FI104376B - Monoclonal antibodies specific to Hb A1c - Google Patents

Abstract

The invention relates to a monoclonal antibody or a fragment thereof containing the combining site of said antibody, at which said antibody or fragment binds specifically to the glucosylated form of the N-terminal peptide sequence in the beta-subunit of human haemoglobin. The invention further relates to hybridoma cell lines producing such monoclonal antibodies.

Description

104376

Hb Alc specific macroclonal antibodies. The present invention relates to hemoglobin glycosylated by dividing it from FI application 854187.

Determining the shape known as Hb Alc. Determining the degree of glycosylation of an individual's blood hemoglobin is a useful index for monitoring glucose levels in diabetes mellitus. The invention provides monoclonal antibodies that specifically recognize glycosylated N-terminal peptide residues in Hb Alc.

Hemoglobin is a protein tetramer consisting of four amino acid chains (subunits) each having about 143 parts and a total molecular weight of about 64,000. At the other end of the molecule (the 6-subunit NH2 terminus) is a valine unit that can react with glucose. Glycosylation of hemoglobin occurs by a non-enzymatic reaction involving glucose and the α-amino group of valine. Between the reagents, Schiff base formation occurs and glucose undergoes an Amadori rearrangement to form 1-deoxifructovalin. This complex is covalent and largely irreversible. The glycosylation reaction is regulated by the concentrations of the reagents, i.e.: hemoglobin and glucose. In normal · 25 (non-diabetic) individuals, approximately 3% of total hemoglobin is glycosylated. Hemoglobin tetramers containing 1-deoxifructovalin at the N-terminus of the β chain are identified as glycosylated, i.e., Alc hemoglobin ...

In diabetes, glucose levels are high enough to increase the rate of glycosylation directly *: *: proportional to the blood glucose level, which reflects the severity of the diabetes mellitus. In hemoglobin, the Alc level has increased to about 5-12%. Because circulating hemoglobin • ·. With a lifetime of approximately 120 days, measurement of glycosylated hemoglobin • 2 104376 provides a value that reflects the average glucose level over this period. In particular, a high-glucose meal is not reflected in high levels of glycosylated hemoglobin or serum albumin. Thus, the determination of glycosylated-5 dun hemoglobin concentration provides a more accurate picture of circulating glucose levels and thus a more accurate picture of the patient's condition over the long term.

U.S. Patent 4,247,533 discloses an analytical method wherein Hb Alc antibodies were obtained from a particular sheep by injecting Hb Alc and absorbing with non-Gly cosylated hemoglobin to obtain polyclonal antibodies that discriminated Hb Alc: n and non-Gly-cosylated Hb. Such antibodies then form the basis for an assay to determine the proportion of glycosylated hemoglobin present in the sample. However, in the assay, a suitably immunized sheep and antibody absorptions are required to obtain the appropriate specificity. Thus, it is expensive and difficult to produce specific polyclonal antibodies. Preparations prepared by this absorption method have been reported to have low titres and affinity. The reproducibility of this method is also "open" because there are no reports of its use in analyzing human hemoglobin containing clinical specimens.

• · · ·: ***; Another method for finding antibodies specific for Hb Alc is disclosed in U.S. Patent 3,378,744. These investigators replaced the conventional hemoglobin molecule with a synthetic peptide immunogen as an immunizing agent.

• · · I,. This substance was injected into an animal which normally does not have Hb Alc in the blood, for example sheep. The synthetic **: peptide immunogen had a glycosylated peptide residue which had an amino acid sequence which: corresponds to the first 4-10 [. amino acids in the hemoglobin sequence at the N-terminus. The following studies show that sheep polyclonal antibodies obtained against synthetic peptide immunogen have no significant specificity. -; blameless for the glycosylated form, Hb Alc.

Definitions 5 Amino Acid_Abbreviation

Arginine Arg

Aspartic acid Asp

Glutamic acid Glu

Lysine Lys 10 Serine Ser

Asparagine Asn

Glutamine Gin

Glysiini Gly

Proline Pro 15 Threonine Thr

Alanine Ala

Histidine His

Cysteine Cys

Methionine Met 20 Valine Val

Isoleucine Ile *

• »I

Leucine Leu

Tyrosine Tyr • f V ·· Phenylalanine Phe 25 Tryptophan Trp This invention makes it possible to specifically define glycosylated proteins such as glycoprotein, glycosylated hemoglobin and albumin. and especially Hb • · · 30 Ale in biological fluids such as blood. Monoclonal antibodies derived from synthetic glycosylated N-terminal peptide residues in Hb Alc - *: ·· i have been found to specifically bind to such residues in glycosylated hemoglobin S-subunit. - t ·. 35 ropes. Antibodies may be prepared by various conventional methods for the preparation of monoclonal antibodies. In principle, antibodies are prepared against a synthetically derived immunogen having a desired glycosylated N-terminal peptide residue chemically linked to an immunogenic carrier such that the glycosylated peptide has at least 2, and preferably 5 to 15, amino acid units corresponding to Hb Alc. The resulting antibodies are specific for the glycosylated synthetic peptide and the corresponding present epitope on the hemoglobin Alc molecule.

The drawings are graphical representations of the experiments described in the examples of the Hb Alc immunoassay.

Figure 1 is a view showing inhibition of the binding of glycopeptide I (PEPTIDI 1) Ab-3 to Alc. The antibody was preincubated with the glycopeptide before being transferred to an Alc coated microtiter plate. The monoclonal antibody that binds Ale was determined using another antibody-enzyme conjugate. The results are plotted in Figure 1 as 20% inhibition, with 0% inhibition being the value obtained in the absence of a competing agent. The 0-0 line represents a non-carbohydrate identical peptide, and this indicates that the carbohydrate is essential for antibody binding. All points are averages of three experiments • i * 25.

• · i i: ***; Figure 2 is a graph showing the inhibition of (PEPTIDI 3) Ab-3 binding to Alc by the glycopeptide 3 ··· · * · * ·. The competing experiment was performed as shown in Figure 1. way.

• · · • «· I,. Fig. 3 is a graph showing the inhibition of the α-binding of Ab-3 and Ab-4 by Alcopoly by glycopeptide 4 (PEPTIDI 4). The competing experiment was performed in Figure 1 brochure *: ··; like this.

'. Figure 4 is a typical standard plot using optimal · · 35 analysis conditions. Whole blood constant was prepared using 5 · 5,737,376 denatured diabetic patients with varying proportions of whole blood containing 12.66% Alc. HPLC ion exchange, and the standard donor (3.83% Alc). All points of the triple measurements are 5 drawn.

Figure 5 is a standard graph using a synthetic peptide constant. The analysis was performed as shown in Figure 4 with the exception that different amounts of synthetic glycopeptide were used as a competitor in place of whole blood. All triple configuration values were plotted.

Figure 6 is a graph illustrating a comparison of an immunoassay method to a boronate affinity method on donors. The results of the triple assays are plotted in the immunoassay coordinate.

Figure 7 is a graph illustrating the expression of the Hb Alc epitope under various denaturation conditions.

Figure 8 is a drawing showing the results of sheep immunization with the synthetic glycopeptide of Example 1 (b).

Figure 9 is a drawing showing that murine monoclonal antibodies are specific for Alc hemoglobin.

Immunoanalytical determination of HB Alc using an antibody reagent of the invention can be performed essentially by any conventional method. These include more classical methods such as immunodiffusion, immunoelectrophoresis, agglutination methods and complement binding, as well as newer methods requiring the use of specifically detectable labels such as radioimmunoassay and non-radioisotope. printing.

• · ·

Performing an immunoassay on Hb Alc according to the invention involves the essential steps of treating an aqueous test sample to effectively denature a significant amount of this protein that may be present therein.

«I

. 6,104,376 and contacting the denatured sample with the antibody reagent, and determining the binding of the antibody reagent to this protein. The assay step will of course vary according to the basic immunoassay method used. A common method used for this assay involves the use of a labeled reagent that reacts with either the analyte or the antibody reagent and is used in a manner that demonstrates or competes with the formation of an immune complex between the analyte and the antibody reagent.

The latter method can be performed in a number of ways, such as competitive binding, in which the labeled reagent is made to compete with the protein analyte for binding to the antibody reagent. The amount of labeled reagent bound to the antibody-15 reagent or the amount of labeled reagent unbound to the free antibody reagent is suitably measured and can be functionally related to the amount of protein analyte in the sample. Because this antibody reagent is targeted to a linear epitope in a protein assay, the labeled reagent may be a labeled form of a denatured protein or denatured fragment thereof, or preferably a peptide containing the amino acid sequence of a linear epitope! the stamped shape of the tide star. The latter preferred reagent can be prepared by the synthetic peptide methods and equipment in use and not by the protein itself; the name molecule needs to be isolated, purified and denatured.

Another useful immunoassay for the detection of protein analytes is the sandwich method. This method employs two antibody reagents, one labeled and the other, to separate the originally labeled antibody reagent bound protein analyte from the unbound antibody reagent. The unlabeled second antibody reagent typically is an immobilized or immobilizable form as is known in the art. s In radioimmunoassays, the free molecules and the bound molecules must be physically distinguishable so that the label can be measured, since the resulting signal is qualitatively the same in both molecules. Such a process is known in the art to be heterogeneous because phase separation is required. Other heterogeneous immunoassay methods are known, such as enzyme-labeled immunoassays, sometimes referred to as ELISAs (see U.S. Patent 3,654,090), and fluorescence immunoassays (see U.S. Patents 4,201,763; 4,133,639 and 3,992,631). ).

Numerous immunoassay methods have recently been developed which avoid the separation step by using Lei-15, whose detectable signal is affected by the binding of the labeled reagent to the binding agent, i.e., the antibody. Such methods have become known as homogeneous methods and, when available, are advantageous in the invention because no separation step or radioisotopes are required. Some such methods include fluorescence quenching and enhancement (see U.S. Patent 4,160,016), energy transfer immunoassay (see U.S. Patent No. 3,996,345) and double antibody - ethereal inhibition * * immunoassay (see U.S. Patents 3,935,074 and 3,998,943). Especially preferred homogeneous immunoassay methods are those employing the enzyme • · ·; * · *; label involved in the catalyzed reaction. Examples include substrate-labeled immunoassay (see U.S. Patent 4, ···, 279,992 and GB Patent Application 1,552,607), prostate-grouped (FAD) -labeled immunoassay (see U.S. Patent No. 4,122,157). 4,238,565), enzyme modulator-labeled immunoassay, for example, the use of inhibitor labels (see U.S. Patents 4,134,972 and 4,273,866) and enzyme-labeled immunoassay (see U.S. Patent 3,817,837).

• · • · β 104376

The antibody reagent of the invention is characterized in that it binds specifically to the linear peptide epitope of Hb Alc. Thus, the term "antibody reagent" as used herein refers to any agent having an antibody binding site specific for such a peptide epitope. The term thus encompasses whole antibodies as well as their appropriate fragments and polyfunctional forms. As a whole antibody, it may belong to any class or subclass of known immunoglobulin bulbs, e.g., IgG, IgM, etc. Any fragment of any such immunoglobulin with specific binding capacity to a peptide epitope may also be used, e.g., usually as Fab, Fab '. fragments of IgG known as F1 and F (ab ') 2. In addition, aggregates, polymers, derivatives, conjugates and hybrids of immunoglobulins or fragments thereof may be used where appropriate.

The immunoglobulin source of the antibody reagent may be obtained by any of the available methods, such as conventional antiserum and monoclonal methods. The antiserum can be obtained by well-defined methods of immunization of an animal such as a mouse, rabbit, guinea pig or similar animal with a suitable immunogen.

• *

Immunoglobulins are also obtained by the somatic cell hybrid-25 foundation, which is usually monoclonal

IMI W

antibodies called antibodies.

• · · .'j *. Monoclonal antibody reagents are particularly preferred. Hybridoma cell lines are made to produce antibodies against only the linear peptide epitope of the protein molecule • · * * ... 30 rather than the whole protein · · · *. and such cell lines and their antibodies are selectively screened for the identification and isolation of monoclonal antibodies reactive with the desired epitope.

• «• · 9 104376

In one method for producing such antibodies, a protein chain fragment that corresponds to and contains a naturally occurring linear peptide epitope sequence is fused to a protein carrier and injected into a laboratory animal to elicit an immune response. Alternatively, the immunogen may contain a linearized or denatured form of the protein or fragment thereof. The lymphocytes of an immunized animal, such as spleen cells, are combined with myeloma cells to produce hybridomas that are cultured and screened for monoclonal antibodies. Monoclonal antibodies are screened for antibodies that are selective for the peptide epitope, and this particular cell line is cloned for use in the production of additional monoclonal antibodies.

To produce antibodies against a synthetic peptide immunogen in a laboratory animal, e.g., BALB / c mice, rats, or the like, a peptide containing the desired epitope is produced and isolated from the naturally occurring protein or chemically synthesized and purified. Such a protein fragment contains all critical amino acids of the desired epitope. acid units and may contain other amino acid units, some or all of which optionally correspond to the amino acid sequence of the protein of interest.

In order to ensure that the epitope-containing peptide fragment is optimally antigenic, it may be advantageously m-linked to an immunogenic carrier. The immunogenic carrier may be any of the commonly known carriers containing · · functional groups available for binding to the peptide * '· ** · residue. In most cases, the carrier is a protein or polypeptide, although other substances such as carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids and the like having sufficient size and immunogenicity may also be used. In most cases, the molecular weights of the immunogenic proteins and polypeptides are in the order of 4,000 to 10,000,000, preferably greater than 15,000 and more generally greater than 50,000. In general, proteins from one animal species are immunogenic when introduced into the bloodstream of another animal species. Particularly useful proteins are albumins, globulins, hemocyanins, glutelins and the like. Other references to current conventional immunogenic carriers and their incorporation into hapten are as follows: Parker, Radioimmu Noassay of Biologically Active Compounds, Prentice-Hall (Englewood Cliffs, New Jersey USA, 1976); Butler, J. Immunol. Meth. (1974) 7: 1-24; Weinryb and Shroff, Drug 15 Metab. Rev. 10; (1974) 271-283; Broughton and Strong,

Clin. Chem. 22; 726-732 (1976); and Playfair et al., Br. Med. Bull. 30; (1974) 24-31.

The number of epitopes linked to a particular immunogenic carrier is limited only by the number of available 20 binding sites on the carrier, and may be several thousand with certain high molecular weight synthetic peptides such as polylysine.

• I

'· *. The epitope density of a particular carrier depends on the molecular weight of the carrier and the density of available binding sites. Optimal epitope densities for optimal immunogen synthesis and repeatability and optimal antibody response are about 10-50% of the available binding groups in the vehicle.

The peptide fragments are attached to the carrier by a suitable coupling method. Functional groups of the *** native amino acids in the fragment or functional groups obtained by chemical conversion of the fragment are usually used for direct coupling or via the bifunctional linkers to the functional groups of the carrier 11114376. It is preferable to design the peptide difrag. having one unique reactive functional group to provide unambiguous binding to the carrier.

Monoclonal antibodies raised against synthetic glycosylated N-terminal Hb Alc peptide residues have been found to specifically bind to such residues in the glycosylated β-subunit of hemoglobin. Antibodies can be prepared by 10 different methods according to standard monoclonal techniques. In principle, antibodies are prepared against a synthetically derived immunogen containing a desired glycosylated N-terminal peptide residue chemically bound to an immunogenic carrier, wherein the glycosylated peptide contains at least 2, and preferably about 5 to 15, amino acid units corresponding to Hb Alc. The resulting monoclonal antibodies are specific for glycosylated synthetic peptides and the corresponding raised epitope in hemoglobin Alc molecule.

. Monoclonal antibodies specific for Hb Alc in human blood are secreted from the hybridoma of the hybridoma derived by combining myeloma cells with «« «· · '. ·: Lymphocytes obtained from an animal immunized with a synthetic peptide immunogen. The synthetic peptide immunogen ··· is preferably of the form: [Glyko- (NH) Val-His-AA-R] n ------ Strain and • · · · · · · Wherein Glyko (NH) Val is a non-enzymatically glycosylated spacer, His is the second amino acid in the native '* β subunit Hb sequence, AA is a bond or one or more amino acid residues, R is a suitable linker, kan -.... and is an immunogenic carrier and n (epitope density) 35 is an average of one to 12,104,376 binding sites available on the carrier. The linker group R may be any desired linking reagent and AA may contain one or more additional amino acid residues corresponding to the N-terminus of the β-subunit of human hemoglobin-5 carbohydrate. Group AA can be selected, for example, from the following amino acid sequences or any of their continuous fragments starting with a leucine unit: -Leu-Thr-Pro-Glu-Glu-Lys-. In addition, the linker group R may contain additional amino acid units that are not present in standard human hemoglobin, but which can be conveniently added by peptide synthesis methods and can function as functional groups for binding to a carrier. A particularly useful linker group is -Tyr-Tyr-Cys, which provides a unique thiol group for the controlled coupling of a glycosylated peptide moiety to carriers.

The Hb Alc monoclonal antibody of the invention is characterized by its specificity for binding to the glycosylated form of the N-terminal peptide sequence of the human hemoglobin β-subunit. This glycosylated residue is a remarkable structural property of Hb Alc. The antibody according to the invention requires an epitope or determinant site having I · | '' at least 1-deoxifructosyl carbohydrate moiety formed by the Amadori rearrangement of the reaction product between glucose and the terminal amine, and an expanding peptide sequence containing at least one: ': *: Hb Alc N-terminal sequence other amino acid units in the peptide sequence that are · · · 30 characteristic of the epitope may be the same or different from the ones in the native Hb Alc sequence **** In this way, the epitope is characterized by at least two contact or junction sites with an antibody unique to the glycosylated N-terminal Hb Alc sequence. specifically binds a glycosylated peptide residue of the following formula

Gluco- (NH) Val-His-AA-5 wherein Gluco- (NH) Val and AA are as defined above. Particularly preferred monoclonal antibodies have been found to be specific for the glycosylated dipeptide residue, regardless of the nature of AA. Antibodies are also obtained which specifically require longer glycosylated peptide sequences when AA is a sequence of 1 to 12, preferably 1 to 6, amino acids corresponding to the N-terminus of the human hemoglobin β subunit. This specificity of the monoclonal antibody allows for the specific detection of a raised glycosylated N-terminal peptide residue in Hb Alc 15, while other glycosylated peptide epitopes of hemoglobin and other proteins and peptides that are present in the human bloodstream remain.

The glycosylated N-terminal peptide residue of the native Hb Alc molecule is made available to the monoclonal antibodies or fragments thereof according to the invention * by appropriate denaturation or degradation of the sample protein to be analyzed. The underlying hypothesis for the success of the preparation of specific antibodies according to the invention in which the earlier: ***: attempts have failed: * · *; is presented below, but its accuracy should not be regarded as critical to the invention of the method of the invention. ·, For humility.

• · · # * .. f 30 The N-terminal sequence of the human hemoglobin β-subunit • · «is very similar to the corresponding mouse hemo * · ** · globin sequence, whereby the first four amino acids' • '' ί are identical. mouse hemoglobin is glycosylated: i. almost identical to human hemoglobin.

• ·. Thus, in the native human hemoglobin molecule, the N-terminal sequence of? 14,144,376 fetuses would be foreign to the mouse and no immune response could be expected. This is a prior art conclusion and accordingly an animal (sheep) with a completely different hemoglobin-5 sequence that is not glycosylated has been selected in the hope of detecting an immune response. However, in the present invention, it has been shown that when a glycosylated N-terminal residue is exposed to the mouse immune system in the form of a synthetic peptide immunogen, the epitope appears as a structure to which the mouse can elicit an immunological response. Somatic cell cloning methods can isolate hybridomas that secrete highly specific antibodies. These antibodies bind to a glycosylated N-terminal peptide residue in the native hemoglobin-15 molecule, if exposed enough to interact with the binding site of the antibody. The mode of exposure of the epitope is described in more detail below.

Sterile access of the antibody reagent to epitope 20 may be accomplished in any effective manner. Exposure of the epitope to the intact protein was understood to occur by physical or chemical denaturation or degradation at least in the epitope region.

Such denaturation or degradation may be localized to the epitope region or may require more general or predominantly complete denaturation of the tertiary and in addition secondary protein structure or partial or complete protein degradation.

Denaturation can be accomplished by a variety of methods, including conventional protein processing. methods by physical methods such as heat, ultrasound, high or low pH, as preferred, by chemical denaturation by decomposition, or by ____: interacting with a chaotropic agent or chaotropic solution. Useful chaosatic agents include, but are not limited to, guanidine, urea, and various detergents, such as sodium dodecyl sulfate (SDS) and others, such as deoxycholate and certain salts of bile acids, 3- (3-Cholamidopropro-5-yl). ) dimethylammonio-1-propanesulfonate, organic solvents such as methanol, propanol and acetonitrile, and certain salts such as potassium thiocyanate. Nonionic detergents such as Triton X, Tween, Nonidet NP-40, and octyl glucosides may also serve as denaturants of the protein. Reagents (eg, mercaptoethanol or dithiothreitol) that reduce disulfide bonds may be effective promoters of the denaturation process. Protein denaturation may be most effective when combinations of chemical and / or chemical and physical methods are used (e.g., guanidine and heat, guanidine and SDS, or guanidine and dithiothreitol). In particular, strong chaotro-pit such as guanidine are particularly preferred. Of course, denaturation conditions that result in significant aggregation, insolubility, or protein precipitation such that inadequate amounts are avoided are avoided. This epitope is available in solution for binding to the antibody. Sufficient denatured protein must remain in solution or suspension in order to achieve useful immuno-binding. The amount of solubility required will depend upon the conditions of the intended or desired binding.

• · ·

A significant amount of the desired protein in a given assay sample can be denatured to elicit a peptide epitope for binding to the antibody, · · ·. ···. 30 by distilling the sample with an aqueous solution of chaotrope in which

• I I

the chaotrope is present in a concentration sufficient to denature the marker, preferably at a concentration greater than 1 molar, with a 3 molar solution being particularly useful. The denaturation process is greatly facilitated by the short-term heating of the alloy. It has been found that denaturation at temperatures below 37 ° C can take from one to several hours, while at temperatures above 50 ° C sufficient denaturation can be achieved in a minute or less. In order to prevent significant denaturation of antibody and other protein reagent 10 to be added, the sample-Kao-tropical mixture is usually diluted in a separate step or by adding reagent solutions to a level at which the chaotropically is not capable of denaturing these reagents. significant renaturation. In the case of guanidine, this preferably requires dilution to a concentration below about 1.0 molar.

Non-limiting examples of proteolytic enzymes for use in the invention are trypsin, chymotrypsin, proline-specific endoprotease, pepsin and papain. When carrying out an immunoassay, an inhibitor of proteolytic enzymes is added to the assay mixture. bitters, as is known in the art, to prevent the degradation of the prophylactic substances.

More specifically, hybridoma cell lines are raised to produce antibodies against only the glycosylated moieties of the hemoglobin molecule, rather than the whole protein, and such cell lines and their antibodies. agents are screened to identify and isolate the monoclonal antibodies, which are then * * * *. reacted selectively with the glycosylated Hb Alc epitope *****.

To produce such antibodies, a protein chain jun fragment which naturally corresponds to · · is combined. 35 occurring glycosylated peptide sequences, protein · »·

: I

17 104376 and injected into a laboratory animal to elicit an immune response. Lymphocytes of the immunized animal, such as spleen cells, are fused with myeloma cells to obtain hybridomas that are grown and screened for the production of mono-5 clonal antibodies. Monoclonal antibodies are distinguished from those that are selective for a glycosylated peptide epitope and a particular cell line is cloned for use in the production of an additional monoclonal antibody.

In order to produce an antibody in a laboratory animal, eg BALB / c mice, rats or similar animals, the glycosylated hemoglobin fragment must either be produced and isolated from naturally occurring human hemoglobin, or be chemically synthesized and purified. The hemoglobin moiety should have a 1-deoxy-fructose residue and at least two amino acid units, preferably 3, 4, 5 or more residues corresponding to the N-terminus of the hemoglobin β-subunit (valine-histidine). Preferably it has from about 5 to about 15 and more preferably from about 20 to about 10 units.

. In order to ensure that the glycosylated peptide fragment is optimally antigenic, it may be advantageously combined with a highly immuno-carrier. a genetic molecule such as bovine serum albumin (BSA) 25 or volcanic haemocyanin (KHL). The fragment should also have an inherently rearranged adduct of the glu-: T: mucose-valine reaction, which may be present from the beginning *, as in the isolated naturally occurring hemoglobin fragment, or preferably may be formed. synthesis peptide during synthesis or before attachment of the peptide to a large protein carrier. The carrier may be added in any manner that does not destroy the antigenicity of the fragment.

The glycosylated fragment can be produced by chemical or enzymatic cleavage of naturally occurring Hb 104376, e.g., Alc. This fragment can be coupled to a carrier using classical coupling methods, e.g., glutaraldehyde or carbodiimide, and the conjugate used as an immunogen.

In a preferred chemical method, the synthesis of a portion of a known hemoglobin sequence requires the addition of one or more amino acid units (not present in the regular sequence) to optimize its antigenicity and coupling properties. The terminal unit si-10 then contains a thiol (SH) group which can be coupled to the ligand in a conventional manner, such as by reaction with a bifunctional coupling reagent, e.g. m-maleimido-benzoyl N-sulfosuccinimide ester (MBS).

According to a preferred embodiment, tyrosine was added to the lysine end of the synthetic Hb 15 fragment containing eight units of NH 2 -valine-histidine-leucine-threonine-proline-glutamic acid-glutamic acid-lysine-COOH and cysteine containing 11 units of cysteine. .

This can be glycosylated in a conventional manner by a non-enzymatic reaction with glucose. Subsequently, the glycopeptide is coupled to a large carrier to give an antigen which is given for the production of antibodies. Lymphocytes from 25 animals producing antibody to the glycosylated peptide epitope are then combined in the usual way to produce hybridomas which are cloned and those which produce monoclonal antibodies of the desired specificity are further cloned. . The cell line (s) having the highest selectivity for the monoclonal antibody to the glycosylated epitope as opposed to the non-glycosylated Hb are then propagated and the antibodies recovered. Reference publications on such monoclonal antibody methods include Lym., J. 35 phocyte Hybridomas, ed. Melchers et al., SpringerVerlag, «104376 (New York 1978), Nature 266: 495 (1977); Science 208: 692 (1980) and Methods in Enzymology 74 (part B): 3-46 (1981).

The antibodies can then be used in the usual manner to react with blood samples containing 5 unknown amounts of glycosylated Hb, and the degree of reaction can be compared by calibrated standards to determine the degree of glycosylation. The measurement may be by fluorescence, immunoassay, or the like, by coupling appropriate measurable groups to monoclonal monoclonal antibodies in a known manner without reducing their binding power to Hb Alc glycosylated epitopes.

Alternatively, a reagent test strip based assay may be used in which a carboxyl-containing material such as carboxymethylcellulose is coated on a wooden or plastic strip. Subsequently, the strip is immersed in a disrupted and denatured unknown blood sample, and thus any glycosylated hemoglobin is adsorbed. Thereafter, the strip is immersed in a solution of monoclonal antibodies of appropriate size. ie labeled (e.g., enzyme, fluorescence, cofactors «· *, etc.) at a site that does not interfere with binding to the Hb Alc epitope. The amount of bound antibody is determined by the amount of label * * in the strip and indicates the amount of glycosylated Hb present in an unknown sample. Binding of the label and its determination are carried out in a conventional manner.

The linking group R in the above formula may have substantially any suitable and stable structure. The full linking group R is usually an aliphatic chain containing from 1 to 20 atoms without hydrogen and including * 'heteroatoms such as nitrogen, oxygen and sulfur. The glycosylated residue may be attached through various groups to form a linking chain R, including methylene, ether, thioether, imino and the like. One skilled in the art will recognize a large number of linker groups which can be selected for the preparation of an immunogen. Typically, a glycosylated peptide is prepared by terminating it in a functional group, such as an amino, carboxyl, thiol, hydroxyl or maleimido group, which is active in the coupling reaction with a suitable group of a carrier molecule.

The following examples illustrate the present invention, but should not be construed as limiting it.

Example 1

Preparation and Characterization of Antibodies to the Hb Alc Glycopeptide Epitope (a) A peptide containing 11 amino acids with 8 N-terminal units of hemoglobin B and two tyrosine units and a cysteine unit was synthesized according to the method of Gutte, B. and R.B. Merrifield; J. Am. Chem. Soc., 91: 2.501 (1969) to give the following peptides: NH2-valine-histidine-leucine-threonine-proline-glutamic acid-glutamic acid-lysine-tyrosine. tyrosine-cysteine COOH.

To glycosylate this peptide, 200 mg of this purified peptide is reacted with 0.25 molar * * glucose in 20 ml of anhydrous pyridine for 48 hours to 25 minutes at room temperature in the dark. The mixture is dried under vacuum. The resulting syrup is resuspended in 20 mM potassium phosphate, pH 2.95, and purified by HPLC.

Fractions containing glycopeptide are dissolved in 0.1 M triethylammonium acetate, pH 8.5, and chromatographed. Affigel-601 on a boronate affinity resin (Biorad), whereby the glycopeptide is selectively adsorbed. The resin is washed with 0.1 M triethylammonium acetate, pH 8.5, and glyco

the peptide is eluted with 0.1 M triethylammonium acetate, pH

• · 35 5.0. The eluate is freeze-dried.

• · 21 104376

The product is resuspended in 1 ml of water, reacted with a 500-fold molar excess of dithiothreitol (to retain the SH-group in cysteine) and the reduced peptide is purified again by HPLC and freeze-dried. This glycopeptide is stored at -20 ° C under nitrogen before further use.

b) KLH-MBS conjugate as previously described by Lerner R. et al., Proc. Natl. Acad. Sci. 78: 3403 (1981), is reacted with the product of (a) to give a double molar ratio of glycopeptide to maleimide in 50 millimolar (mM) potassium phosphate, pH 7.2, for one hour at room temperature.

c) The solution of b) is mixed with an equal volume of Freund's complete adjuvant to form a water-in-oil emulsion and 200 µg of conjugate is injected into BALB / cBy mice. Mice are boosted on days 30 and 60, killed, and their spleens used for fusion according to Kohler and Milstein, Nature 256: 495 (1975), 20 to obtain numerous hybridomas. The hybridoma. dooms are screened to identify those that produce specific monoclonal antibodies. fusion to a glycosylated peptide epitope.

The screening for monoclonal antibodies specific for Alc is performed using an ELISA method, where the antibody is absorbed onto the surfaces of polystyrene microtiter plates: Linbro. Antigens are purified by man

Alc and non-glycolysed Ao hemoglobins. Alc is purified from human erythrocyte hemolysate using two different ···, 30 chromatographic methods. In the first purification, glycosylated hemoglobin is bound to the surface of a boronate affinity resin, Pierce Chemical Co., Rockford,

Illinois, USA, Item No. 42,000, as described by the manufacturer. Typically, 1-5 g of hemoglobin per 100 µl · 35 ml per boronate resin is used and the bound (glycohemoglobin • 22 104376 µl) fraction is eluted as disclosed in Pierce Chemical Co., Glyco-Test Bulletin, Item No. 42,000. The eluted glycolemoglobin fraction is equilibrated in a low ionic strength buffer and chromatographed on an ion-5 exchange resin such as McDonald, M. et al., J. Biol. Chem., 253: 2327-2332 (1978). The Alc "peak" is analyzed by isoelectric focusing and carbohydrate analysis using thiobarbituric acid analysis and the results show that this purification method yielded 10 ultra pure Alc hemoglobin because the purified material contains both carbohydrate and conventional Ao-hemoglobin isoglobin. Similarly, Ao-hemoglobin was purified on the basis of its non-binding affinity to boronate affinity 15 resin, and then chromatographed by ion exchange as Ao "peak" on ion exchange chromatography purification. Pure Alc and Ao hemoglobins are adsorbed onto separate microtiter plates (2 pg / 100 μΐ PBS / well) overnight at 4 ° C. The free binding sites of the plates are filled with 1% BSA in PBS for 60 minutes at room temperature and then washed four times with PBS. Super-. . ·. natant from each hybridoma cell line is added to Alc and * · ·

Ao plate and incubate at room temperature for 60 · · ·! I minutes. Plates are washed four times with PBS and the second antibody (rabbit anti-mouse IgG peroxidase, Miles Labo-

Ml ratories, Inc., Elkhart, Indiana, USA at a dilution of 1: 5 · 1 · 000 in 1% BSA in PBS) is added and the plates are incubated for 60 minutes at room temperature. Plates are washed four times with PBS and 200 μΐ substrate solution (24.3 mM sit-30 tartaric acid, 51.4 M sodium phosphate, pH 5.3 containing 2.2 mM o-phenylenediamine and 5.2 mM hydrogen peroxide) is added. '. one hundred. After 20 minutes, quench the reaction with 50 μΐ of 8 M H2SO4 and read the product of the peroxidase reaction at 492 nm.

Of the parent hybridomas producing antibodies to hemoglobin, nine (9) are identified as specific for the Alc epitope, while 191 react with both Alc and non-glycosylated hemoglobin. Since pre-immunized mouse serum has no detectable antibody response to human A0 or Alc hemoglobin by ELISA, the primary immune response is against eight peptide sequences which are equally common to Alc and Ao. Because the synthetic peptide immunogen consists of eight amino acid residues of the 10 hemoglobin sequences, the mice 'immune response is mainly directed against the peptide rather than the carbohydrate (191,200 hybridomas react with both Ao and Alc). As expected, immunized mouse serum also has extensive cross-reactive antibodies that react with both Ao and Alc, indicating that specificity for Alc is not achieved unless the hybridoma is screened for Alc and Ao. -Hemo-globin for reactivity. Preferred hybridomas that produce antibodies against Hemoglobin Alc rather than Hemoglobin Ao were deposited with the ATCC identified by accession number ATCC HB 8639. and ATCC HB 8869, October 11, 1984, and July 10, 1985, respectively.

• i · r <* ·! d) Identification of peptides competing for antibody binding to Alc.

• · ·

The following peptides are prepared by enzymatic digestion of the 11 amino acid starting glycosylated

IM

·. · · Of the peptide GlykoVal-His-Leu-Thr-Pro-Glu-Glu-Lys-Tyr-Tyr-

Cys (Glycopeptide 1). All peptide fragments are purified by HPLC and quantitated by the amino acid sequence.

: * · *; Tryptic digestion of the parent peptide produced pep- *. thidi Glyko-Val-His-Leu-Thr-ProGlu-Glu-Lys (Glycopeptide 2). Proline-specific endoprotease produces the peptide Glyko-Val-His-Leu-Thr-Pro (Glycopeptide 3). Glyco-Peptide: 35 Val-His-FAD (Glycopeptide 4), wherein the dipeptide is combined with 24 104376 N6-aminohexyl FAD and then glycosylated, is prepared by the method of Carrico and Johnson, US. U.S. Patent No. 4,255,566, issued to Dr. Kin Yip * East and Dr. R. Buckler, Ames Division, Miles 5 Laboratories, Inc., Elkhart, Indiana USA.

In a typical competition assay, each peptide, eight nanomoles to eight picomoles, in 100 µl PBS in which 7.2 mM Na2HPO4, 2.8 mM NaH2PO4, and 127 mM NaCl, pH 7.4, is incubated with 100 μ1 of monoclonal cell-10 culture supernatant for 60 minutes at room temperature. This mixture is added to a polystyrene microtiter plate coated with 1 pg Alc hemoglobin / well. If the peptide competes with the antibody, the antibody is not free to bind immobilized Alc. The plate is washed four 15 times with PBS. Another antibody rabbit anti-mouse IgG coupled to horseradish peroxidase) is added for 30 minutes and the plate is washed with PBS. The substrate (2.2 mM o-phenylenediamine) and hydrogen peroxide (0.012%) are added and the resulting color is measured at 492 nm. The amount of product 20 refers to the degree of competition e.g. if no product is present, it means that the competing peptide completely prevented antibody binding to immobilized Alc. The results show that all four glycopeptides I · ·, * I previously presented, including Gluko-Val-His-FAD,? 25 are effective competitors. Glycopeptides 1-4 inhibit • »» ···! binding of one of the antibodies, Ab-4, to A * - * (see Figures 1-3). Glycopeptides 2-3, but not Glyco · · · V · peptide 4, inhibit another antibody, Ab-3 (see Figures 1-4).

30 Peptides lacking carbohydrate do not competitively inhibit, indicating that carbohydrate is •. essential epitope component and produces specificity for the recognition of antibodies to Alc hemoglobin (see Figure 1).

• • * 25 104376

Example 2

Competitive Immunoassay for Hb Alc This competitive immunoassay is based on the use of a certain amount of the hapten label (as shown in Example 5) that competes with the digested whole blood Alc to bind the immobilized antibody. Because the antibody recognizes both Alc and hapten, the amount of Alc in the sample determines the amount of hapten label that binds to the antibody. Because the antibody is immobilized, all unbound reagent can be removed by a simple washing step. The bound label can then be assayed and compared to a standard for the determination of Alc in the original blood samples.

The assay was developed using whole blood as sample 15 and the method can be divided into the following steps: (1) Cell lysis - denaturation of hemoglobin

Because the final assay requires less than 0.3 μΐ of whole blood, accurately pipette 20 volumes of blood (5 to 50 μΐ from fingertip or whole blood) into a denaturing solution (3 M guanidine-HCl, 10 mM Tris-HCl, pH

. 7.5) and the solution is heated at 56 ° C for 2-15 minutes.

• · ·

Lower temperatures can also be used, but then · · i! more time is needed to completely denature the sample. Denaturation (a) inactivates the coagulation mechanism in the absence of samples prepared with anticoagulants; (b) lysing red blood cells; (c) denature proteases,

M1 · · enzymes, etc., and optimally expands the Alc epitope of hemoglobin; (d) either sterilizes or: T: 30 inhibits the growth of microorganisms in a denatured blood sample, even if the sample is not prepared by hand *. aseptically (e.g., fingertip blood) and (e) provides a stable clinical specimen that can be stored for days at room temperature without affecting the final assay.

• · 26 104376 (2) Dilution and Competition

A small amount of denatured whole blood is pipetted to 10 times the volume of the buffer containing the hapten label. This effectively dilutes the hemo-5 globin to an appropriate concentration for analysis and dilutes to a low concentration of denaturing agent that does not interfere with the antibody or enzyme activity. The antibody-coated bead is then added for a period of time during which the antibody binds to either Alc hemoglobin 10 or hapten HRP.

(3) Washing and reading

After competing incubation, the bead is washed and the label read after addition of a suitable substrate. The signal is then compared to a standard and the amount of Alc in the original whole blood sample is determined.

The details of the analysis are summarized below:

Pearl Coating Method:

Polystyrene beads (0.63 cm diameter, mirror-shaped 20 surface finish) manufactured by Precision Ball Company, Chicago,

Illinois, USA. Batches are screened to obtain beads of la exhibits the least variation in several im-

I I

, /. munoanalyysimäärityksissä. Before coating beads • ·! wash with absolute methanol and then with water. Washing with methanol appeared to significantly reduce the coefficient of variation of several determinations of the same sample. This • ·

* ··. * Antibody solution (5 pg antibody / 100 μΐ 0.2 M

• · · V · sodium borate, pH 8.5 with 0.02% sodium azide) is added to the moist beads and the beads are spun overnight: T: 30 at 4 ° C. The beads are then washed, blank binding sites filled with 1% BSA in PBS 0.02% *. sodium azide. Typically, 500-1000 | beads at one time and used for weeks without evidence of a decrease in antibody activity. Top:: ··: 35 knock-out experiments using radioactive antibodies • 27 to 104376 showed that 0.5 µg of antibody binds per bead.

Beads are used in this immunoassay because they are capable of binding relatively large amounts of protein.

The hydrophobic absorption of a protein on the surface of a polystyrene is practical, but could certainly be replaced by one of the many methods of covalently attaching proteins to polystyrene, functionalized resins, or silica. Polystyrene can also be of 10 designs in a tube or cuvette.

The working procedure is as follows: (a) Dilute 50 μΐ whole blood 1.0 with any denaturing solution (3 M guanidine HCl, 10 mM Tris, pH 7.5), heat at 56 ° C for 15 minutes, further dilute with 15 100 µl 1.0 any denaturing solution.

(b) Add 50 μΐ of the above solution 0.5 to any phosphate buffered saline (PBS) pH 7.5 containing hapten-HPR. Incubations, washes and enzymatic reactions are conveniently performed on 48-well polystyrene tissue culture plates.

(c) Add antibody coated bead and incubate for 30 minutes at room temperature with shaking.

Wash beads with buffer (PBS) (usually 3 washes in 1 mL portions).

• · · ’/ 25 (e) Add o-Phenylenediamine substrate and hydrogen · · · ···· peroxide.

· *** (f) Stop reaction and read product after 20 minutes. The above analysis is used to determine the clinical results presented below. 30 and is shown in Figure 4. Competition with Glycopeptide I is shown in Figure 5. Evaluation of normal and diabetic donors is shown in Figure 6. Boronate affinity assays are performed exactly by Pierce Chemical Co. (GlycoTest, product). # 42,000): ··: 35.

• · 28 104376

Example 3

Optimal presentation of the Alc epitope

Optimal reactivity of the human Alc epitope is obtained by treating native hemoglobin (whole blood 5 or hemolysate) with methods or reagents that bring the epitope to the antibody binding site. Optimal display of the epitope can be achieved by physical denaturation (heat, ultrasound, etc.), a chemical method using 10 classical denaturants (urea, guanidine, SDS, protease) or a combination of physical and chemical methods. The most effective is the method of adding whole blood (50 μΐ) to 1 ml of a solution of 3 M guanidine hydrochloride and 10 mM Tris-HCl, pH 7.4 and heating the solution to 56 over one minutes. The resulting sample will work optimally in the following Alco-epitope immunoassays. The solution can be diluted to one-tenth with buffer, whereby guanidine is diluted to a concentration of 0.3 M, which has little or no effect on normal antibody-antigen interactions and enzyme densities, and is a suitable medium for subsequent immunoassays.

The competitive immunoassay of Example 2 is used. Competitor is Whole Diabetes mellitus «« · / 25 Alc. The whole blood sample is introduced into 3.0 M guanidine at 20 ° C, m 2 at 37 ° C, or 56 ° C for periods of 0 to 320 minutes.

* · .. * The results (Figure 7) show that over time ··· · at 20 ° C or 37 ° C, the epitope appears and thus competes effectively with the hapten-HRP conjugate. At 56 ° C, the epitope appears within a maximum of five: * · *: minutes, the earliest point determined * in this assay. The results show that in the native hemoglobin Alc tetramer the epitope is inward and inaccessible and does not compete with the linear synthetic glycopeptide-HPR conjugate. However, if denatured by hemoglobin Ale, the newly exposed epitope becomes an effective competitor for the linear glycopeptide-enzyme conjugate.

Example 4 Comparison of Antibody Specificity: A Sheep Polyclonal Antibody vs. a Mouse Monoclonal Response

The sheep is immunized, at five sites, in IM Freund's complete adjuvant with the Gly-10 copeptide-KHL conjugate of Example 1 (b) (4 mg). Booster injections are given in the same manner after 30 and 60 days. The 60-day booster dose is in Freund's incomplete excipient. Pre-immunized serum and immune serum are determined for their AA1c and Ao specificity by ELISA analysis as shown in Example 1 (c). The results (see Figure 7) show that the synthetic glycopeptide stimulates an immune response against human hemoglobin, but immunoglobulins are not specific for AA1c hemoglobin. In contrast, mouse monoclonal antibodies to 20 Alc are quite specific for Alc as measured by the same assay (ELISA assay of Example 1 (c); see Figure 8).

; Attempts to purify the antiserum of sheep by immuno «i ·. ·. : affinity antibody that is specific for Aleys,

• I I

. * failed.

• »I

Example 5 Preparation of hapten-label conjugates Glycopeptide 1 (HPR) conjugate was prepared.

The hapten-HPR conjugate was prepared by reacting 15 mg of horseradish peroxidase (HPR) with a 10-fold: 30 molar excess of MBS (see Example 1b) in 50 mL of sodium phosphate, 1 mM EDTA, pH 7.0. The MBS-HRP conjugate was purified by gel filtration (using the buffer above) and 0.34 mg of glycopeptide hapten (Pe. Ptide 1) was added. The final hapten-HPR conjugate '·' · 35 was purified by gel filtration by HPLC and used as a &quot; 104376 30 dilution of 1: 1,000 to 1: 100,000 in the competitive immunoassay of Example 3.

Example 6

Test strip immunoassay for Hb Alc 5 a) 1 mg of the monoclonal antibody of Example 1 (c) in 0.1 M sodium borate buffer, pH 8.5, can be mixed with 200-fold molar excess of fluorescein isothiocyanate (FITC) and reacted for 30 min. minutes at room temperature. The fluorine-labeled mono-10 clonal antibody can be purified by gel filtration.

(b) The test strip (polystyrene, cellulose, etc.) containing COOH groups is immersed in 0.5 ml of anhydrous denatured hemolysate pH 7.5. The strip is flushed with a pus-15 filter at pH 7.5 and immersed in a buffered solution of fluorescein-labeled monoclonal antibody of (a) for five minutes at room temperature. The strip is rinsed again and the degree of fluorescence of the strip represents the degree of Hb Alc of an unknown sample. Example 7

Coupling of a monoclonal antibody with a reagent. and its use in immunoassay «« ·; Whatman 1 filter paper (7 cm) is taken up in 20 ml of ice-cold d-H2O and the pH of the solution is adjusted to 10.5 - 1 * 25 with 11.5 M NaOH. The solution is monitored throughout the activation and the pH is maintained between 10.5 and 11.5 by the dropwise addition of 5 M NaOH. A small mixing rod is introduced into the bottom of its decanter ··· V: The filter beaker is then placed in an ice-filled petri dish, ·. · * 30 is either placed on a magnetic stirrer. 1 g of solid BrCN is added to the beaker and incubated for 20 minutes (on ice) with stirring. solution and washed with 100 ml of ice-cold distilled water (d-H 2 O), then washed with ice-cold 35 0.2 M Na 2 HPO 4, citric acid buffer, pH 6.8. 1mg / ml in 0.2M Na2HPO4-citric acid buffer, pH 6.8) is added and antibody coupling is allowed for one hour Ethanolamine (10ml in 1mM solution) is added to fill the unreacted sites (15min) and pa-5 peri pes Phosphate buffered saline (PBS, 10 mM NaH 2 PO 4, 140 mM NaCl, pH 7.5) to remove unreacted components.

This reagent strip is immersed in a standardized amount of an unknown, denatured hemolysate having 10 labeled antibody binding competitors for Ale hemoglobin. A suitable competitor is a glycopeptide (Glycopeptide 1) covalently attached to horseradish peroxidase (hapten-HRP). The strip is removed and rinsed with PBS. The amount of hemoglobin bound to the strip is measured (inversely proportional to Alc of the sample and Alc hemoglobin can be quantified by comparison with standard samples).

Example 8

Alc ELISA Assay 20 volumes of denatured blood hemolysate (100 μΐ) are added to polystyrene microtiter plates and. allow to bind for 60 minutes at room temperature. Plate

I I I

, ·,; washed four times with PBS containing 0.05% Tween-20

I I

^ (PBST). The monoclonal antibody (bound to horseradish / riperoxidase) is added (100 μΐ, 1 μg / ml) in PBST and the reaction is allowed to react for 30 minutes at room temperature. Excess antibody · · * ··· * is removed by washing four times with V * PBST. The substrate (o-Phenylenediamine, 2.2 mM) and H 2 O 2 (0.012%) in PBS are added and the reaction product is measured at 492 nm. The color intensity describes the amount of Alc in the hemolysate compared to standard values.

^ *. Example 9 ««

Radioimmunoassay of Ale 100 μΐ of denatured blood hemolysate (200 nmoles *.: 35 hemoglobin) is mixed with 7 nmoles of iodinated pep- * · 32 104376 tide Glyko-Val-His-Leu-Thr-Pro-Glu-Glu-Lys- Tyr-Tyr-Cys (500,000 pulses per minute / 7 nmoles). The monoclonal antibody is added in an amount sufficient to bind 50% of the glycosylated peptide if 5 normal (about 3%) levels of Alc-hemoglobin are present in the blood hemolysate. Higher hemoglobin Alc values compete with the peptide while reducing the total number of antibody-bound pulses. The antibody can be recovered by immunoprecipitation with another antibody (rabbit anti-mouse IgG) or by absorption on protein A coated particles. The amount of iodinated peptide bound to the antibody can be determined in a gamma isotope counter and gives the amount of Alc in the blood hemolysate when compared to the standards.

It is to be understood that the description and examples are illustrative but not limiting of the present invention and other embodiments within the spirit and scope of the invention will be apparent to those skilled in the art.

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Claims (10)

1. A monoclonal antibody or fragment thereof containing the binding site of the antibody, characterized in that it binds specifically to the glycosylated N-terminal peptide sequence in the β-subunit of human hemoglobin. Monoclonal antibody or fragment thereof according to claim 1, characterized in that it specifically binds to a glycosylated peptide residue of the formula Glyco- (NH) Val-His-AA wherein Glyco- (NH) Vai represents a non-enzymatic glycosylated valine residue. and AA is a bond or one or more additional amino acid residues. The monoclonal antibody or fragment thereof according to claim 2, characterized in that AA is a sequence containing 1-12 amino acids and corresponding to the N-end of the β-subunit of human hemoglobin. 4. A monoclonal antibody or fragment thereof: according to any of claims 1-3, characterized in that it is formed against an immunogen which contains a glycosylated peptide chemically linked to an immunogenic carrier, wherein the glycosylated β peptide has at least two amino acid units corresponding to the N- end of the β-subunit of the hemoglobin, or to a denatured or cleaved protein. 5. A monoclonal antibody or fragment thereof according to any one of claims 1-4, characterized in that it binds specifically to said ....: glycosylated, N-terminal peptide sequence. , when sufficiently produced, so that the entry of the antibody into it is sterically possible. A monoclonal antibody or fragment thereof according to claim 5, characterized in that said glycosylated peptide sequence has been produced for the antibody by physical or chemical denaturation or digestion. A monoclonal antibody or fragment thereof according to claim 5 or 6, characterized in that said glycosylated peptide sequence has been produced for the antibody by chemical denaturation by contacting it with a chaotropic substance, presumably guanidine, urea, thiocyanate or a detergent. Monoclonal antibody or fragment thereof according to any one of claims 1-4, characterized in that it binds specifically to said glycosylated N-terminal peptide sequence or hemoglobin adsorbed to a solid surface presumably made of polystyrene or cellulose. A hybridoma cell line that secretes monoclonal antibodies that bind specifically to the glycosylated N-terminal peptide sequence in the β subgroup of the hemoglobin and whose ATCC deposit number is HB 8639 or 8869. 10. Monoclonal antibodies to hemoglobin AiC produced by hybridomas, whose ATCC deposit number is HB 8639 or 8869. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · • * * «f (|