WO1996034290A1 - Haemoglobin standards - Google Patents

Abstract

The present invention relates to a complex of glycosylated haemoglobin with a ligand, for use as a standard reference material in the determination of glycosylated haemoglobin, wherein in said complex, the ligand displaces the oxygen of glycosylated oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe2+ therein to Fe3+, while not substantially changing the absorption spectrum of glycosylated oxyhaemoglobin.

Description

Haemoglobin Standards

The present invention relates to haemoglobin derivatives for use as standard reference or control materials, ie. as "standards" or "controls", in the measurement or assay of haemoglobin. In particular, the invention relates to carbonmonoxy derivatives of glycosylated haemoglobin.

As oxygen-transporter, haemoglobin is a physiologically very important protein. A sufficient concentration of haemoglobin in blood is essential for adequate transport of oxygen and carbon dioxide between lungs and other tissues. Blood haemoglobin concentration may be diminished as a consequence of haemorrhage or haemolysis, or as a result of impaired blood formation in the bone marrow. Conversely, blood haemoglobin concentration may rise as a compensatory measure when there is impaired gas exchange through the lungs, or in a variety of other disorders. It is therefore important to assess blood haemoglobin concentration to detect anaemia (diminished haemoglobin concentration) or erythrocytosis (increased erythrocyte count and haemoglobin concentration) . Determination of blood haemoglobin concentration is consequently one of the most commonly performed determinations in clinical laboratories today.

Haemoglobin is present in erythrocytes largely in unglycosylated form, although a minor portion may be glycosylated, primarily by glucose. This glycosylation takes place substantially by non-enzymatic processes and is dependent on the concentration of glucose in the blood; the level of glycosylation of haemoglobin is therefore a reflection of circulating blood glucose concentrations, and since haemoglobin is relatively long-lived in red blood cells (the average life of erythrocytes is about 120 days) , the amount of glycosylated (or glycated) haemoglobin provides a more accurate indication of the control of glucose concentration during an extended period of time, than does measuring glucose concentration in the blood directly. This is particularly important in the diagnosis and monitoring of diseases or disorders where control of blood glucose levels is affected, most notably diabetes. The determination of glycosylated haemoglobin in blood is therefore another important clinical diagnostic assay.

A variety of assays for haemoglobin, and for the glycosylated fraction, exist. Haemoglobin has a strong absorption peak and is conveniently assayed directly, by spectrophotometric measurement of blood samples . The iron atom in the haem prosthetic group of haemoglobin may be in the ferrous (+2) or ferric (+3) oxidation states. Only the "ferro" (+2) form may bind oxygen, and this is the usual form of haemoglobin in the blood. The absorbance spectrum of haemoglobin is altered on the binding of oxygen; oxyhaemoglobin demonstrates two absorbance peaks at about 576-578 nm and 540-542 nm respectively. Deoxyhaemoglobin has a single broad peak at about 555 nm.

On exposure to air or oxygen, the Fe²⁺ atom of haem oxidises readily to Fe³⁺, forming the oxidised form of haemoglobin known as methaemoglobin. This has a characteristic absorption spectrum with peaks at about 415 nm, and characteristically at 630-635 nm.

For determination of haemoglobin, an international standard has been agreed. This is the cyan ethaemoglobin reference method. Cyanmethaemoglobin (HbFe (III) CN) is formed by reaction of methaemoglobin with KCN (methaemoglobin being prepared by oxidation of haemoglobin with ferricyanide) , and has an absorbance peak at 540 nm. Cyanmethaemoglobin standards (usually 80 mg/dl in serum) are certified by an appropriate certifying agency. Blood samples to be determined are reacted with Drabkin' s solution (containing K₃Fe(CN)6, KCN and NaHC0₃ to lyse the cells and convert haemoglobin in the sample to cyanmethaemoglobin and the resulting absorbance is read and compared against the cyanmethaemoglobin standard.

For determination of glycosylated haemoglobin, a variety of methods exist, relying for example, on the separation of the glycosylated and non-glycosylated fractions by ion exchange chromatography or on affinity chromatography utilising the specific binding of boronic acid derivatives to glycosyl moieties, to isolate specifically the glycosylated fraction. The glycosylated haemoglobin may be detected in a number of ways, for example by use of labelled boronic acid derivatives, conjugated to coloured or fluorescent dyes.

An effective and convenient assay for glycosylated haemoglobin using such labelled boronic acid conjugates is described by us in WO90/13818. In this assay, both glycosylated and non-glycosylated haemoglobins are separated together from the sample, for example by precipitation, and the glycosylated fraction is detected by means of labelled boronic acid derivatives which bind specifically to the glycosyl moieties. The conjugates may be labelled in a variety of ways, including by means of radioactive, fluorescent, chemiluminescent enzymic, or coloured labels. The use of coloured dyes as signal- forming labels is simple and convenient, and the assay was found to work particularly effectively with the dye N- (resorufin-4-carbonyl) -piperidine-4-carboxylic acid - N-hydroxysuccinamide-ester) (hereinafter abbreviated to RESOS) . RESOS has an absorption maximum at 575 nm.

To avoid interference with the absorption of haemoglobin itself, the assay may be improved by using dyes with an absorption maximum above 600 nm. Such dyes are described in WO92/08722.

As standards in such assay methods for glycosylated haemoglobin, are used glycosylated haemoglobin preparations having given glycosylated haemoglobin contents. Such glycosylated or glycated haemoglobin forms may be prepared by reaction of haemoglobin with glucose under reducing or non-reducing conditions, as described in Methods of Enzymology, volume 231, p. 66. No international standard has yet been agreed.

However, haemoglobin is not stable to storage, and gradual oxidation to methaemoglobin results in a shift in the absorbance spectrum as mentioned above, which leads to reliability problems with the standards. Attempts have been made to circumvent this by including reducing agents in the standard preparations. However, such preparations also have problems with long term stability. Alternatively, the preparations may be lyophilised, although again this solution is not entirely problem-free, since lyophilisation of standards is not widely accepted and it is also difficult to avoid methaemoglobin being formed.

Since it is difficult to avoid the oxidation of haemoglobin to methaemoglobin in air, many standards, or control reference materials for glycosylated haemoglobin comprise partially glycosylated haemoglobin, and are unstable preparations vulnerable to oxidation.

As mentioned above, methaemoglobin has an absorption peak at about 630-635 nm. This unfortunately overlaps with the absorption peaks of the coloured dyes which advantageously are commonly used in glycosylated haemoglobin determinations. It is therefore desirable to avoid methaemoglobin formation in standards to be used for this purpose. A need thus exists for alternative haemoglobin control reference materials to be used as standards in haemoglobin determinations, and especially determinations of glycosylated haemoglobin. The present invention addresses this need.

More particularly, we have found that derivatives of glycosylated haemoglobin may be prepared, which are resistant to oxidation, and which exhibit an absorption spectrum close to that of oxyhaemoglobin, ie . the absorption peaks of which are in the "red" area of the spectrum below 600 nm. Such derivatives are particularly useful as standards for glycosylated haemoglobin determinations.

In one aspect, the present invention thus provides the use of a glycohaemoglobin-ligand complex as a standard reference material in haemoglobin determinations, wherein in said complex, the ligand displaces the oxygen of oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe²⁺ therein to Fe³⁺, while not substantially changing the absorption spectrum of oxyhaemoglobin.

As used herein the term "glycohaemoglobin" refers to glycosylated haemoglobin.

As mentioned above, such a standard is particularly useful in determination of glycosylated haemoglobin. However, its utility is not limited and it may also be used in determinations of "total" (ie. glycosylated and non-glycosylated) haemoglobin concentration. Nonetheless, the use of such a glycosylated standard as a control reference material in glycosylated haemoglobin determinations is particularly preferred.

In another aspect the invention thus provides a complex of glycosylated haemoglobin with a ligand, for use as a standard reference material in determinations of glycosylated haemoglobin, wherein in said complex, the ligand displaces the oxygen of glycosylated oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe²⁺ therein to Fe³⁺, while not substantially changing the absorption spectrum of glycosylated oxyhaemoglobin.

Alternatively viewed, this aspect of the invention also provides a standard reference material for use in determinations of glycosylated haemoglobin, comprising a complex of glycosylated haemoglobin with a ligand, wherein in said complex, the ligand displaces the oxygen of glycosylated oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe²⁺ therein to Fe³*, while not substantially changing the absorption spectrum of glycosylated oxyhaemoglobin.

The term "determinations" as used herein in relation to haemoglobin or glycosylated haemoglobin assays, encompasses both qualitative and quantitative, as well as semi-quantitative assessments of haemoglobin or glycosylated haemoglobin concentrations. Quantitation is included in the sense of both obtaining an absolute value for the amount of glycosylated or "total" haemoglobin in a sample, and also obtaining an index, ratio, percentage or similar indication of the level of glycosylated haemoglobin, for example relative to the total haemoglobin concentration or content of the sample.

As mentioned above, an important feature of the ligand in the complexes of the invention is that the absorption spectrum of oxyhaemoglobin is not substantially affected. The important criterion here, is to prevent a bathochromic (red) shift in the absorption peaks ie. to prevent the development of absorption peaks above about 600 nm; minor shifts of the two absorption peaks of oxyhaemoglobin at about 540 and 576-575 nm may be tolerated.

Complexes of glycosylated haemoglobin with the ligand carbon monoxide represent a particularly preferred embodiment of the invention. Such complexes, or derivatives are termed herein "carbonmonoxy glycohaemoglobin" .

Carbonmonoxy haemoglobin, the complex of non- glycosylated haemoglobin with carbon monoxide, or carboxy haemoglobin as it is also termed, is known and the interaction between carbon monoxide and haemoglobin has been studied; carbon monoxide binds reversibly to haem in a manner almost identical to oxygen but with significantly higher affinity, thereby displacing oxygen from oxyhaemoglobin. Carbon monoxide is not readily displaced from haemoglobin except at high oxygen tension. It is therefore a serious poison. In normal, non-smoking individuals, levels of carbonmonoxy haemoglobin in the blood are very low. In heavy smokers, the levels may rise to up to 8-9% saturation of haemoglobin with carbon monoxide .

As will be described in more detail below, carbonmonoxy glycohaemoglobin may readily be formed by exposure of oxyhaemoglobin to carbon monoxide gas with glycosylation taking place before or after this step. Preferably, the standard reference material of the invention comprises at least 60% (w/w) carbonmonoxy glycohaemoglobin, relative to total haemoglobin, preferably at least 80%, and more preferably at least 90

In normal human subjects, the glycosylated haemoglobin content is about 4% of total. Levels may rise up to about 15-20% in diabetics. The level of glycosylation of the standard reference material of the invention may vary according to choice, but generally is selected to match natural levels.

Most preferably, the standard reference material comprises up to 50% glycosylated haemoglobin (relative to total haemoglobin) , preferably 1 to 15% glycosylated haemoglobin.

It may, according to the assay in which the standards is to be used, be desirable to provide a range of standard reference materials, with varying glycosylated haemoglobin contents.

The glycosyl moiety of the glycosylated haemoglobin fraction may comprise any of the known glycosyl moieties or related cis-diol containing moieties with which haemoglobin may react, for example sugars and sugar aldehydes such as glucose, galactose, fructose, sucrose, glycoaldehydes, etc. Glucose is however strongly preferred as this reflects more closely the native state of haemoglobin glycosylation.

The haemoglobin may be any of the haemoglobins known in animal species, but preferably it is from a mammalian species eg. human, bovine or porcine. Human haemoglobin is preferred for use in assays specific for human glycohaemoglobin. In other situations, non-human materials may be preferred. However, all forms of oxygen-transporting proteins are included, including analogous proteins such as myoglobin, and all are subsumed under the general heading "haemoglobin" as used herein. The term is also used herein to include mutated or derivatised forms of native haemoglobin proteins, for example containing amino acid sequence deletions, additions and substitutions as well as chemical modifications of individual amino acids.

The carbonmonoxy glycohaemoglobin complexes of the invention are preferably provided as standard reference materials in solution form. Whilst any appropriate medium may be used as the carrier of diluent for the complex eg. buffer or aqueous saline, advantageously the medium is serum or a medium physiologically similar to serum, for example artificial serum, defibrinogenated plasma, anticoagulated plasma, or a solution of albumin in aqueous saline or similar solution, so as to approximate most closely the native situation of blood. Plasma may be used, but is less advantageous due to the presence of fibrinogen.

It is desirable, in preparing the standard solutions to minimise the content of agents which may promote or accelerate oxidation of haemoglobin, for example metal ions, especially Cu²⁺, ions such as fluoride, azide, cyanate, thiocyanate, or imidazole .

Conveniently, the content of the carbonmonoxy glycohaemoglobin complex in the standard solution is 5 to 500 g/1, preferably 15 to 300 g/1 more preferably 30- 225 g/1.

Although this is less preferred, the complexes may also be lyophilised.

Carbonmonoxy glycohaemoglobin may readily be prepared by simple exposure to carbon monoxide gas using methods known per se, for example by passing a stream of gas over, or bubbling the gas through a haemoglobin solution, preferably contained in a dialysis bag or tube, submerged in an appropriate buffer solution, or alternatively, a suspension of intact erythrocytes. Thus for example, a sample of whole blood may be washed to remove plasma and then resuspended in appropriate medium eg. buffer or aqueous saline. Anticoagulants such as EDTA may be added. At this stage of the reaction, the haemoglobin may be glycosylated or non- glycosylated. The carbon monoxide itself may be obtained commercially or may readily be prepared for use as required, for example by decarboxylation of formic acid by H₂S0₄. It is desirable during the "carbonmonoxylation" step to exclude oxygen or air in order to minimise the risk of methaemoglobin formation. Thus, the reactions are advantageously performed under anaerobic conditions. Reagents such as dithionite may also be added which react with dissolved oxygen and thus help to promote complete CO-transformation of the haemoglobin.

For the "carbonmonoxylation" step, the haemoglobin may be present as oxy- or deoxyhaemoglobin or it may be present as a derivative thereof. Deoxyhaemoglobin may be prepared for example by adding dithionite to the haemoglobin solution or erythrocyte preparation under anaerobic conditions. Oxyhaemoglobin is however the preferred substrate for carbonmonoxylation.

For the preparation of the glycosylated carbonmonoxy haemoglobin, the haemoglobin may be subjected to a glycosylation step before or after the CO-transformation. It is however preferred to glycosylate following CO-transformation.

Glycosylation may also be performed using methods well known in the art and described in the literature for non-enzymatic glycosylation of proteins. The glycosylation step may also be performed on intact erythrocytes or on a haemoglobin solution as desired, although the use of intact erythrocytes is preferred, since formation of methaemoglobin may more easily be avoided.

Glycosylation may be achieved simply by incubating the haemoglobin or the erythrocytes in the presence of the appropriate sugar or sugar aldehyde or other glycosyl donor as required. Thus for example, a suspension of erythrocytes in a suitable buffer or medium, desirably but not necessarily already subjected to CO-transformation, may be incubated, preferably under physiological conditions, for a period of time until glycosylation has been effected. Incubation times may vary, eg. from several hours up to several days, for example from 1 hour up to 15 days, preferably up to 10 days . Suitable incubation times have been found to include for example, 12 to 24 hours or periods of 10 days. Incubation may generally be performed at any temperature, for example 0°C to 37°C but reaction times are of course faster at the higher temperatures . Incubations at from about room temperature to about 37°C have generally been found to be suitable. A temperature of about 25°C is preferred. At higher temperatures unwanted oxidation reactions etc. may take place, and such conditions are therefore best avoided. Physiologically acceptable non-nucleophilic buffers or media may be used for the incubation, eg phosphate buffer, aqueous saline etc.

The concentration of the glycosyl moiety may vary according to choice, for example depending on the level of glycosylation required, the nature of the sugar etc. Concentrations of from 0.01 to 10 M have been found to be effective, preferably 0.05 to 2 M.

Under such conditions a so-called non-reductive glycation reaction takes place. For a sugar aldehyde such as glucose, the product of such a reaction is an aldimine or Schiff ' s base which is labile. This then undergoes Amadori rearrangement into a stable ketamine adduct (in the case of glucose, 1-amino-l-deoxyfructose derivative of haemoglobin is formed) .

A faster reaction may be obtained under reducing conditions (so-called reductive non-enzymatic glycation) . In this reaction the haemoglobin or the cells are incubated with the glycosyl reagent in the presence of a reducing agent, or the reducing agent is added after some time, once the initial Schiff's base adduct has formed. In the case of the latter a mixture of products is formed, including the stable ketamine adduct, and a stable aldamine derivative (eg. 1-amino-l- deoxy-glucose) . Where the reducing agent is present from the onset, the stable aldamine product predominates. Mild reducing agents are preferred, and NaCNBH₃ or NaBH₄ have been found to give particularly good results. For rapid glycation borane-pyridine- complex and borane-dimethylamine-complex may be used advantageously.

As mentioned above, haemoglobin preparations are susceptible to oxidation and precautions should be taken during the various processing steps, including washing and incubation steps etc. to avoid or minimise such unwanted side reactions. Reagents should therefore preferably be used with the lowest possible metal ion contents, and chelating agents eg. EDTA, may beneficially be added. Autoxidation also increases at high temperature, at low pH and in the presence of high salt concentrations. It is therefore best for such conditions to be avoided, particularly during the more prolonged glycosylation steps.

It is also important to avoid the presence of ligands which may bind to haemoglobin and cause spectral shifts, for example azide, fluoride and cyanate ions etc .

Following glycosylation, further processing depends on the desired end-product etc. In the case of haemoglobin solutions, these may optionally be washed if desired, and a desired carrier or diluent eg. serum may be added.

One particular embodiment of the invention thus comprises a solution of a complex of glycosylated haemoglobin with a ligand, in serum, wherein in said complex, the ligand displaces the oxygen of oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe²⁺ therein to Fe³⁺, while not substantially changing the absorption spectrum of oxyhaemoglobin.

Preferably said serum solution comprises carbonmonoxy glycohaemoglobin.

In the case of erythrocytes, these may be washed and lysed to release the haemoglobin product which may then be processed as mentioned above. However, lysis is not an essential step and the product of the invention may be presented as a preparation of erythrocytes containing a complex of glycosylated haemoglobin with a ligand, wherein in said complex, the ligand displaces the oxygen of oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe²⁺ therein to Fe³⁺, while not substantially changing the absorption spectrum of oxyhaemoglobin. Preferably, the ligand is carbon monoxide. The erythrocytes may be suspended in an appropriate medium, for example serum or a physiologically acceptable buffer as discussed above optionally containing a detergent eg. Triton X-100^® or Tween^®.

To effect haemolysis to release the haemoglobin complexes, any standard haemolysis techniques may be used, for example lysis in hypotonic buffer, use of detergents and freezing. Such techniques are well known and widely described in the literature. Following haemolysis, the haemoglobin may be separated and recovered from the cellular debris, for example by centrifugation, washed if desired and appropriate carriers or diluents added as discussed above. At this stage, antibiotics such as ciprofloxacin, genta icin sulphate, penicillin or streptomycin may be added and the resulting hemoglobin solution filtered through a 0.22 μm tangential (cross flow) -filtration unit. Although experimentation has shown that this is not essential, chemicals to reduce methemoglobin formation, for instance thiol compounds and catalytic amounts of metal ions as described by Masukawa and Iwata, Life Sciences. 1977, 21(5) , p. 695, or enzymes like superoxide dismutase, peroxidase, catalase, could also be added.

As mentioned above, the complexes of the invention have particular utility as standard haemoglobin control reference materials. Whilst this utility is not limited to particular assays or haemoglobin reference systems, the reference materials have, as mentioned above, been found to be especially useful in the assay for glycosylated haemoglobin described in WO 90/13818.

This assay is based on separation of both glycosylated and non-glycosylated haemoglobin from a sample, followed by selective labelling of the glycosylated component using derivatives of boronic acid conjugated to a signal-forming label, generally a coloured dye. The materials required to perform this assay may conveniently be supplied in the form of a kit. Such a kit may conveniently also contain the standard glycosylated haemoglobin reference material of the present invention.

Thus, a further aspect of the present invention also provides and analytical test kit for assessment of glycosylated haemoglobin in a sample, said test kit comprising:

(a) a reagent comprising a signal-forming molecule comprising a conjugate of one or more dihydroxyboryl residues or salts thereof linked to a signal-forming label;

(b) means for the separation of haemoglobin from a sample ;

(c) a glycosylated haemoglobin standard control reference material according to the invention, as defined above; optionally in combination with one or more buffer salts or solutions.

The means for separation of the haemoglobin may conveniently comprise a selective precipitating agent, such as metallic cations, especially zinc ions, organic solvents or haemoglobin-specific antibodies as described in WO 90/13818.

The signal-forming agent is preferably a coloured dye, including advantageously the coloured dyes described in WO 92/08722. Exemplary dyes include cyanines, oxazines, thiazines, and triphenylmethines .

The invention will now be described in more detail in the following non-limiting Examples.

Example 1

Preparation of human carbonmonoxy σlvcohaemoαlobin by non-reductive αlvcation at room temperature

5 ml whole human blood using EDTA as anticoagulant is washed using 0.155 M Na Cl ( sonicated and N₂-bubbled) . Initial glycation level GHb 5% (determined by boronic acid affinity chromatography) . Washed erythrocytes lery) are CO-treated completely to transform oxyHb to the much more stable CO derivative (CO produced in the laboratory by decarboxylation of formic acid by H₂S0₄) . Transformation is followed by spectroscopy (photometric spectrum scan in the 400-650 nm range) . The ery •containing HbCO) are incubated at room temperature with 5 volumes of 0.2M glucose in 50mM phosphate buffer, pH 7.4, incubation time 10 days. After incubation the ery are washed, lysed and analysed for GHb. GHb level recorded: 10%. Methaemoglobin formation and spectrum changes are not detectable.

Example 2

Preparation of human carbonmonoxy αlγcohaemoσlobin by non-reductive σlvcation at 37°C

5 ml whole human blood using EDTA as anticoagulant is washed using 0.155 M Na Cl ( sonicated and N₂-bubbled) . Initial glycation level GHb 5% (determined by boronic acid affinity chromatography) . Washed erythrocytes

(ery) are CO-treated completely to transform oxyHb to the much more stable CO derivative (CO produced in the laboratory by decarboxylation of formic acid by H₂S0₄) . Transformation is followed by spectroscopy (photometric spectrum scan in the 400-650 nm range) . The ery

(containing HbCO) are incubated at 37°C with 5 volumes of 0.2M glucose in 50mM phosphate buffer, pH 7.4, incubation time 18 hours. After incubation the ery are washed, lysed and analysed for GHb.

GHb level recorded: 8.8%. Methaemoglobin level increases approximately by 2%.

Example 3

Preparation of human carbonmonoxy σlvcohaemoαlobin bv reductive glycation at 37°C

5 ml whole human blood using EDTA as anticoagulant is washed using 0.155 M Na Cl ( sonicated and N₂-bubbled) Initial glycation level GHb 5% (determined by boronic acid affinity chromatography) . Washed erythrocytes (ery) are CO-treated completely to transform oxyHb to the much more stable CO derivative (CO produced in the laboratory by decarboxylation of formic acid by H₂S0₄) . Transformation is followed by spectroscopy (photometric • spectrum scan in the 400-650 nm range) . The ery (containing HbCO) are incubated at 37°C with 5 volumes of 0.2M glucose, 20mM NaCNBH₃ in 50mM phosphate buffer, pH 7.4, incubation time 18 hours. After incubation the ery are washed, lysed and analysed for GHb. GHb level recorded: 39.8%. Methaemoglobin level increases approximately by 2%.

Example 4

Preparation of bovine carbonmonoxy glycohaemoglobin

(a) Lysis by detergent

200 ml of washed bovine erythrocytes were centrifuged at 1500g for 10 minutes, then CO-treated. The cells were lysed by adding Triton X100 to a concentration of 0.3% (v/v) . The haemoglobin was glycated by reductive glycation in dialysis bags (cut-off<15000) , 36 hours at 20-25° C, using at least 5x volume (compared to vol. of lysate) of glycation buffer (sonicated and N₂-bubbled) . Glycation buffer: 50mM phosphate, 0.155M NaCl, 0.5M glucose, 40 mM NaCNBH₃, pH 7.4. Finally the glycated protein was dialysed extensively against 50mM phosphate, 0.15 M NaCl, pH 7.4. Glycation level 20% (measured by using boronic acid affinity columns, Pierce Chemical Company) . (b) Lvsis by freezing

200 ml of washed bovine erythrocytes were centrifuged at 1500g for 10 minutes, then CO-treated and frozen. Lysed cells were thawed (in a refrigerator) , then glycated by reductive glycation in dialysis bags (cut-of<15000) , 36 hours at 20-25 ° C, using at least 5x volume of glycation buffer (sonicated and N₂-bubbled) . Glycation buffer: 50mM phosphate, 0.15 M NaCl, 0.5M glucose, 40 mM NaCNBH₃, pH 7.4. Finally the glycated protein was dialysed extensively against 50mM phosphate, 0.15M NaCl, pH 7.4. Glycation level 20%, measured by using boronic acid affinity columns, (Pierce Chemical Company) .

Example 5

General procedure for preparing carbonmonoxy glγcohaemoglobin standards

THE PROCESS

All work should be conducted under aseptic conditions, ie. glassware and plastics, should be autoclaved or optionally washed in 70% ethanol prior to use.

1. Collect blood.

2. Centrifugation step - remove plasma. The blood is filled in centrifugation vials of 50 ml. Centrifuge at 3000 rpm (1500 rcf) for 10 minutes at 5°C.

3. Wash the erythrocytes in 0.155 M NaCl, 1 mM EDTA (the solution must be sterile filtered, treated with ultrasound and bubbled with N₂-gas) . Repeat five times. 4. Treat the blood with CO-gas for about 45 minutes. Divide washed/treated erythrocytes into to equal volumes .

5. Depending upon whether the blood used is of human or bovine origin, add 200 mM glucose/20 mM NaCNBH₃ (for human blood) or 400 mM glucose/80 mM NaCNBH₃ (for bovine blood) in 50 mM phosphate buffer, 0.155 M NaCl, pH 7.4

(ultrasound treated, N₂-bubbled and sterile filtered) to the CO-Hb that are going to be glycated. Do this directly in the CO-container by filling it up with buffer. Put 3 x 1 ml of the solution in small vials for testing purposes.

6. Incubate the solution at 27-28°C or at room temperature for a period of between 16-72 hours e.g. 16- 24 hours. Test the glycation level by using boronic acid affinity columns (Pierce Chemical Company) . Stop at 10-15% GHb. After the glycation, wash the erythrocytes in 0.155 M NaCl, 1 mM EDTA ("sterile"/ ultrasound/N₂-bubbled) - 4 times in sterile 50 ml centrifugation vials.

7. Hemolyze both solutions by adding Triton X to 0.3% v/v. Spin down the cell fragments or ghosts.

8. Add 400 mM sodiumphosphate, 0.15 M NaCl pH 7.4 to a final concentration of 80 mM phosphate buffer (the solution must be sterile filtered, treated with ultrasound and bubbled with N₂-gas) . Antibiotics may be added ie . (penicillin 100 U/ml, streptomycin 0.1 mg/ml, amphotericin 0.25 μg/ml, gentamicin 50 μg/ml and ciprofloxacin 5 μg/ml - all together or in combinations) . The solution is made up with buffer, or optionally serum, to a concentration of not less than 120 mg/ml. Finally add serum to a concentration of haemoglobin of not less than 120 mg/ml. Mix approximately 1/3 of the two solution to make a third medium %GHb solution.

9. Filter the Hb-solution through a 0.22 μm sterile filter.

10. Treat the solution with CO-gas. Transfer the Hb- solution to appropriate glass vials and finally seal the vials under inert gas.

CENTRIFUGATIONS :

Centrifuge the blood at 2500-3000 rpm for 10 minutes in

5°C.

ASEPTIC CONDITIONS:

Work under good aseptic conditions - (LAF-bench) (glassware baked at 90-100°C for 2 hours, plastics washed with 70% alcohol) .

SOLUTIONS : Washing (step 3)

- 0.155 M NaCl, 1 mM EDTA ( "sterile"/ultrasound/N₂- bubbled)

Glycation buffer (step 6) :

- 200 mM Glucose/20 mM NaCNBH₃ in 50 mM phosphate buffer, 0.155 M NaCl, pH 7.4 ( "sterile"/ultrasound/N₂- bubbled

Washing (step 7) :

- 0.155 M NaCl, 1 mM EDTA ( "sterile"/ultrasound/N₂- bubbled) .

Claims

1. Use of a glycohaemoglobin-ligand complex as a standard reference material in haemoglobin determinations, wherein in said complex, the ligand displaces the oxygen of oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe ⁺ therein to Fe³⁺, while not substantially changing the absorption spectrum of oxyhaemoglobin.

2. Use as claimed in claim 1 wherein the haemoglobin to be determined comprises both glycosylated and non- glycosylated haemoglobin.

3. Use as claimed in claim 1 wherein the haemoglobin to be determined is glycosylated haemoglobin.

4. A complex of glycosylated haemoglobin with a ligand, for use as a standard reference material in the determination of glycosylated haemoglobin, wherein in said complex, the ligand displaces the oxygen of glycosylated oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe²⁺ therein to Fe³⁺, while not substantially changing the absorption spectrum of glycosylated oxyhaemoglobin.

5. A standard reference material for use in determinations of glycosylated haemoglobin, comprising a complex of glycosylated haemoglobin with a ligand, as defined in claim 4.

6. Use or a complex or a standard reference material as claimed in any preceding claim wherein the ligand is carbon monoxide .

7. Use or a standard reference material as claimed in claim 6 wherein at least 60% (w/w) of the total haemoglobin in the standard reference material is carbonmonoxy glycohaemoglobin.

8. Use or a standard reference material as claimed in any preceding claim wherein up to 50% of the total haemoglobin in the standard reference material is glycosylated haemoglobin.

9. Use or a complex or a standard reference material as claimed in any preceding claim wherein the glycosyl moiety of the glycosylated haemoglobin is glucose .

10. Use or a complex or a standard reference material as claimed in any preceding claim wherein the haemoglobin is mammalian haemoglobin.

11. Use or a complex or a standard reference material as claimed in claim 10 wherein the haemoglobin is human haemoglobin.

12. Use or a standard reference material as claimed in any preceding claim wherein said complex is in solution.

13. Use or a standard reference material as claimed in claim 12 wherein said solution comprises buffer, aqueous saline, plasma, serum or a medium physiologically similar to serum.

14. Use or a standard reference material as claimed in claim 12 or claim 13 wherein said complex is present at a concentration of 5 to 500 g/1.

15. Use or a standard reference material as claimed in any preceding claim wherein said complex is lyophilised.

16. A preparation of erythrocytes comprising a complex of glycosylated haemoglobin with a ligand, wherein in said complex, the ligand displaces the oxygen of oxyhaemoglobin and binds with the haem moiety of the haemoglobin so as to prevent oxidation of the Fe²⁺ therein to Fe³⁺, while not substantially changing the absorption spectrum of oxyhaemoglobin.

17. An analytical kit for the assessment of glycosylated haemoglobin in a sample, said test kit comprising:

(a) a reagent comprising a signal-forming molecule comprising a conjugate of one or more dihydroxyboryl residues or salts thereof linked to a signal-forming label;

(b) means for the separation of haemoglobin from a sample,-

(c) a glycosylated haemoglobin standard control reference material as defined in any one of claims 5 to 16.