtain antibodies are then immunized with this immunogen according to methods known to a person skilled in the art. It is preferable to use rabbits or sheep or, in the case of the production of monoclonal antibodies, mice. The polyclonal antibodies can either be used directly or preferably after chromatographic purification on DEAE or after immunosorbtive purification. Monoclonal antibodies are obtained in the usual manner by immortalizing the spleen cells of the immunized animals, cloning those immortalized cells which produce the desired antibody and isolating the antibody according to known methods.

SIt is also preferred that a mixture of at least two immunogens be used for the immunization which contains fructose-Val-His, fructose-Val-His-Leu, fructose-Val- His-Leu-Thr and fructose-Val-His-Leu-Thr-Pro as the hapten component.

Those immortalized cells which produce the desired antibody are identified in the usual manner by means of an ELISA test to detect binding to HbAlc, HbS1c and if desired, HbClc.

10 The invention in addition concerns a process for the production of antibodies which recognize HbAlc as well as HbSlc and HbClc by immunization with an immunogen which contains the glycated oligopeptide fructose-Val- His, fructose-Val-His-Leu or fructose-Val-His-Leu-Thr- Pro as the hapten or a mixture of at least two such immunogens and isolation of the antibody from the serum of the immunized animals according to known methods.

A preferred subject matter of the invention is a process for the production of monoclonal antibodies which recognize HbAlc, HbSlc and HbClc, by immunization with an immunogen which contains the glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu or fructose-Val- His-Leu-Thr-Pro as the hapten or a mixture of at least two such immunogens, immortalization of the spleen cells of the immunized animals, cloning those immortalized cells which produce the desired antibody and isolation of the antibody from the cloned cells according to known methods.

A particularly preferred subject matter of the invention is such a process according to the invention for the production of polyclonal or monoclonal artibodies which recognize HbAlc, HbS1c and HbC1c in which an immunogen is used in which the hapten component is bound to KLH as "a carrier protein.

The invention in addition concerns the use of an antibody according to the invention for the immunological determination of the content of glycated haemoglobin in a method for the simultaneous immunological determination of the content of HbAlc as 11 well as glycated haemoglobin variants such as e.g. HbSlc and HbC1c in a blood sample.

The antibodies according to the invention additionally recognize further glycation variants of Hb such as e.g.

HbA21c and HbElc.

The invention in addition concerns a reagent for the immunological determination of the content of N-terminally glycated haemoglobin which contains at least one antibody according to the invention as well as a method for the simultaneous immunological determination of HbAlc, HbSIc and HbClc using the antibodies according to the invention.

The invention is elucidated in more detail by the following examples.

6 -L L-ULl LI 12 Example 1: Production of immunogens for obtaining antibodies against glycated haemoglobins of the HbAlg type 1.1 Use of B-galactosidase as carrier -otein The peptide fructose-Val-His-Leu-Thr-Lys-OH is synthesized according to the description in EP-A o 329 994 by means of solid phase synthesis on a semiautomated peptide synthesizer from the Labortec Company (Bubendorf, Switzerland) and bound to B-galactosidase.

For this 10 mg fructose-Val-His-Leu-Thr-Lys-OH is taken up in 1 ml 0.1 mol/l potassium phosphate buffer pH 7 and added to 6.2 mg maleimidohexanoic acid-Nhydroxysuccinimide ester in 2 ml ethanol. The reaction solution is stirred for 14 hours at room temperature and subsequently purified on Polycosil C18 and the fractions which are pure according to HPLC are lyophilized.

*0 In order to produce the immunogen, 48 mg B-galactosidase (8-Gal) (EIA quality, Boehringer Mannheim GmbH, Catalogue No. 570 079) is dissolved under an argon atmosphere in 2 ml 0.1 mol/l potassium phosphate buffer pH 7.0 gassed with argon and 5 mg of the lyophilized peptide fructose-Val-His-Leu-Thr-Lys(MH)-OH is added in the absence of oxygen and stirred for 1 hour at room temperature. Subsequently the entire reaction solution is applied to an AcA202 column (2 x 24 cm, Pharmacia, Sweden) which was equilibrated with argon-saturated 0.09 sodium chloride solution. Subsequently it is eluted with the same equilibration buffer and the protein fractions which contain the desired immunogen are 13 collected. The coating of the B-galactosidase with the hapten groups can be determined by reacting a sample with Ellman's reagent.

The immunogens fructose-Val-His-Lys-(MH)-BGal, fructose- Val-His-Leu-Lys-(MH)-BGal and fructose-Val-His-Leu-Thr- Pro-Lys-(MH)-BGal are produced in an analogous manner.

1.2 Use of KLH as carrier protein The hapten fructose-Val-His-Leu-Thr-Cys-OH is synthesized according to the description in EP-A 0 329 994 by means of a solid phase synthesis on a semiautomated peptide synthesizer from the Labortec Company (Bubendorf, Switzerland).

The carrier protein keyhole limpet haemocyanin (KLH) is reacted with maleimidohexanoyl-N-hydroxy3uccinimide (MHS). For this 2 g KLH is dissolved in 0.1 mol/l sodium bicarbonate pH 8.35. The insoluble protein fraction is separated by centrifugation. Subsequently the pH value of the solution is adjusted to pH 8.30 with 0.1 mol/l NaOH. 370 mg maleimidohexanoyl-N-hydroxysuccinimide dissolved in 3 ml DMSO is added to this protein solution. After a 15 minute reaction time at room temperature, the pH value is adjusted to pH 7.0 with 0.1 mol/l HC1 and the product is purified by means of gel permeation chromatography on Ultrogel AcA 202 (2x24 cm, Pharmacia, Sweden). The number of maleimidohexanoyl groups in the product obtained is determined according to EP-A 0 329 994 (Example 4) using Ellman's reagent. As a rule a loading of 600 900 maleimidohexanoyl groups per mol KLH is achieved.

-I L 14 The KLH derivative obtained in this way is purged for ca. 15 min. with argon. Subsequently 2 mol of the peptide hapten is added per mol maleimidohexanoyl group and incubated for 3 hours at room temperature. The purification is then carried out by separating the nonreacted peptide by gel permeation chromatography on Ultrogel AcA 202 (2 x 24 cm, Pharmacia, Sweden).

15 Example 2: Production of polyclonal antisera against HbA1l 2.1 Immunization sheep are immunized with the immunogen produced according to example 1.1 in Freund's adjuvant. The dosea in each case is 500 Ag per animal for the first and each successive immunization. 5 additional sheep were immunized in the same manner but in each case with 1 mg immunogen per animal and immunization.

sheep are immunized in the same manner with an analogous immunogen but in which the hapten was bound to KLH (produced according to example 1.2) and also with 500 gg or 1 mg per animal and immunization.

goes After 5 months (KLH immunogen) or 6 months (B-Gal immunogen), serum samples are taken from all animals and the serum titre of the antisera obtained is determined by means of a turbidimetric test.

Antiserum test Reagents used: Haemolysis reagent: 20 mM MES pH 1

SDS

0.02 potassium hexacyanoferrate (III) 0.1 NaN 3 Brij 16 Reaction buffer: mM MES pH 150 mM NaCI Brij 0.1 NaN 3 3 PEG 6000 Polyhapten solution: mM MES pH 150 mM NaCl Brij 0.1 NaN 3 6 PEG 6000 0.1 BSA polyhapten 30 gg/ml Preparation of the antisera for the measurement The sera are diluted 1+1 with twice concentrated reaction buffer, incubated overnight at 4 0 C and the precipitate which forms is removed by centrifugation.

The supernatant is incubated for 1 hour in an ice bath, filtered through a 0.22 pm filter and the pH value is o adjusted to 6.0. The antibody solution formed in this way can be used directly in the test or diluted f:rther with reaction buffer.

Measurement In order to assess the antiserum titre, antibody solution and polyhapten solution are mixed and the turbidity produced is measured photometrically. The measurement is carried out at 370C on a Hitachi 704 automated analyser. The procedure is as follows: 17 8 /l haemolysis reagent and 350 pl antibody solution are mixed and incubated for 5 min. Subsequently 70 pl polyhapten solution is added and it is incubated for a further 5 min. The turbidity which forms in this period is measured at 340 nm (using the reference wavelength 700 nm) as an absorbance.

2.3 Results The results are summarized in Tables 1 (B-Gal immunogen) and 2 (KLH immunogen). Many animals show measured signals of 500 mA and more at low antisera dilutions (1:2 At an antiserum dilution of 1:8 this only applies to one animal from the B-Gal immunization whereas this is the case for 8 animals immunized with KLH. Most of these sera still generate very high measured signals even at this dilution and can apparently still be used at high dilutions for the measurement of glycated haemoglobins. The dose of the immunogen used for the immunization does not have any recognizable influence on the serum titre.

*e oe* *eoe* *ge 18 Table 1 Maximum measured signal in a competitive immunoassay when using a polyclonal antiserum which was obtained by immunization with a peptide hapten bound to B-galactosidase.

Animal Dose of Measured Measured Measured the s3gnal mA signal mA signal mA immunogen at at at (mg) antiserum antiserum antiserum dilution dilution dilution 1:2 1:4 1:8 1 1.0 435 12 0 2 1.0 1261 309 0 3 1.0 77 2 0 4 1.0 205 2 0 1.0 887 305 4 6 0.5 1000 417 67 7 0.5 1289 992 650 8 0.5 1100 239 0 9 0.5 848 253 0 0.5 228 3 2 see..: g* sob* 19 Table 2 Maximum measured signal in a competitive immunoassay when using a polyclonal antiserum which was obtained by immunization with a peptide hapten bound to KLH.

Animal Dose of Measured Measured Measured the signal mA signal mA signal mA immunogen at at at (mg) antiserum antiserum antiserum dilution dilution dilution 1:2 1:4 1:8

S

9..4 .00.

000*0 .06.

0 60460: 0 1.0 1.0 1.0 0.5 0.5 1580 1094 1044 1306 1620 1194 1435 1467 1158 1486 1591 532 844 1548 1707 973 1531 1598 1301 1575 1575 99 356 1619 1696 572 1394 1444 630 1608 Lr- _I 20 Example 3: Isolation of HbAo and HbS 0 and in vitro glycation of HbS 0 to form Hb81, Commercially available HbS (Sigma, Catalogue. No. H0392) or human erythrocyte haemolysate are separated chromatographically and the HbSO purified in this way is reacted in vitro with glucose to form HbS1c.

3.1 Isolation of HbS 0 A HbS preparation from the Sigma Company was dissolved in 50 mmol/l MES pH 6.2 and subsequently dialysed "*against the same buffer for 12 hours. The dialysate was applied to a S-Sepharose HP chromatography column from the Pharmacia Company equilibrated with the above buffer. The elution was carried out by a LiCl gradient.

The HbSO fraction was isolated. It contained no glycated haemoglobins as could be demonstrated by means of HPLC S analysis using a MonoSHBAlc column from the Pharmacia Company and their instructions for the determination of HbAlc.

3.2 Production of glycated HbS Part of the HbSo fraction was dialysed against a 100 mmol/l phosphate buffer pH 6.5 and subsequently reacted with a 2600-fold molar excess of glucose. After hours at 37 0 C the reaction was stopped and the incubation mixture was dialysed extensively to remove glucose. The dialysate was also analysed by means of

~I

21 HPLC. The chromatogram showed a portion of 11.1 glycated HbS.

3.3 Isolation of HbAO Haemoglobins present in human erythrocytes were released by lysis of PBS-washed human erythrocytes in distilled water. Cell debris and erythrocyte ghosts were removed by centrifugation. The haemolysate obtained in this way was dialysed against 50 mM MES pH 6.2. The dialysate was subsequently applied to a S-Sepharose HP chromatography column from the Pharmacia Company equilibrated with the above buffer. The elution was carried out by a LiCI gradient. The HbA 0 fraction was isolated. It contained no glycated haemoglobins as could be demonstrated by means of HPLC analysis using a MonoS/HbAlc column from .".the Pharmacia Company and their instructions for the determination of HbAlc.

go *oo *ooo•> *oo *oo

I

22 Example 4: Simultaneous determination of HbAlg and HbSB1 Whole blood samples were firstly haemolyzed with a special haemolysis reagent and subsequently measured on a photometer (BM/Hitachi 717 of the Boehringer Mannheim GmbH) in a two channel procedure. The immunological determination of HbAlc in g/dl according to the TINIA test principle was carried out in one channel by a turbidimetric measurement at 340 nm while the total haemoglobin content was determined in the other channel by photometric measurement at 570 nm. The content of HbAlc in percent is calculated according to the following formula: HbA1 [g/dl] HbA x 100 total haemoglobin [g/dl] SThe following reagents were used: S1) Haemolysis reagent: 20 mM sodium phosphate pH 7.4 0.9 TTAB (tetradecyltrimethylammonium bromide) 2) Antibody solution (RI HbAlc) mmol MES buffer pH 6.2 (2-(N-morpholino)-ethanesulfonic acid) 150 mmol NaC1 PEG 6000 (polyethylene glycol MW ca. 6000) Brij® t- 23 mg/ml PAb <HbAlc>S-IgG(DE) (polyclonal sheep antibody against HbAlc), produced using the immunogen fructose-Val- His-Leu-Thr-MH-BGal 3) Polyhapten solution (R2 HbAlc): mmol MES buffer pH 6.2 150 mmol NaCl PEG 6000 Brij® pg/ml polyhapten 1 1) Polyhapten: fructose-Val-His-Leu-Thr, coupled via Cys-maleimidohexanoic acid to dextran as described in the German patent application P 41 40 142.5.

4) Buffer solution for the total haemoglobin determination (R1 Hb): mM sodium phosphate pH 7.4 S150 mmol/1 NaCl e *9 5) Calibrators a e: 20 mmol sodium phosphate pH 7.4 0.9 TTAB 1.4 mg/ml sheep haemoglobin 0, 0.05, 0.1, 0.16, 0.3 human HbAlc 4.1 Determination of HbAlc The haemolysis reagent was added to the sample in a ratio of 1+100 and incubated for ca. 5 minutes at 25 0

C.

1~ 24 250 Al antibody solution (Ri HbAlc) was added by pipette to 10 Al haemolyzed sample. After 5 minutes incubation at 37 0 C, the sample blank value was measured bichromatically at 700/340 nm Ca. 20 seconds after the measurement, 50 Al polyhapten solution was added, stirred immediately and it was incubated for a further minutes at 370C. Afterwards the turbidity was measured bichromatically at 700/340 nm (A2).

The sample-specific difference in absorbance is calculated according to the formula AA A 2 K-Al in which K represents the volume correction factor.

K Vsamp I

VRI

*aaple a

K

V total A calibration curve was established using the calibrators a e which contained increasing concentrations of HbAlc and the unknown HbAlc concentration of the sample in g/dl can be read off via the sample-specific difference in absorbance AA.

4.2 Determination of the total haemoglobin concentration The haemolysate obtained in 4.1 is used for the determination of the total haemoglobin concentration.

Haemoglobin is oxidized by the reagents present in the haemolysis reagent and a characteristic haemoglobin chromophore is obtained by complexation of the detergent

I

25 molecule. 20 pl of this haemolysate is pipetted together with 230 A1 buffer (R1 Hb) into the cuvette, stirred briefly and after 5 minutes incubation at 37 0 C, the absorbance is measured bichromatically at 660/570 nm. A calibration curve is established using the calibrator a and physiological NaCl solution (zero standard) and the concentration is read via the absorbance of the unknown sample.

The percentage HbAlc content is calculated by means of the formula HbA1 [g/dl] HbAle x 1 00 Hb [g/dl] o «o ooo 26 Example Determination of the specificity of the antibodies according to the invention A HbAlc determination is carried out on a Hitachi 717 according to example 4 in order to determine the specificity of the antibodies obtained according to example 2. For this, HbA 0 (purified from human blood according to example HbSO (Sigma H0392 purified according to example sample containing HbSlc (produced in vitro according to example 3) and two whole blood controls with known HbAlc values (BioRad, Lyphochek® Diabetes Control Level 1 and 2, Order No.

740) are measured as samples. The result is summarized in Table 3.

Table 3 Table 3 a a *9 t Sample HbAlc/HbSlc Hb HL" c/HbSlc [g/dl] [g/dl] HbA 0 0 20 0 HbSO 0 13.9 0 hbS 0 HbSlc 1.33 12.3 10.8 (HPLC: 11.1 Lyphochek level 1 0.75 14.3 5.2 (target value 5.6 Lyphochek level 2 0.98 9.1 10.8 (target value 10.4 I CL a C__ 27 It follows from this that the antibodies according to the invention recognize HbAlc as well as HbSlc but not the corresponding non-glycated haemoglobins HbA 0 and HbS 0 Example 6: Simultaneous determination of HbAlc and HbS1, in a heterozygous HbAS sample The heterozygous HbAS sample (30 HbS) was measured analogously to example 4 on a Hitachi 717. As a comparison, the HbAlc content was determined with a high resolution HPLC method according to Bissd Wieland J. Chromatogr. 434, 1988, 95 110 (elution profile see Figure 1) and tha glycohaemoglobin content was determined using the affinity chromatographic Glyc-Affin method of determination of the IsoLab Company (Order No.

SG 6200). The results are summarized in Table 4.

Table 4 HbAlc HbSIc HbAlc HbSlc Immunoassay -5.6 HPLC 3.3 Glyc-Affin -5.4 A HbAlc value of 3.3 is measured using the HPLC method according to Biss& and Wieland. The value for glycated C -I t'i I 28 HbS (HbSlc) cannot be given since the elution time of the HbSlc peak is not exactly known. It could be one of the peaks at 31 33 minutes elution time.

Due to the detection of HbS1c, the Tinaquant test measures approximately 2.3 higher than the HPLC method. 7.25 glycohaemoglobin is measured using the affinity chromatography method (detection of glycohaemoglobin HbAlc HbSIc HbAla HbAlb elysine-glyc. Hb). Since these other glycated haeroglobin species are also detected, the Glyc-Affin test r.-.aures higher than the described immunological test. 7n a method comparison with normal samples between Tinaquant and Glyc-Affin, a line of correlation of HbAlc (immunoassay) 0.66 x glycohaemoglobin (affinity chromatography) 0.6 0. is therefore obtained with a very good correlation (Figure If the Glyc-Affin value is corrected using the formula for the lines of correlation for the detection of HbAla, HbAlb etc. then this results in an Salmost identical measured value for HbAlc HbS1c using the immunoassay.

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Claims (5)

1. Use of antibodies which simultaneously recognise HbA1, HbSl and HbCl c and are obtainable by immunization with at least one immunogen which contains the glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu, fructose-Val-His-Leu-Thr or fructose-Val-His-Leu-Thr-Pro as a hapten component, for the simultaneous immunological determination of HbAlc, HbSl, and HbCl.

2. Use as claimed in claim 1, wherein the immunological determination is carried out according to an agglutination test principle or according to CEDIA, EMIT, FPIA, or ELISA technique.

3. Use as claimed in any one of claims 1 or 2, wherein the antibody is a polyclonal or monoclonal antibody. e

4. Use as claimed in any one of claims 1 to 3, wherein the content of HbAl,, HbSl, and HbCl, in blood samples is determined.

5. Use as claimed in claim 1, substantially as hereinbefore described with reference to the Examples. Dated this 18th day of 'pril, 1995 Boehringer Mannheim GmbH By its Patent Attorneys Davies Collison Cave 950418,q:\ opr\jms,50590-po.018.29 Abstract The invention relates to a method for the immunological determination of the content of glycosylated haemoglobin in a blood sample in which an antibody is used which recognizes HbAlc, HhSic and HbClc, to the antibody used in this process as well as to a process for the production of such an antibody. e o oo*o* *oo o o o