CA2103318A1 - Simultaneous determination of hba1 and haemoglobin variants with a glycation analogous to hba1 - Google Patents
Simultaneous determination of hba1 and haemoglobin variants with a glycation analogous to hba1Info
- Publication number
- CA2103318A1 CA2103318A1 CA002103318A CA2103318A CA2103318A1 CA 2103318 A1 CA2103318 A1 CA 2103318A1 CA 002103318 A CA002103318 A CA 002103318A CA 2103318 A CA2103318 A CA 2103318A CA 2103318 A1 CA2103318 A1 CA 2103318A1
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- Prior art keywords
- antibody
- val
- fructose
- leu
- hbalc
- Prior art date
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
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- Urology & Nephrology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
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, HbSlc and HbClc, to the antibody used in this process as well as to a process for the production of such an antibody.
Description
2 ~ ~ ~ 3 ~
,~
,,.
imultaneGus aet~rmin~tion of HbAl~ an~ haemoglobi~
~ari~nt~ ~ith a glyc tion ~alogou~ to HbA1c ,,, 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 HbA1c, HbS1c and HbC1c as well as a process ~- for th~ production of such antibodies. -~,t ~
Haemoglobin which transports oxygen and C02 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 HbAo. ~ -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, anoth'er pathological haemoglobin, also differs from HbA by an amino acid substitution at position 6 of the B chain. In ~ , ;,'".i''', `
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- 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 hy a non-enzymatic reaction -~
wi~h 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 -~
fre2 amino acid of the haemoglobin. The aldimine formed -in this process rearranges by means of an Amadori rearrangement to form a N~ deoxy-D-fructose-1-yl) residue. The glycated haemoglobin is sta~le in this rearranged form.
The glycated haemoglobin which u~;ually forms is denoted HbA1c. The aforementioned glycated haemoglobin variants are denoted HbS1c 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 B chain of haemoglobin. The various haemoglobin -~
variants are glycated in this process to the same extent as normal HbAo (J- Sosenko et al., Diabetes Care (1980), 590 ~ 593).
!
The concentration of glycated haemoglobins in blood depends on the blood glucose concentr~tion. The proportion of glycated haemoglobins in rPlation to total haemoglobin is noxmally in the range of 3 - 6 % in adults. When the blood sugar level is increased this proportion increases up to 15 %. The determination of ~ ;
: .
: :.
,~
,,.
imultaneGus aet~rmin~tion of HbAl~ an~ haemoglobi~
~ari~nt~ ~ith a glyc tion ~alogou~ to HbA1c ,,, 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 HbA1c, HbS1c and HbC1c as well as a process ~- for th~ production of such antibodies. -~,t ~
Haemoglobin which transports oxygen and C02 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 HbAo. ~ -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, anoth'er pathological haemoglobin, also differs from HbA by an amino acid substitution at position 6 of the B chain. In ~ , ;,'".i''', `
~ 5~
~ ~33~ ~
- 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 hy a non-enzymatic reaction -~
wi~h 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 -~
fre2 amino acid of the haemoglobin. The aldimine formed -in this process rearranges by means of an Amadori rearrangement to form a N~ deoxy-D-fructose-1-yl) residue. The glycated haemoglobin is sta~le in this rearranged form.
The glycated haemoglobin which u~;ually forms is denoted HbA1c. The aforementioned glycated haemoglobin variants are denoted HbS1c 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 B chain of haemoglobin. The various haemoglobin -~
variants are glycated in this process to the same extent as normal HbAo (J- Sosenko et al., Diabetes Care (1980), 590 ~ 593).
!
The concentration of glycated haemoglobins in blood depends on the blood glucose concentr~tion. The proportion of glycated haemoglobins in rPlation to total haemoglobin is noxmally in the range of 3 - 6 % in adults. When the blood sugar level is increased this proportion increases up to 15 %. The determination of ~ ;
: .
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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 Gf 120 days, the determination of glycated haemoglobin in blood provides a parameter for the blood glucose level which is independent of a short-term incrsase e.g. after a carbohydrate-rich meal.
~'' The determination of glycated haemoylobin in blood is ~l thus of great importance for the diagnosis and monitoring of diabetes mellitus. A number of , chromatographic and electrophoretic methods for the ~i detection of HbA1c have therefore been developed.
However, the glycated haemoglobin variants cannot be determined simultaneously with HbA1c using these methods (J. Sosenko et al., Diabetes Care 3 (1980), 590 - 593, D. Goldstein et al., C~C Critical Reviews in Clinical Laboratory Sciences 21 (1984), 187 - 225 and Allen et al., Annual Clinical Biochemistry 29 (1992), 426 429).
~l Although, in addition to Hb.~lc glycated haemoglobin l variants such as HbS1c or HbC1c, are also determined simultaneously using affinity chromatographic methods, this method is not speci~ic for glycation at the N-terminus of the B chain since all glycations of the ~I haemoglobin molecule te.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. Thase prior-art methods can therefore not be used for the diagnosis 'i''"~.
~l ,., ~` `:
-~ 4 ~ 2~ 3~'l3 :~:
:~ and monitoring of diabetes mellitus in blood samples of ~ :
patients with haemoglobinopathies.
It has now surprisingly turned out that by using il. antibodies which can be obtained by immunization with an :~:
il immunogen containing the glycated oligopeptide fructose-~1 Val-His-Leu-Thr-Pro or a part thereof as the hapten ;1 component, antibodies are obtained which recognize ~:~
;- HbAlC, HbSlC and HbC1C and are suitable for use in immunological methods of determination for HbA1c, HbSlC
j and HbC1c. ~-~
J;," Accordingly the invention concerns the use of antibodies ;' which recognize HbA1c, HbS1c and HbC1c 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 -~-:
f, component for the immunological, simultaneous ~:
~;.1 determination of HbA1c, HbS1c and HbC1c.
~ i 1 The fact that antibodies produced in this way recognize HbA1c, HbS1c and HbC1c is also par~icularly 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 ahtibodies which are obtained with the aforementioned immunogen would dif~erentiate between HbA1c, HbS1c and HbC1c.
The simultaneous immunological determination of N-terminally glycated haemoglobin HbA1c, ~bS1c and HbCl~
can be achieved with these antibodies by means of all , .~....
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,~.
current immunoassays such as e.g. ELISA, ~luorescence immunoassay, radioimmunoassay, fluorescence-polarisation immunoassay (FPIA), cloned enzyme donor immunoassay `-~ (CEDIA) or enzyme multiplied immunoassay techni~ue (EMIT~. The test can be carried out in this process as a hcmogeneous tes~, 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 -q or latex particle-enhanced immunoassay (LPIA). These are ;~;
~i~ 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 speci~ic antibody. The hapten used for the '~ immunization (preferably fructose-Val-His-~eu-Thr-Pro) ~`~
,( is preferably usPd as the hapten which is present bound ~`
`.,,`1 to a high molecular carrier. In the absence of antigen ti from the sample this immunogen i~3 cross-linked by the antibodies used in the test to form large ag~regates 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 ~j of glycated haemoglobin, it is possible to determine of the amount of glycated haemoglobin (HbA1c, HbS1c and ~bC1c) in the sample solution. For this HbA1c, HbSlc or HbC1c can be used alone or as mixtures for the standard.
~`1 In addition it is preferred that the method according to ~; the invention is carried out according to the CEDIA, EMIT, FPI~ or ELISA technique.
' - 6 - 21~331~
~.
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 ~nzymatically 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. ~lowever, 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 ~rom 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.-~l Rubenstein, Biochemical and Biophysical Research ,.- J
~ 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 wavelenqth in a period of about 1o-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-~luorophore 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., 30urnal 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. -j :, ,`' The invention in addition concerns monoclonal and ;
polyclonal antibodies which recognize HbA1c, HbS1c and HbC1c and are obtainable by ir~unization with an `" ," ''`'~
,~
s~
1: 2 ~ O ~ 3 ~
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 j~ antibodies ~rom 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 :
v~ ~ mixture of an immunogen which contains fructose-Val-His-Leu-Thr as the hapten and with at least one further ~
~,5',~1 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 o~ the invention are monoclonal antibodies which recognize HbA1c, HbSlc and HbClc and are obtainable by immunization with the said :~:
.1 immunogen, immortalization of the spleen cells of the :~
immunized animals, cloning those immortalized cells ~: which produce the desired anti~ody and isolation of the antibody from the cells producing the antibodies according to known methods.
~he immunogen can be produced analogously to the method described in EP-A O 329 994 (which .is a subject matter of the disclosure in the present patent application). In -~
this process the hapten is bound t~ a carrier protein such as e.g. KLH (keyhole l mpet hemocyanin), B-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- ~a~3~
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 us~d 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-~eu-Thr-Pro as the hapten component.
Those immortalized cells which produce the desired antibo~y are identi~ied in the usual manner by means of an ELISA test to detect bindin~ to HbAlc, HbS1c and if desired, HbC1c.
`~
~, :
,~ , .
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- lo- 2 :' The invention in addition concerns a process for the production of antibodies which recognize HbA1c as well a~ HbSlC and ~bClC 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, HbS1C and HbC1C, by immunization with an immunogen which contains the glycated oliyopeptide 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, HbS1c and HbC1c in which an immunogen is used in which the hapten component is bound to KLH as a carrier proteinO
The invention in addition concerns the use of an antibody according to the invention for the .
i~munological det~rmination of the content of glycated haemoglobin in a method for the simultaneous . .
immunological determination of the content of HbA1c as . . .-,.:
' ~
:
a 3 3 .~
well as glycated haemoglobin variants such as e.g. HbS1c and HbClc in a blood sample.
The antibodies according to the invention additionally recognize further glycation variants of Hb such as e.g.
HbA21C and HbE1c 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 HbC1c using the antibodies according to the invention.
The invention is elucidated in more detail by the following examples. -~' , : - 12 - ~ ~ 3 3 ~ ~
., .
Ex~mpl~ 1:
Pro~uatio~ o~ i~mu~oge~s ~or obt~i~ing antibo~ie~
ag~i~st gly~ate~ h~e~oglobins of the ~bA1~ type e o$ B-galacto ~ e a~ carrier protein ~- ~
The peptide fructose-Val-His-Leu-Thr-Lys-OH is ~ :`
synthesized according to the description in ~P-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 khis 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-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 (B-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 ~olumn (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 - 2~ ~33 ~ ~
- .
collected. The coating of the B-galactosidase with the hapten groups can be determined hy reacting a sample with Ellman's reagent.
, .
- .:
~he immunogens fructose-Val-His-Lys-(MH)-BGal, fructose-Val-His-Leu-Lys-(MH)-BGal and ~ructose-Val-His-Leu-Thr-Pro-Lys-(MH)-BGal are produced in an analogous manner.
. ., 1.2 U~e of KLH as oarrisr 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) i5 reacted with maleimidohexanoyl-N-hydroxysuccinimide (MHS). For this 2 g KLH is dissolved in 0.1 mol/l sodium bicarbonate p~ 8.35. The insoluble protein fraction is separated by centrifugation. Sublsequently 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 HCl 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 ~a. 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 `~b ~ purification is then carried out by separating the non~
'~!'';; reacted peptide by gel permeation chromatography on ~;j., Ultrogel AcA 202 (2 x 24 cm, Pharmacia, Sweden).
~'` ` .
"',',,,.:""
~.~
~` - 15 - 21Q331~
." .
E~ample 2:
;~ Pro~u~tio~ o~ polyolonal ~tisera agai~t EbAl . .
~t~i 2 7 1 Immunization t"~
','f 5 sheep are immunized with the immunogen produced -, according to example 1.1 in Freund's adjuvant. The dose im in each ca~e is 500 ~g per animal for the first and each ,~, successive immunization. ~ additional sheep were immunized in the same manner but in each case with 1 mg -~
~' immunogen per animal and immunization.
~,i l .",.
.f 10 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 ,`'f,~ 500 ~g or 1 mg per animal and i~nunization.
f~
~l After 5 months (KLH immunogen) or 6 months (~-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 6.0 1 % SDS
0.02 % potassium hexacyanoferrate (III) 0.1 ~ NaN
0.5 % Brij 35 "",'' ~
2 ~r ~ 3 3 1 g ;
Reaction buffer~
, 20 mM MES pH 6.0 150 mM NaCl 0.5 % Brij 35 ~,' O. 1 ~6 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 ~g/ml , Preparation of the antisera for the measurement ~ . ' ~ ~
The sera are diluted 1~1 with twice concentrated reaction buffer, incubated overnight at 4C 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 ~m filter and the pH value is adjusted to 6Ø The antibody solution formed in this way can be used directly in the test or diluted further ~
with reaction buffer. ;;
. , ',';
Measurement ~c-~i In order to assess the antiserum titre, antibody solution and polyhapten solution are mixed and the ~, turbidity producPd is measured photometricallyO The measurement is carried out at 37C on a Hitachi 704 automated analyser. The procedure is as follows:
- 17 - 2 ~
, 8 ~1 haemolysis reagent and 350 ~1 antibody solution are mixed and incubated for 5 min. Subsequently 70 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 ~1' 700 nm) as an absorbance.
r~
~' 2.3 ~sult~ ~
~, 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 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 o~ these sera still generate very high measured signals even at this dilution and can apparently still be used at high dilutions for the meaisurement of glycated haemoglobins. The dose of the immunogen used for the immunization does not have any recognizable in~luence on the serum titre.
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- 18 - 2 ~ a ~
, . .
., ~
.. ,, ,.~.- .. ~.
~::' T~ble 1 : .
- i .::
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
~r~ immunogen at at at .
(mg) antis~rum antiserum antiserum :-~:
dilution dilution dilution 1:2 1:4 1-8 , . ~- ~
7 1 1~0 435 12 0 ~
2 1.01261 309 0 . :
3 1.0 77 2 ~ -:
1.0205 2 0 :~:
~, 5 loO887 305 4 :
~1 .
6 0.51000 417 67 7 0.51289 992 650 ` 8 0.51100 239 0 ~.
9 0.5848 253 0 0,5Zz~ 2 `-~:
.,. .:
: . .,:
`~
~ '.'`','.
I" . , E, -- 19 -- 2 ~ ~ ~ 3 1 ~ `
-Tabl~ 2 ,''',' '.' .
Maximum measured signal in a competitive immunoassay ~
when using a polyclonal ankiserum 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 ~394 18 0.5 1467 1598 1444 19 0~5 1158 1301 630 -.:
¦ ) 5 ¦ 1486 ¦1575 ¦1605 "'''~, , :~
';~
i ~3'~1~
~, EYamP1~ 3:
, ol~tion o~ ~bAo an~ Hb~ an~ in vitro gly~tio~ of ~bBn to fo~m ~b~15 Commercially available HbS (Sigma, Catalogue. No. H0392) or human erythrocyte haemolysat~ are separated , chromatographically and the HbSo purified in this way is reacted in vitro with glucose to form HbS1c.
.`'~'. . .
3.1 Isolatio~ of ~b~O
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 equilibratled 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 MonoSHBAlc column from the Pharmacia Company and their instructions for the determination of HbAlC. ~ ~ ' .-"...,~.
~ 3.2 Pro~u~tion of gly~ate~ ~bR ; -~
' ~
Part o~ 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 20 hours at 37C the reaction was stopped and the incubation mixture was dialysed extensively to remove glucose. The dialysate was also analysed by means of ~ ~;
- 21 - 2~
~PLC. The chromatogram showed a portion of 11.1 %
glycated HbSo !, i - 3.3 Iisolation of ~bAo i ~ .
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 th~ Pharmacia Company equilibrated with the above bufEer. The elution was carried out by a LiCl gradient. The HbAo 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 HbA1c.
~ , ~
~ ''.
f - 22 - 2 1 ~3 ", ~
., ~x~mpl~ 4:
8imultaneou~ ~e~er~in~tio~ o~ ~bA1c ~nd ~bS1¢
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 immunolo~ical determination of HbA1C in g/dl according to the TINIA --test principle was carried out in one channel hy a -~
turbidimetric measurement at 340 nm while the total ~-haemoglobin content wa~ determined in the other channel by photometric measurement at 570 nm. The content of HbA1c in percent is calculated according to the following formula:
% HbA1~ a _ HhA1~ [g/dl]
total haemoglobin [g/dl]
The following reag~nts were used:
1) Haemolysis reagent~
20 mM sodium phosphate pH 7.4 ~1 O.9 % TTAB (tetradecyltrimethylammonium bromide) 2) Antibody solution (R1 - HbA1c)~
20 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 " ,:
, r~
- 23 - ~033~8 -- ~
" .
- 1.0 mg/ml PAb <HbA1c>S IgG(DE~ (polyclonal sheep s antibody against HbA1c), ', produced using the ;~ :
;cl immuno~en fructose-Val~
-i ~is-~eu-Thr-MH-BGal . ~-, .~ :
-. :' 3) Polyhapten solution (R2 - HbA1c)~
20 mmol MES buffer pH 6.2 150 mmol NaCl ~: ~
6.0 % PEG 6000 --.-0.5 % Brij~ 35 --30 ~glml polyhapten~
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.
. ,~
..
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 Gf 120 days, the determination of glycated haemoglobin in blood provides a parameter for the blood glucose level which is independent of a short-term incrsase e.g. after a carbohydrate-rich meal.
~'' The determination of glycated haemoylobin in blood is ~l thus of great importance for the diagnosis and monitoring of diabetes mellitus. A number of , chromatographic and electrophoretic methods for the ~i detection of HbA1c have therefore been developed.
However, the glycated haemoglobin variants cannot be determined simultaneously with HbA1c using these methods (J. Sosenko et al., Diabetes Care 3 (1980), 590 - 593, D. Goldstein et al., C~C Critical Reviews in Clinical Laboratory Sciences 21 (1984), 187 - 225 and Allen et al., Annual Clinical Biochemistry 29 (1992), 426 429).
~l Although, in addition to Hb.~lc glycated haemoglobin l variants such as HbS1c or HbC1c, are also determined simultaneously using affinity chromatographic methods, this method is not speci~ic for glycation at the N-terminus of the B chain since all glycations of the ~I haemoglobin molecule te.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. Thase prior-art methods can therefore not be used for the diagnosis 'i''"~.
~l ,., ~` `:
-~ 4 ~ 2~ 3~'l3 :~:
:~ and monitoring of diabetes mellitus in blood samples of ~ :
patients with haemoglobinopathies.
It has now surprisingly turned out that by using il. antibodies which can be obtained by immunization with an :~:
il immunogen containing the glycated oligopeptide fructose-~1 Val-His-Leu-Thr-Pro or a part thereof as the hapten ;1 component, antibodies are obtained which recognize ~:~
;- HbAlC, HbSlC and HbC1C and are suitable for use in immunological methods of determination for HbA1c, HbSlC
j and HbC1c. ~-~
J;," Accordingly the invention concerns the use of antibodies ;' which recognize HbA1c, HbS1c and HbC1c 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 -~-:
f, component for the immunological, simultaneous ~:
~;.1 determination of HbA1c, HbS1c and HbC1c.
~ i 1 The fact that antibodies produced in this way recognize HbA1c, HbS1c and HbC1c is also par~icularly 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 ahtibodies which are obtained with the aforementioned immunogen would dif~erentiate between HbA1c, HbS1c and HbC1c.
The simultaneous immunological determination of N-terminally glycated haemoglobin HbA1c, ~bS1c and HbCl~
can be achieved with these antibodies by means of all , .~....
.:
~ ~ 5 ~ ~ ~ 33~
,~.
current immunoassays such as e.g. ELISA, ~luorescence immunoassay, radioimmunoassay, fluorescence-polarisation immunoassay (FPIA), cloned enzyme donor immunoassay `-~ (CEDIA) or enzyme multiplied immunoassay techni~ue (EMIT~. The test can be carried out in this process as a hcmogeneous tes~, 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 -q or latex particle-enhanced immunoassay (LPIA). These are ;~;
~i~ 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 speci~ic antibody. The hapten used for the '~ immunization (preferably fructose-Val-His-~eu-Thr-Pro) ~`~
,( is preferably usPd as the hapten which is present bound ~`
`.,,`1 to a high molecular carrier. In the absence of antigen ti from the sample this immunogen i~3 cross-linked by the antibodies used in the test to form large ag~regates 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 ~j of glycated haemoglobin, it is possible to determine of the amount of glycated haemoglobin (HbA1c, HbS1c and ~bC1c) in the sample solution. For this HbA1c, HbSlc or HbC1c can be used alone or as mixtures for the standard.
~`1 In addition it is preferred that the method according to ~; the invention is carried out according to the CEDIA, EMIT, FPI~ or ELISA technique.
' - 6 - 21~331~
~.
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 ~nzymatically 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. ~lowever, 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 ~rom 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.-~l Rubenstein, Biochemical and Biophysical Research ,.- J
~ 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 wavelenqth in a period of about 1o-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-~luorophore 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., 30urnal 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. -j :, ,`' The invention in addition concerns monoclonal and ;
polyclonal antibodies which recognize HbA1c, HbS1c and HbC1c and are obtainable by ir~unization with an `" ," ''`'~
,~
s~
1: 2 ~ O ~ 3 ~
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 j~ antibodies ~rom 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 :
v~ ~ mixture of an immunogen which contains fructose-Val-His-Leu-Thr as the hapten and with at least one further ~
~,5',~1 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 o~ the invention are monoclonal antibodies which recognize HbA1c, HbSlc and HbClc and are obtainable by immunization with the said :~:
.1 immunogen, immortalization of the spleen cells of the :~
immunized animals, cloning those immortalized cells ~: which produce the desired anti~ody and isolation of the antibody from the cells producing the antibodies according to known methods.
~he immunogen can be produced analogously to the method described in EP-A O 329 994 (which .is a subject matter of the disclosure in the present patent application). In -~
this process the hapten is bound t~ a carrier protein such as e.g. KLH (keyhole l mpet hemocyanin), B-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- ~a~3~
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 us~d 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-~eu-Thr-Pro as the hapten component.
Those immortalized cells which produce the desired antibo~y are identi~ied in the usual manner by means of an ELISA test to detect bindin~ to HbAlc, HbS1c and if desired, HbC1c.
`~
~, :
,~ , .
~ ...
- lo- 2 :' The invention in addition concerns a process for the production of antibodies which recognize HbA1c as well a~ HbSlC and ~bClC 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, HbS1C and HbC1C, by immunization with an immunogen which contains the glycated oliyopeptide 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, HbS1c and HbC1c in which an immunogen is used in which the hapten component is bound to KLH as a carrier proteinO
The invention in addition concerns the use of an antibody according to the invention for the .
i~munological det~rmination of the content of glycated haemoglobin in a method for the simultaneous . .
immunological determination of the content of HbA1c as . . .-,.:
' ~
:
a 3 3 .~
well as glycated haemoglobin variants such as e.g. HbS1c and HbClc in a blood sample.
The antibodies according to the invention additionally recognize further glycation variants of Hb such as e.g.
HbA21C and HbE1c 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 HbC1c using the antibodies according to the invention.
The invention is elucidated in more detail by the following examples. -~' , : - 12 - ~ ~ 3 3 ~ ~
., .
Ex~mpl~ 1:
Pro~uatio~ o~ i~mu~oge~s ~or obt~i~ing antibo~ie~
ag~i~st gly~ate~ h~e~oglobins of the ~bA1~ type e o$ B-galacto ~ e a~ carrier protein ~- ~
The peptide fructose-Val-His-Leu-Thr-Lys-OH is ~ :`
synthesized according to the description in ~P-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 khis 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-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 (B-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 ~olumn (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 - 2~ ~33 ~ ~
- .
collected. The coating of the B-galactosidase with the hapten groups can be determined hy reacting a sample with Ellman's reagent.
, .
- .:
~he immunogens fructose-Val-His-Lys-(MH)-BGal, fructose-Val-His-Leu-Lys-(MH)-BGal and ~ructose-Val-His-Leu-Thr-Pro-Lys-(MH)-BGal are produced in an analogous manner.
. ., 1.2 U~e of KLH as oarrisr 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) i5 reacted with maleimidohexanoyl-N-hydroxysuccinimide (MHS). For this 2 g KLH is dissolved in 0.1 mol/l sodium bicarbonate p~ 8.35. The insoluble protein fraction is separated by centrifugation. Sublsequently 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 HCl 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 ~a. 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 `~b ~ purification is then carried out by separating the non~
'~!'';; reacted peptide by gel permeation chromatography on ~;j., Ultrogel AcA 202 (2 x 24 cm, Pharmacia, Sweden).
~'` ` .
"',',,,.:""
~.~
~` - 15 - 21Q331~
." .
E~ample 2:
;~ Pro~u~tio~ o~ polyolonal ~tisera agai~t EbAl . .
~t~i 2 7 1 Immunization t"~
','f 5 sheep are immunized with the immunogen produced -, according to example 1.1 in Freund's adjuvant. The dose im in each ca~e is 500 ~g per animal for the first and each ,~, successive immunization. ~ additional sheep were immunized in the same manner but in each case with 1 mg -~
~' immunogen per animal and immunization.
~,i l .",.
.f 10 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 ,`'f,~ 500 ~g or 1 mg per animal and i~nunization.
f~
~l After 5 months (KLH immunogen) or 6 months (~-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 6.0 1 % SDS
0.02 % potassium hexacyanoferrate (III) 0.1 ~ NaN
0.5 % Brij 35 "",'' ~
2 ~r ~ 3 3 1 g ;
Reaction buffer~
, 20 mM MES pH 6.0 150 mM NaCl 0.5 % Brij 35 ~,' O. 1 ~6 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 ~g/ml , Preparation of the antisera for the measurement ~ . ' ~ ~
The sera are diluted 1~1 with twice concentrated reaction buffer, incubated overnight at 4C 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 ~m filter and the pH value is adjusted to 6Ø The antibody solution formed in this way can be used directly in the test or diluted further ~
with reaction buffer. ;;
. , ',';
Measurement ~c-~i In order to assess the antiserum titre, antibody solution and polyhapten solution are mixed and the ~, turbidity producPd is measured photometricallyO The measurement is carried out at 37C on a Hitachi 704 automated analyser. The procedure is as follows:
- 17 - 2 ~
, 8 ~1 haemolysis reagent and 350 ~1 antibody solution are mixed and incubated for 5 min. Subsequently 70 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 ~1' 700 nm) as an absorbance.
r~
~' 2.3 ~sult~ ~
~, 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 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 o~ these sera still generate very high measured signals even at this dilution and can apparently still be used at high dilutions for the meaisurement of glycated haemoglobins. The dose of the immunogen used for the immunization does not have any recognizable in~luence on the serum titre.
'. :
P~
~ {
~-.`,,~.
r,~
f., ~
- 18 - 2 ~ a ~
, . .
., ~
.. ,, ,.~.- .. ~.
~::' T~ble 1 : .
- i .::
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
~r~ immunogen at at at .
(mg) antis~rum antiserum antiserum :-~:
dilution dilution dilution 1:2 1:4 1-8 , . ~- ~
7 1 1~0 435 12 0 ~
2 1.01261 309 0 . :
3 1.0 77 2 ~ -:
1.0205 2 0 :~:
~, 5 loO887 305 4 :
~1 .
6 0.51000 417 67 7 0.51289 992 650 ` 8 0.51100 239 0 ~.
9 0.5848 253 0 0,5Zz~ 2 `-~:
.,. .:
: . .,:
`~
~ '.'`','.
I" . , E, -- 19 -- 2 ~ ~ ~ 3 1 ~ `
-Tabl~ 2 ,''',' '.' .
Maximum measured signal in a competitive immunoassay ~
when using a polyclonal ankiserum 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 ~394 18 0.5 1467 1598 1444 19 0~5 1158 1301 630 -.:
¦ ) 5 ¦ 1486 ¦1575 ¦1605 "'''~, , :~
';~
i ~3'~1~
~, EYamP1~ 3:
, ol~tion o~ ~bAo an~ Hb~ an~ in vitro gly~tio~ of ~bBn to fo~m ~b~15 Commercially available HbS (Sigma, Catalogue. No. H0392) or human erythrocyte haemolysat~ are separated , chromatographically and the HbSo purified in this way is reacted in vitro with glucose to form HbS1c.
.`'~'. . .
3.1 Isolatio~ of ~b~O
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 equilibratled 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 MonoSHBAlc column from the Pharmacia Company and their instructions for the determination of HbAlC. ~ ~ ' .-"...,~.
~ 3.2 Pro~u~tion of gly~ate~ ~bR ; -~
' ~
Part o~ 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 20 hours at 37C the reaction was stopped and the incubation mixture was dialysed extensively to remove glucose. The dialysate was also analysed by means of ~ ~;
- 21 - 2~
~PLC. The chromatogram showed a portion of 11.1 %
glycated HbSo !, i - 3.3 Iisolation of ~bAo i ~ .
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 th~ Pharmacia Company equilibrated with the above bufEer. The elution was carried out by a LiCl gradient. The HbAo 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 HbA1c.
~ , ~
~ ''.
f - 22 - 2 1 ~3 ", ~
., ~x~mpl~ 4:
8imultaneou~ ~e~er~in~tio~ o~ ~bA1c ~nd ~bS1¢
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 immunolo~ical determination of HbA1C in g/dl according to the TINIA --test principle was carried out in one channel hy a -~
turbidimetric measurement at 340 nm while the total ~-haemoglobin content wa~ determined in the other channel by photometric measurement at 570 nm. The content of HbA1c in percent is calculated according to the following formula:
% HbA1~ a _ HhA1~ [g/dl]
total haemoglobin [g/dl]
The following reag~nts were used:
1) Haemolysis reagent~
20 mM sodium phosphate pH 7.4 ~1 O.9 % TTAB (tetradecyltrimethylammonium bromide) 2) Antibody solution (R1 - HbA1c)~
20 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 " ,:
, r~
- 23 - ~033~8 -- ~
" .
- 1.0 mg/ml PAb <HbA1c>S IgG(DE~ (polyclonal sheep s antibody against HbA1c), ', produced using the ;~ :
;cl immuno~en fructose-Val~
-i ~is-~eu-Thr-MH-BGal . ~-, .~ :
-. :' 3) Polyhapten solution (R2 - HbA1c)~
20 mmol MES buffer pH 6.2 150 mmol NaCl ~: ~
6.0 % PEG 6000 --.-0.5 % Brij~ 35 --30 ~glml polyhapten~
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 :~
de~ermination (Rl - Hb):
20 mM sodium phosphate pH '7.4 150 mmol/l NaCl S) Calibrators a -~e~
20 mmol :sodium:phosphate pH 7.4 0.9 ~ TT~B
1.4 mg/ml sheep haemoglobin O, 0.~5, O.l, 0.16, 0.3 bu=an HbAl~
t~ atioD o~ ~AlC~
The haemolysis reagent was added to the sample in a raSio of l+lOO and incubated for ca. 5 minutes at 25C.
:~ - 24 ~ 3~
:. 250 ~1 antibody solution (R1 - HbAlc) was added by pipette to 10 ~1 haemolyzed sample~ After 5 minutes ~:
incubation at 37OC, the sample blank value was measured -:; bichromatically at 700/340 nm (A1). Ca. 20 seconds after - the measurement, 50 ~1 polyhapten solution was added, ::
: stirred immediately and it was incubated for a further 5 ; ::
., .~ .
:. minutes at 37C. Afterwards the turbidity was measured bichromatically at 700/340 nm (A2).
The sample-specific difference in absorbance is calculated according to the formula ~::
, ......................................................................... .. .
in which K represents the volume correction factor.
" K Vsample ~ ~Rl ~:
~total ::
A calibration curve was established using the calibrators a e which contained increasing . concentrations of HbAlc and the unknown HbAlG
concentration of the sample in g/dl can be read off via the sample-specific difference in absorbance ~Ao :~
~"~ ...
4O2 D~termination oP the tot~l haemoglobin co~centr~tion 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 .
2 `~ ~
- 25 ~ 33~ ~
: -, - molecule. 20 ~1 of this haemolysate is pipetted toyether with 230 ~1 buffer (R1 - Hb) into the cuvette, stirred ~i briefly and after 5 minutes incubation at 37C, the -~
-, absorbance is measured bichromatically ak 660/570 nm. A
~, calibration curve is established using the calibrator a and physiological NaCl solution (zero standard) and the i concentration is read via the absorbance of the unknown `, sample.
~¦ The percentage HbA1C content is calculated by means of ~ -~
the formula HbA1 [g/dl]
~! % HbA1 = c x 100 ~;3 i ~ - 26 ~
` .~ 2 ~ ~ 3 3 1 ~
.
,, Ex mpl~ 5: :~
,~ :
Det~rmination o~ ~h~ spe~ y of the ~ntibo~ies ,~? ~aording to th~ i~v~tion !,~, , , ,: - .:
A HbAlc determination is carried out on a Hitachi 717 according to example 4 in order to determine the specificity o~ the antibodies obtained according to example 2. For this, HbAo (purified from human blood according to example 3), HbSo (Sigma H0392 purified according to example 3), sample containing HbSlc (produced in vitro according to example 3) and two whole blood controls with known HbA1c values (BioRad, ~-Lyphochek~ Diabetes Control Level ~ and 2, Order No.
740) are measured as samples. The result is summarized :~:-in Table 3.
,.~
Ta~l~ 3 Sample _ HbAlc/Hbslc Hb HbAlc/HbSlc . [g/dl]tg/dl] t~]
HbAo 20 .
HbSo O 13.9 0 :
HbSo + HbSlc 1.3312.3 10.8 (HPLC: 11.1 %) .
Lyphochek level 1 0.7514.3 5.2 (target value 5.6 %) Lyphochek level 2 0.989.1 10.8 (target value 10.4 ~) _ ''.
.'~,.
~ - 27 ~ 3 1:l8 .~^ 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 HbSo.
..
.j Ex~mpl~ 6s ... .
8imultan~ous determi~ tion o~ ~bAlc ~a Hb81~ in ~
heterozygou~i ~bA~ ~iample ~ -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 Bissé 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 me~hod o~ determination o~ the IsoLab Company (Order No.
SG 6200). The results are summarized in Table 4.
,-~ TAble 4 :~
~ ' ,~
; HbAlc HbSlc HbAl~ + HbSl Immunoassay ; : 5.6 %
HPLC 3.3 % _ _ ~Glyc-Affin ~ _ ¦
. A HbAlc value of 3.3 % is measured using the HPLC method `~
according to Bisse and Wieland. The value for glycated :::
- 28 ~ 31~
," ., ,~,, HbS (HbS1c) cannot be given since the elution time of the HbS1c 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 - HbA1C + HbSlc + HbAla + HbAlb +
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 ~'''11 ~ ,.
% HbAlc (immunoassay) = 0.66 x ~sl % glycohaemoglobin (affinity chromatography) + 0.6 is there~ore obtained with a ~ery 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 ~tc. then this results in an r ¦ almost identical measured value for HbA1c + HbSlc using the immunoassay.
~ '' '"' ' ' i, :`
~`3 '`f~ s~ f"~ f~"~
de~ermination (Rl - Hb):
20 mM sodium phosphate pH '7.4 150 mmol/l NaCl S) Calibrators a -~e~
20 mmol :sodium:phosphate pH 7.4 0.9 ~ TT~B
1.4 mg/ml sheep haemoglobin O, 0.~5, O.l, 0.16, 0.3 bu=an HbAl~
t~ atioD o~ ~AlC~
The haemolysis reagent was added to the sample in a raSio of l+lOO and incubated for ca. 5 minutes at 25C.
:~ - 24 ~ 3~
:. 250 ~1 antibody solution (R1 - HbAlc) was added by pipette to 10 ~1 haemolyzed sample~ After 5 minutes ~:
incubation at 37OC, the sample blank value was measured -:; bichromatically at 700/340 nm (A1). Ca. 20 seconds after - the measurement, 50 ~1 polyhapten solution was added, ::
: stirred immediately and it was incubated for a further 5 ; ::
., .~ .
:. minutes at 37C. Afterwards the turbidity was measured bichromatically at 700/340 nm (A2).
The sample-specific difference in absorbance is calculated according to the formula ~::
, ......................................................................... .. .
in which K represents the volume correction factor.
" K Vsample ~ ~Rl ~:
~total ::
A calibration curve was established using the calibrators a e which contained increasing . concentrations of HbAlc and the unknown HbAlG
concentration of the sample in g/dl can be read off via the sample-specific difference in absorbance ~Ao :~
~"~ ...
4O2 D~termination oP the tot~l haemoglobin co~centr~tion 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 .
2 `~ ~
- 25 ~ 33~ ~
: -, - molecule. 20 ~1 of this haemolysate is pipetted toyether with 230 ~1 buffer (R1 - Hb) into the cuvette, stirred ~i briefly and after 5 minutes incubation at 37C, the -~
-, absorbance is measured bichromatically ak 660/570 nm. A
~, calibration curve is established using the calibrator a and physiological NaCl solution (zero standard) and the i concentration is read via the absorbance of the unknown `, sample.
~¦ The percentage HbA1C content is calculated by means of ~ -~
the formula HbA1 [g/dl]
~! % HbA1 = c x 100 ~;3 i ~ - 26 ~
` .~ 2 ~ ~ 3 3 1 ~
.
,, Ex mpl~ 5: :~
,~ :
Det~rmination o~ ~h~ spe~ y of the ~ntibo~ies ,~? ~aording to th~ i~v~tion !,~, , , ,: - .:
A HbAlc determination is carried out on a Hitachi 717 according to example 4 in order to determine the specificity o~ the antibodies obtained according to example 2. For this, HbAo (purified from human blood according to example 3), HbSo (Sigma H0392 purified according to example 3), sample containing HbSlc (produced in vitro according to example 3) and two whole blood controls with known HbA1c values (BioRad, ~-Lyphochek~ Diabetes Control Level ~ and 2, Order No.
740) are measured as samples. The result is summarized :~:-in Table 3.
,.~
Ta~l~ 3 Sample _ HbAlc/Hbslc Hb HbAlc/HbSlc . [g/dl]tg/dl] t~]
HbAo 20 .
HbSo O 13.9 0 :
HbSo + HbSlc 1.3312.3 10.8 (HPLC: 11.1 %) .
Lyphochek level 1 0.7514.3 5.2 (target value 5.6 %) Lyphochek level 2 0.989.1 10.8 (target value 10.4 ~) _ ''.
.'~,.
~ - 27 ~ 3 1:l8 .~^ 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 HbSo.
..
.j Ex~mpl~ 6s ... .
8imultan~ous determi~ tion o~ ~bAlc ~a Hb81~ in ~
heterozygou~i ~bA~ ~iample ~ -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 Bissé 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 me~hod o~ determination o~ the IsoLab Company (Order No.
SG 6200). The results are summarized in Table 4.
,-~ TAble 4 :~
~ ' ,~
; HbAlc HbSlc HbAl~ + HbSl Immunoassay ; : 5.6 %
HPLC 3.3 % _ _ ~Glyc-Affin ~ _ ¦
. A HbAlc value of 3.3 % is measured using the HPLC method `~
according to Bisse and Wieland. The value for glycated :::
- 28 ~ 31~
," ., ,~,, HbS (HbS1c) cannot be given since the elution time of the HbS1c 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 - HbA1C + HbSlc + HbAla + HbAlb +
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 ~'''11 ~ ,.
% HbAlc (immunoassay) = 0.66 x ~sl % glycohaemoglobin (affinity chromatography) + 0.6 is there~ore obtained with a ~ery 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 ~tc. then this results in an r ¦ almost identical measured value for HbA1c + HbSlc using the immunoassay.
~ '' '"' ' ' i, :`
~`3 '`f~ s~ f"~ f~"~
Claims (14)
1. Use of antibodies which recognize HbAlc, HbSlc and HbClc and are obtainable by immunization with at least one immunogen which contains a glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu, fructose-Val-His-Leu-Thr-Pro as a hapten component, for an immunological simultaneous determination of HbAlc, HbSlc and HbClc.
2. Use as claimed in claim 1, wherein the immunological determination is carried out according to an agglutination test principle or according to a CEDIA, EMIT, FPIA, or ELISA technique.
3. Antibody which recognizes HbAlc, HbSlc and HbClc and is obtainable by immunizing a mammal with at least one of an immunogen which contains a glycated oligopeptide fructose-Val-His, fructosa-Val-His-Leu and fructose-Val-His-Leu-Thr-Pro as a hapten component and isolating said antibody from a serum of said immunized mammal.
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 a hapten and with at least one further immunogen that contains at least one of fructose-Val-His, fructose-Val-His-Leu and fructose-Val-His-Leu-Thr-Pro as a hapten component and isolating said antibody from serum of said immunized mammal.
5. Antibody as claimed in claim 3 or 4, wherein said antibody is a monoclonal antibody further obtainable by immortalizing spleen cells of said immunized mammal, cloning those immortalized cells which produce a desired antibody and isolating said antibody.
6. Process for producing an antibody which recognizes HbAlc, HbSlc and HbClc by immunizing a mammal with an immunogen that contains at least one of a glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu and fructose-Val-His-Leu-Thr-Pro as a hapten component or with a mixture of two of said immunogens and isolating said antibody from serum of said immunized mammal.
7. Process for producing a monoclonal antibody which recognizes HbAlc, HbSlc and HbClc by immunizing a mammal with an immunogen that contains at least one of a glycated oligopeptide fructose-Val-His, fructose-Val-His-Leu and fructose-Val-His-Leu-Thr-Pro as a hapten component or with a mixture of two of said immunogens/ immortalizing spleen cells of said immunized mammal, cloning those immortalized cells which produce a desired antibody and isolating said antibody.
8. Process as claimed in claim 6 or 7, wherein an immunogen is used in which the hapten component is bound to KLH as a carrier protein.
9. Use of an antibody as claimed in claim 3 or 4 for an immunological determination of N-terminally glycated haemoglobin content in a method for a simultaneous determination of HbAlc, HbSlc and HbClc content in a blood sample.
10. Use of an antibody as claimed in claim 5 for an immunological determination of N-terminally glycated haemoglobin content in a method for a simultaneous determination of HbAlc, HbSlc and HbClc content in a blood sample.
11. Reagent for an immunological determination of N-terminally glycated haemoglobin containing at least one antibody as claimed in claim 3 or 4.
12. Reagent for an immunological determination of N-terminally glycated haemoglobin containing at least one antibody as claimed in claim 5.
13. Method for an immunological simultaneous determination of HbAlc, HbSlc and HbClc using antibodies as claimed in claim 3 or 4.
14. Method for an immunological simultaneous determination of HbAlc, HbSlc and HbClc using antibodies as claimed in claim 5.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4238705.1 | 1992-11-17 | ||
| DE4238705 | 1992-11-17 | ||
| DEP4310500.9 | 1993-03-31 | ||
| DE4310500A DE4310500A1 (en) | 1992-11-17 | 1993-03-31 | Simultaneous determination of HbA1¶c¶ and hemoglobin variants with HbA1¶c¶-analogous glycation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2103318A1 true CA2103318A1 (en) | 1994-05-18 |
Family
ID=25920507
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002103318A Abandoned CA2103318A1 (en) | 1992-11-17 | 1993-11-17 | Simultaneous determination of hba1 and haemoglobin variants with a glycation analogous to hba1 |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP0598329B1 (en) |
| JP (1) | JPH06225790A (en) |
| CN (1) | CN1094061A (en) |
| AT (1) | ATE213069T1 (en) |
| AU (1) | AU661207B2 (en) |
| CA (1) | CA2103318A1 (en) |
| CZ (1) | CZ244493A3 (en) |
| ES (1) | ES2172511T3 (en) |
| FI (1) | FI935078A (en) |
| HU (1) | HUT67126A (en) |
| IL (1) | IL107622A0 (en) |
| NO (1) | NO934139L (en) |
| NZ (1) | NZ250198A (en) |
| PL (1) | PL301068A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013029115A1 (en) * | 2011-09-01 | 2013-03-07 | Newsouth Innovations Pty Limited | Electrochemical competition sensor |
| WO2013029114A1 (en) * | 2011-09-01 | 2013-03-07 | Newsouth Innovations Pty Limited | Electrochemical affinity sensor |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1015028A2 (en) | 1997-09-19 | 2000-07-05 | Serex, Inc. | Methods to improve immunogenicity of antigens and specificity of antibodies |
| WO2000070350A1 (en) * | 1999-05-12 | 2000-11-23 | Cme Telemetrix Inc. | METHOD AND APPARATUS FOR RAPID MEASUREMENT OF HbA¿1c? |
| JP3949854B2 (en) * | 1999-10-01 | 2007-07-25 | キッコーマン株式会社 | Method for measuring glycated protein |
| US7449339B2 (en) | 1999-11-23 | 2008-11-11 | Nir Diagnostics Inc. | Spectroscopic method and apparatus for total hemoglobin measurement |
| USRE46130E1 (en) | 2000-07-14 | 2016-08-30 | Arkray, Inc. | Method of selectively determining glycated hemoglobin |
| JP3801133B2 (en) | 2000-07-14 | 2006-07-26 | 松下電器産業株式会社 | Peptide fructose and its protein conjugate |
| JP4323429B2 (en) * | 2002-10-23 | 2009-09-02 | 積水メディカル株式会社 | Novel fructosyl peptide oxidase and its use |
| WO2019045052A1 (en) * | 2017-08-31 | 2019-03-07 | キッコーマン株式会社 | Glycated hemoglobin oxidase variant and measurement method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4478744A (en) * | 1982-01-25 | 1984-10-23 | Sherwood Medical Company | Method of obtaining antibodies |
| CA1339952C (en) * | 1984-10-29 | 1998-07-14 | William J. Knowles | Immunoassays for denatured protein analytes, particularly hb alc, and monoclonal antibodies thereto |
| DE3439610A1 (en) * | 1984-10-30 | 1986-04-30 | Boehringer Mannheim Gmbh, 6800 Mannheim | Immunogens, process for their preparation and antibodies against glycosylated haemoglobin obtained therewith |
| DK145385D0 (en) * | 1985-03-29 | 1985-04-01 | Novo Industri As | MONOCLONAL ANTIBODY FOR IMMUNKEMIC ANALYSIS |
| DE3806198A1 (en) * | 1988-02-04 | 1989-08-17 | Boehringer Mannheim Gmbh | IMMUNOGENS AND ITS USE TO OBTAIN ANTIBODIES AGAINST HBA (DOWN ARROW) 1 (DOWN ARROW) (DOWN ARROW) C (DOWN ARROW) |
-
1993
- 1993-11-10 AU AU50590/93A patent/AU661207B2/en not_active Ceased
- 1993-11-11 AT AT93118251T patent/ATE213069T1/en not_active IP Right Cessation
- 1993-11-11 ES ES93118251T patent/ES2172511T3/en not_active Expired - Lifetime
- 1993-11-11 EP EP93118251A patent/EP0598329B1/en not_active Expired - Lifetime
- 1993-11-15 IL IL10762293A patent/IL107622A0/en unknown
- 1993-11-15 NZ NZ250198A patent/NZ250198A/en unknown
- 1993-11-16 HU HU9303256A patent/HUT67126A/en unknown
- 1993-11-16 PL PL93301068A patent/PL301068A1/en unknown
- 1993-11-16 CZ CZ932444A patent/CZ244493A3/en unknown
- 1993-11-16 FI FI935078A patent/FI935078A/en not_active Application Discontinuation
- 1993-11-16 NO NO934139A patent/NO934139L/en unknown
- 1993-11-17 JP JP5287954A patent/JPH06225790A/en active Pending
- 1993-11-17 CN CN93120541.7A patent/CN1094061A/en active Pending
- 1993-11-17 CA CA002103318A patent/CA2103318A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013029115A1 (en) * | 2011-09-01 | 2013-03-07 | Newsouth Innovations Pty Limited | Electrochemical competition sensor |
| WO2013029114A1 (en) * | 2011-09-01 | 2013-03-07 | Newsouth Innovations Pty Limited | Electrochemical affinity sensor |
Also Published As
| Publication number | Publication date |
|---|---|
| NZ250198A (en) | 1994-12-22 |
| HUT67126A (en) | 1995-02-28 |
| EP0598329A3 (en) | 1995-06-14 |
| ES2172511T3 (en) | 2002-10-01 |
| EP0598329A2 (en) | 1994-05-25 |
| CZ244493A3 (en) | 1994-05-18 |
| AU5059093A (en) | 1994-06-16 |
| PL301068A1 (en) | 1994-05-30 |
| AU661207B2 (en) | 1995-07-13 |
| FI935078A0 (en) | 1993-11-16 |
| FI935078A (en) | 1994-05-18 |
| HU9303256D0 (en) | 1994-03-28 |
| ATE213069T1 (en) | 2002-02-15 |
| IL107622A0 (en) | 1994-02-27 |
| EP0598329B1 (en) | 2002-02-06 |
| JPH06225790A (en) | 1994-08-16 |
| CN1094061A (en) | 1994-10-26 |
| NO934139D0 (en) | 1993-11-16 |
| NO934139L (en) | 1994-05-18 |
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| EEER | Examination request | ||
| FZDE | Discontinued |