NZ250198A - Antibodies and their use in determining glycated haemoglobin content in blood - 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

New Zealand Paient Spedficaiion for Paient Number £50198 2 5 0 Tff5 i Priority D3tc(c): . .0..<T?7 Complete Specification Filed: J.$\7/V93. Class: aq>n£-! s W .69.1*^13 2. 2 2 DEC 1994* Publication Date: .. P.O. Journal, Mo: . jsn N.Z. No.

NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION SIMULTANEOUS DETERMINATION OF HBAIC AND HAEMOGLOBIN VARIANTS WITH A GLYCATION ANALOGOUS TO HBAIC We, BOEHRINGER MANNHEIM GMBH, a Company of the Federal Republic of SaAclhofV SW". Ilfe^ D-4>%a.9£ Germany of^68298 Mannheim, Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- -1 - (Followed by 1A) - 1A - 0 ig $ aommsmmmmsmmmtimtmm mm? Simultaneous determination of HbAlc and haemoglobin variants with a glycation analogous to HbAlc 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, HbSlc and HbClc as well as a process for the production of such antibodies.

Haemoglobin which transports oxygen and CO2 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().

Variants of the a chain as well as of the 6 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 the B chain. In 2 5 0 •' o this case glutamic acid is substituted by lysine. Apart from these, numerous further haemoglobin variants are known with a defined amino acid substitution (e.g. 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 HbSlc or HbClc. 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 6 chain of haemoglobin. The various haemoglobin variants are glycated in this process to the same extent as normal HbAg (J. Sosenko et al., Diabetes Care 3 (1980), 590 - 593) .

The concentration of glycated 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 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 determination 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 (J. 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). Although, in addition to HbAlc glycated haemoglobin variants such as HbSlc or HbClc, 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 (e.g. on lysine residues or on the a chain) are measured equally (D. Goldstein et al., CRC Critical 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 prior-art methods can therefore not be used for the diagnosis n Vy 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, HbSlc and HbClc and are suitable for use in immunological methods 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, HbSlc and HbClc.

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 6 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, HbSlc 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 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 HbClc 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. 0 •' r - 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. 6-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, antigen-enzyme donor 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 antigen-enzyme donor complex and thus enables formation of the active enzyme.

In the enzyme multiplied immunoassay technique (EMIT), the hapten to be detected is coupled covalently to the marker enzyme in such a way that the enzymatic activity is retained. However, after an antibody binds to the hapten component, the substrate binding to the enzyme is 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 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 complexes are formed and the less fluorescence polarisation can be measured (W. Dandliker et al., Journal of Exp. Med. 122 (1965), 1029).

In immunoassays based on the ELISA principle, binding of an enzyme-label led 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, HbSlc and HbClc and are obtainable by immunization with an u ■] 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 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). I this process the hapten is bound to a carrier protein such as e.g. KLH (keyhole limpet hemocyanin), fi-galactosidase or edestin. 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 K n fc- %if u: 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 (1:8, 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.

It 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, HbSlc and if desired, HbClc.

A 1 c a 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 antibodies which recognize HbAlc, HbSlc and HbClc 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 9 I 0 v n well as glycated haemoglobin variants such as e.g. HbSlc and HbClc in a blood sample.

The antibodies according to the invention additionally recognize further glycation variants of Hb such as e.g. HbA2ic 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, HbSlc and HbClc using the antibodies according to the invention.

The invention is elucidated in more detail by the following examples. 0 1 n Example 1: Production of immunogens for obtaining antibodies against glycated haemoglobins of the HbAlc type 1.1 Use of fi-galactosidase as carrier protein The peptide fructose-Val-His-Leu-Thr-Lys-OH is synthesized according to the description in EP-A 0 329 994 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/1 potassium phosphate buffer pH 7 and added to 6.2 mg maleimidohexanoic acid-N-hydroxysuccinimide 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.

In order to produce the immunogen, 48 mg B-galactosidase (£-Gal) (EIA quality, Boehringer Mannheim GmbH, Catalogue No. 570 079) is dissolved under an argon atmosphere in 2 ml 0.1 mol/1 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 0 ': Of *'ij 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-hydroxysuccinimide (MHS). For this 2 g KLH is dissolved in 0.1 mol/1 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/1 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/1 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.

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 non-reacted peptide by gel permeation chromatography on Ultrogel AcA 202 (2 x 24 cm, Pharmacia, Sweden). 0 ^9 Example 2: Production of polyclonal antisera against HbAlc 2.1 Immunization sheep are immunized with the immunogen produced according to example 1.1 in Freund's adjuvant. The dose in each case is 500 fig 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 ng or 1 mg per animal and immunization.

After 5 months (KLH immunogen) or 6 months (6-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: mM MES pH 6.0 1 % SDS 0.02 % potassium hexacyanoferrate (III) 0.1 % NaN3 0.5 % Brij 35 Reaction buffer: 20 mM MES pH 6.0 150 mM NaCl 0.5 % Brij 35 0.1% NaN3 3 % PEG 6000 Polyhapten solution: 20 mM MES pH 6.0 150 mM NaCl 0.5 % Brij 35 0.1 % NaN3 6 % PEG 6000 0.1 % BSA polyhapten 30 /ig/ml Preparation of the antisera for the measurement The sera are diluted 1+1 with twice concentrated reaction buffer, incubated overnight at 4°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 /xm filter and the pH value is adjusted to 6.0. The antibody solution formed in this way can be used directly in the test or diluted further 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 37°C on a Hitachi 704 automated analyser. The procedure is as follows: 8 nl haemolysis reagent and 350 Ml antibody solution are mixed and incubated for 5 min. Subsequently 70 (il 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 l (fl-Gal immunogen) and 2 (KLH immunogen). Many animals show measured signals of 500 mA and more at low antisera dilutions (1:2 / 1:4). 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.

Table l 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 signal 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 o • H 887 305 4 6 0.5 1000 417 67 7 0.5 1289 992 650 8 0.5 1100 239 0 9 in • o 848 253 0 in • o 228 3 2 0 1 9 Q 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 11 1.0 1580 1591 1575 12 1.0 1094 532 99 13 1.0 1044 844 356 14 1.0 1306 1548 1619 1.0 1620 1707 1696 16 0.5 1194 973 572 17 0.5 1435 1531 1394 18 0.5 1467 1598 1444 19 0.5 1158 1301 630 0.5 1486 1575 1608 2 j 0 '] § 0 Example 3: Isolation of HbA<j and HbSo and in vitro glycation of HbS0 to form HbSlc 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 HbSlc. 3.1 Isolation of HbSg A HbS preparation from the Sigma Company was dissolved in 50 mmol/1 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 analysis using a MonoSjjBAic column from the Pharmacia Company and their instructions for the determination of HbAlc. 3.2 Production of glycated HbS Part of the HbSg fraction was dialysed against a 100 mmol/1 phosphate buffer pH 6.5 and subsequently reacted with a 2600-fold molar excess of glucose. After 20 hours at 37°C the reaction was stopped and the incubation mixture was dialysed extensively to remove glucose. The dialysate was also analysed by means of ■I MA v' i -1 *" O' HPLC. The chromatogram showed a portion of 11.1 % glycated HbS. 3.3 Isolation of HbAg Haemoglobins present in hiaman 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 LiCl gradient. The HbAg 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. 0 Example 4: Simultaneous determination of HbAlc and HbSlc 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: HbAlc [g/dll % HhAlc = —=L = x 100 total haemoglobin [g/dlj The following reagents were used: 1) Haemolysis reagent: mM sodium phosphate pH 7.4 0.9 % TTAB (tetradecyltrimethylammonium bromide) 2) Antibody solution (R1 - HbAlc): mmol MES buffer pH 6.2 (2-(N-morpholino)-ethane- sulfonic acid) 150 mmol NaCl 3.0 % PEG 6000 (polyethylene glycol MW ca. 6000) 0.5 % Brij® 35 OH 1.0 mg/ml PAb <HbAlc>S-IgG(DE) (polyclonal sheep antibody against HbAlc), produced using the immunogen fructose-Val-His -Leu-Thr-MH-6Ga 1 3) Polyhapten solution (R2 - HbAlc): mmol MES buffer pH 6.2 150 mmol NaCl 6.0 % PEG 6000 0.5 % Brij® 35 jug/ml polyhapten1) !) 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 7.4 150 mmol/1 NaCl ) Calibrators a - e: 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°C.

Oi g 250 jul antibody solution (R1 - HbAlc) was added by pipette to 10 (il haemolyzed sample. After 5 minutes incubation at 37°C, the sample blank value was measured bichromatically at 700/340 nm (Al). Ca. 20 seconds after the measurement, 50 /il polyhapten solution was added, stirred immediately and it was incubated for a further 5 minutes at 37°C. Afterwards the turbidity was measured bichromatically at 700/340 nm (A2).

The sample-specific difference in absorbance is calculated according to the formula aa = a2 - k ax in which K represents the volume correction factor. ^sample + K — ^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 Oi 0 8 molecule. 20 fil of this haemolysate is pipetted together with 230 /il buffer (R1 - Hb) into the cuvette, stirred briefly and after 5 minutes incubation at 37°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 % HbAlc P ^ o ^ j / Example 5: 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, HbAg (purified from human blood according to example 3), HbSg (Sigma H0392 purified according to example 3), sample containing HbSlc (produced in vitro according to example 3) and two whole blood controls with known HbAlc values (BioRad, Lyphochek® Diabetes Control Level l and 2, Order No. 740) are measured as samples. The result is summarized in Table 3.

Table 3 Sample HbAlc/HbSlc [g/di] Hb [g/di] HbAlc/HbSlc [%] HbA0 0 0 HbS0 0 13.9 0 HbS0 + HbSlc (HPLC: 11.1 %) 1.33 12.3 .8 Lyphochek level 1 (target value 5.6 %) 0.75 14.3 .2 Lyphochek level 2 (target value 10.4 %) 0.98 9.1 .8 p I? ^ , It follows from this that the antibodies according to the invention recognize HbAlc as well as HbSlc but not the corresponding non-glycated haemoglobins HbAo and HbSg.

Example 6: Simultaneous determination of HbAlc and HbSlc 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 Bisse E., Wieland H., J. Chromatogr. 434, 1988, 95 - 110 (elution profile see Figure 1) and the 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 HbSlc HbAlc + HbSlc Immunoassay - - .6 % HPLC 3.3 % - - Glyc-Affin - - .4 % A HbAlc value of 3.3 % is measured using the HPLC method according to Biss£ and Wieland. The value for glycated

Claims (14)

1. 25 0 * c);- 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 HbSlc, 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 + HbSlc + HbAla + HbAlb + e-lysine-glyc. Hb). Since these other glycated haemoglobin species are also detected, the Glyc-Affin test measures higher than the described immunological test. In 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;is therefore obtained with a very good correlation (Figure 2). 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 almost identical measured value for HbAlc + HbSlc using the immunoassay.;- 29 -;WHAT WE CLAIM IS:;1. 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, HbSlc and HbClc.-;
2. 2. Use as claimed in claim 1, wherein the immunological determination is carried out ^ >;according to an agglutination test principle or according to the CEDIA, EMIT, FPIA, or ELISA technique.;
3. 3. Antibody which recognizes HbAlc, HbSlc and HbClc and is obtainable by immunizing a mammal with an immunogen which contains the glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu and/or fructose-Val-His-Leu-Thr-Pro as the hapten component and isolating the antibody from the serum of the immunized animals.;
4. 4. Antibody which recognizes HbAlc, HbSlc and HbClc and is obtainable by immunizing a mammal with a mixture of an immunogen that contains fructose-Val His-Leu-Thr as the hapten and with at least one further immunogen that contains fructose-Val-His, fructose-Val-His-Leu and/or fructose-Val-His-Leu-Thr-Pro as the hapten component and isolating the antibody from the serum of the immunized animals;S"1 ri;J e-., f) /!;£ vj j f*j - 30 -
5. 5. Antibody as claimed in claim 3 or 4, wherein it is a monoclonal antibody and is obtainable by immunizing with one of the immunogens mentioned in claim 3 or 4, 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.
6. 6. Process for the production of antibodies which recognize HbAlc, HbSlc and HbClc by immunizing a mammal with an immunogen that contains the glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu or fructose-Val-His-Leu-Thr-Pro as the hapten component or with a mixture of two such immunogens and isolating the antibody from the serum of the immunized animals.
7. 7. Process for the production of monoclonal antibodies which recognize HbAlc, HbSlc and HbClc by immunizing a mammal with an immunogen that contains the glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu or fructose-Val-His-Leu-Thr-Pro as the hapten component or with a mixture of two such immunogens, 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.
8. 8. Process as claimed in one of the claims 6 or 7, wherein an immunogen is used in which the hapten component is bound to KLH as a carrier protein. 25 0 t 98 31
9. 9. Use of an antibody as claimed in claims 3 to 5 for the immunological determination of the content of N-terminally glycated haemoglobin in a method for the simultaneous determination of the content of HbAlc, HbSlc and HbClc in blood samples.
10. 10. Reagent for the immunological determination of N-terminally glycated haemoglobin containing at least one antibody as claimed in one of the claims 3 to 5.
11. 11. Method for the immunological simultaneous determination of HbAlc, HbSlc and HbClc using antibodies as claimed in claims 3 to 5.
12. 12. A use according to claim 1 substantially as herein described or exemplified.
13. 13. An antibody according to claim 3 or 4, substantially as herein described or exemplified.
14. 14. A process according to claim 6 or 7, substantially as herein described or exemplified. BOEHRINGER MANNHEIM GMBH By their Attorneys HENRY HUGHES LTD Per: (\lA/r\ ,