WO2014033258A1

WO2014033258A1 - Glycated haemoglobin assay method

Info

Publication number: WO2014033258A1
Application number: PCT/EP2013/067986
Authority: WO
Inventors: Erling Sundrehagen
Original assignee: Axis-Shield Asa
Priority date: 2012-08-31
Filing date: 2013-08-30
Publication date: 2014-03-06
Also published as: GB201215571D0

Abstract

A method for determining a relative concentration of glycated haemoglobin to total haemoglobin in a blood derived sample, where the method involves; i) contacting the sample with a detectable non-immobilised selective binding agent for glycated haemoglobin; ii) subsequently contacting the sample with an immobilised binding agent for both glycated and non-glycated haemoglobin; iii) exposing the immobilised haemoglobin sample to conditions allowing for the independent detection of signals corresponding to total haemoglobin and glycated haemoglobin; and iv) relating the signals to the relative concentrations of glycated haemoglobin and total haemoglobin in the sample. A device suitable for carrying out the method of the invention, as well as the use of such a device in the inventive method are also provided.

Description

Glycated Haemoglobin Assay Method

The present invention relates to methods of assaying for glycated haemoglobin. In particular, the present invention relates to methods for fast, convenient and/or reliable assaying of glycated haemoglobin, particularly at the point-of-care or for home use.

Proteins in solution in body fluids are continually subjected to glycation processes. Glucose reacts with the proteins by non-enzymatic reactions to form glycoproteins, and in many cases the rate and/or level of glycoprotein formation is proportional to the glucose concentration in the body fluid in question. For proteins not initially synthesized as glycoproteins, the fraction of a protein present in glycated form is therefore a function of the life-time of the protein in the organism and the glucose concentration to which the protein has been exposed.

Unlike measurements of glucose concentrations in blood, plasma or urine, which only give information about the glucose concentration at the time of sampling, the amount of a protein present in glycated form gives an indication of the organism's degree of control of glucose concentration over longer periods of time.

Erythrocytes (red blood cells) have a mean lifetime of approximately 120 days and contain large amounts of haemoglobin. The fraction of glycated haemoglobin within an erythrocyte thus provides a good indication of the degree of control that a patient with diabetes mellitus has over their blood glucose levels. The amount of glycated haemoglobin within an erythrocyte is a function of the average glucose concentration in the blood of the patient in the weeks prior to a blood sample being taken for analysis.

In US-A-5242842 it was proposed to assay for glycated haemoglobin using as a reporter-labelled reagent a boronic acid conjugate, e.g. a conjugate of N-(resorufin-4- carbonyl)piperidine-4-carboxylic acid-N-hydroxysuccinimide ester (RESOS) or fluorescein isothiocyanate (FITC) with aminophenyl boronic acid. The anionic B(OH)₃ ^" component of the reagent binds to the cis-diol groups of the glycated haemoglobin so labelling the glycated haemoglobin with the reporter label (e.g.

RESOS or FITC). In US-A-5631364, a group of particularly effective reporter-labelled boronic acid conjugates were described, including the xylene-cyanole-phenyl boronic acid conjugate referred to therein as XC-DAPOL-CPBA. The chromophore label gives this conjugate an absorption maximum at 616 nm (i.e. it is blue in colour) and the proportion of haemoglobin in a sample that is glycated can be determined by measuring sample absorbance at 616 nm and at 415 nm, where haemoglobin has an absorption maximum.

A diagnostic assay for glycated (i.e. glycosylated) haemoglobin using the above system is available commercially from Axis-Shield Pic as the NycoCard® HbA1 c test. In operation of this test a slightly alkaline aqueous reagent solution is mixed with a blood sample and the mixture is applied to a porous membrane which is rinsed before light reflectance by the membrane (i.e. by the haemoglobin trapped on the membrane) is determined. The reagent solution comprises a surfactant (to lyse the erythrocytes and release haemoglobin), XC-DAPOL-CPBA to bind to glycated haemoglobin and zinc ions to precipitate the haemoglobin. The precipitated haemoglobin, both glycated and non-glycated is caught by the membrane while other glycated proteins and excess boronic acid conjugate are washed off the membrane by the rinsing step.

A further refinement of this system was described in US 7,374,943 wherein is described an improved arrangement of reagents such that highly effective assaying of glycated haemoglobin is combined with optimised storage stability.

The above described methods are highly effective for the accurate assessment of the proportion of haemoglobin which is glycated within a blood sample. However, all of these methods are primarily conducted in bulk solution and require a sequence of pipetting, measuring, mixing and separation steps. Although these are

straightforward for trained analytical staff or specialised automated apparatus, such methods are not easily adapted to either automated or manual point-of-care methods and cannot be undertaken by patients in home-testing situations.

In particular, the current most accurate and reliable methods for assay of glycated haemoglobin in a blood derived sample measure total haemoglobin and glycated haemoglobin in the same sample. However these methods require use of accurate pipetting for both blood sample measurement and washing steps. Furthermore, the precipitation of haemoglobin (both glycated and non-glycated) is conducted in a solution step separate from the separation vessel. Thus solution measuring, handling and transfer steps are required in these methods which are not easily adapted to point-or-care automation or use by personnel not trained in analytical methods.

It would thus be a considerable advantage to provide a method for the accurate measurement of glycated haemoglobin in a blood derived sample wherein such a method could be conducted without metering steps for the sample and/or any additional reagents and/or without use of multiple solution vessels, such as separate vessels for precipitation and separation.

The present inventors have now established that by careful choice of reagents, fluid flow and regions of immobilised and non-immobilised reagents, a method may be provided which can accurately assay for glycated haemoglobin in a blood sample without requiring accurate metering steps and/or without the need for precipitation in a vessel separated from the binding and separation vessel.

In a first embodiment, the present invention thus provides a method for determination of a relative concentration of glycated haemoglobin to total haemoglobin in a blood derived sample, said method comprising; i) contacting a sample with a detectable non-immobilised selective binding agent for glycated haemoglobin, optionally within an absorbent substrate, whereby to generate a detectable glycated haemoglobin; ii) subsequently contacting the sample with an immobilised binding agent for both glycated and non-glycated haemoglobin within said absorbent substrate whereby to generate an immobilised haemoglobin sample, iii) exposing the immobilised haemoglobin sample to conditions allowing for the independent detection of a first signal corresponding to total haemoglobin and second signal corresponding to detectable glycated haemoglobin either sequentially or simultaneously; and iv) relating the intensity of the first signal and second signal to the relative concentrations of glycated haemoglobin and total haemoglobin in the sample. In a typical embodiment of the invention, the blood derived sample is applied to a sample application region of an absorbent substrate.

In one embodiment, the blood derived sample may be pre-treated with at least one non-immobilised selective binding agent for glycated haemoglobin prior to application to the sample application region of the absorbent substrate.

Alternatively, said blood derived sample may be applied to the sample application region before contact with a non-immobilised selective binding agent for glycated haemoglobin and may contact the non-immobilised selective binding agent within the absorbent substrate.

In all embodiments, the blood-derived sample may be contacted with at least one pre-treatment reagent and/or diluent prior to contact with a non-immobilised selective binding agent for glycated haemoglobin. A pre-treatment reagent will typically include at least one buffer solution. Since a blood derived sample contains erythrocytes which in turn contain haemoglobins, the pre-treatment reagent may also include at least one lysing agent which serves to lyse the blood sample and thereby release haemoglobin (both glycated and non-glycated haemoglobin) from

erythrocytes in the sample. No precipitation of haemoglobin will be performed by any pre-treatment reagent and thus no binding agent for both glycated and non-glycated haemoglobin will be comprised in any pre-treatment reagent.

Preferred non-immobilised selective binding agents for glycated haemoglobin comprise at least one a boronic acid conjugate, particularly a photometrically detectable boronic acid conjugate as described herein. Such boronic acid conjugates are applicable to all aspects of the present invention.

Preferred immobilised binding agents for both glycated and non-glycated

haemoglobin for use in all aspects of the present invention include zinc and/or zinc- containing compounds as described herein.

Generally, the detection method for first and second signals will comprise irradiation with at least one wavelength of light whereby to cause at least one absorption, reflection, scattering and/or fluorescence from the detectable ligand and at least one other absorption, reflection, scattering and/or fluorescence from all haemoglobin (glycated and non-glycated haemoglobin) in the immobilised haemoglobin sample. In a further embodiment, the present invention provides a device for the assessment of the relative concentration of glycated haemoglobin to total haemoglobin in a blood derived sample, the device comprising: i) an absorbent substrate having a first major surface;

ii) a washing region at, or in fluid communication with, a proximal end of the absorbent substrate;

iii) a sink region at or in fluid communication with a distal end of the absorbent substrate;

iv) a sample application region on the first major surface disposed between the washing region and sink region;

v) a capture region of absorbent substrate disposed between the sample application region and sink region;

vi) optionally a binding region of absorbent substrate disposed between the sample application region and capture region; wherein the capture region contains at least one binding agent for both glycated and non-glycated haemoglobin immobilised to the absorbent substrate.

A device of the invention may optionally include a haemolysis region. This will generally be between the sample application region and the binding region. Such a haemolysis region will typically contain at least one haemolysing reagent.

In one highly preferred embodiment of the methods of the invention, such methods may be conducted by use of a device as described herein. Such a method may comprise, for example, application of a (optionally pre-treated) blood derived sample to the sample application region of the device of the invention. The method may further comprise detection of glycated haemoglobin and total haemoglobin on or within the device. Such detection will generally take place at the capture region.

Correspondingly, the devices of the present invention may be for or suitable for use in any of the methods of the invention as described herein.

In all embodiments, after application of the blood-derived sample to the sample application region of the absorbent substrate, at least one washing solution may be added to the sample application region or preferably to a washing region of the absorbent substrate (or to a washing region in fluid communication with the absorbent substrate, such as a fluid reservoir). The washing solution will typically be an aqueous buffer solution and will comprise no selective binding agent for glycated haemoglobin or binding agent for both glycated and non-glycated haemoglobin. The washing solution serves primarily to transport the blood derived sample through the absorbent substrate so as to contact that sample with the immobilised binding agent for both glycated and non-glycated haemoglobin and/or to remove any unbound detectable binding agent. A sink region will typically be provided on or in fluid communication with the absorbent substrate such that excess washing solution and any materials not retained by the immobilised ligand may be absorbed.

In one preferred embodiment, the blood derived sample and/or any washing solutions may be applied without accurate metering. That is to say, the samples need not be applied with accurately calibrated pipettes. Generally, an "unmetered" sample or volume as indicated herein will conform to an approximate measurement, such as being between 0.25 and 4 times a nominal volume. An "unmetered" sample or volume as indicated herein will not typically be measured to an accuracy of greater than ± 20%. This allows for samples or reagents to be applied, for example, by drops from a supplied disposable pipette. Thus it is not necessary to use calibrated equipment or such careful techniques in such application when compared with prior art assay systems.

In one embodiment, the relative concentrations of glycated and non-glycated haemoglobin determined by the methods and/or determined by utilising the devices of the invention will differ by no more than 10%, preferably no more than 5%, more preferably no more than 2%, when the volume of fluid added varies by up to 10%, preferably up to 20%, more preferably up to 50%. The fluid may be e.g. washing fluid(s) and/or sample fluid. The same may additionally or alternatively apply to the variation of the ratio of the first and second signals with fluid volume in all

embodiments.

The level of glycated haemoglobin in the sample is measured by assessment of the amount of detectable ligand captured on the immobilised binder by means of binding to glycated haemoglobin. Thus it is desirable to remove any excess (unbound) detectable ligand prior to detection of a first signal. This is typically achieved by means of at least one washing step. The methods of the invention may thus preferably include at least one washing step between the steps of contacting the sample with a detectable non-immobilised selective binding agent for glycated haemoglobin and exposing the immobilised haemoglobin to conditions allowing for the independent detection of a first signal corresponding to the total amount of haemoglobin and a second signal corresponding to the amount of glycated haemoglobin present in the sample, and preferably between steps of contacting the sample with an immobilised binding agent for both glycated and non-glycated haemoglobin and exposing the immobilised haemoglobin to conditions allowing for the independent detection of a first signal corresponding to the total amount of haemoglobin and a second signal corresponding to the amount of glycated haemoglobin present in the sample. Such a step may comprise application of a washing solution, such as a buffer, to a washing region of the absorbent substrate such that the solution flows past at least the immobilised ligand (such as in a capture regions).

As used herein, the term "fluid communication" is used to indicate that two components, regions etc may each contain fluid and that such fluid is free to flow between the two parts. Such flow will typically be gravity flow and/or capillary flow. The flow may alternatively be pumped flow.

Preferred materials for formation of the absorbent substrates for use in all aspects of the present invention may be any substrate materials allowing capillary flow of an aqueous fluid therethrough. These include polyester pads, glass-fibre pads (e.g. of borosilicate glass fibre), and cellulose membranes, such as regenerated cellulose membranes or nitrocellulose membranes. Membranes will typically have pore sizes in the region 0.01 to 1000 μηι, preferably 0.5 to 100 μηι. The absorbent substrate as described herein may be formed from a single material but will typically comprise two or more materials in fluid communication. In such an embodiment any one or more of the application, washing, binding, haemolysis and/or sink regions may comprise at least one section of material in fluid communication with the sections of material constituting the neighbouring region(s). In this way, the materials for each region can be selected to best fit the specific purpose of that region. Thus, for example, the application, washing and binding regions may be formed from at least one cellulose membrane and the binding region may be formed from a polyester pad. Thus, in one embodiment, the absorbent substrate is formed from at least two materials in fluid communication. Any reagents present in a region of the absorbent substrate, such as a non- immobilised selective binding agent for glycated haemoglobin, an immobilised binding agent for both glycated and non-glycated haemoglobin and/or a haemolysing reagent, will typically be provided in dry form.

Wherein more than one reagent is provided in more than one region of the absorbent substrate, such as in at least two of a binding region, a capture region and a haemolysis region, then such regions will typically be non-overlapping regions of the absorbent substrate. Such regions may be adjacent or may optionally be separated by a region in which no reagent or binder is present.

The detectable non-immobilised selective binding agent for glycated haemoglobin as described herein in all aspects and embodiments is preferably a boronic acid conjugate. The boronic acid conjugate may for example be any of the chromophore- boronic acid conjugates described in US-A-5242842, US-A-5631364 and US-A- 5739318; however XC-DAPOL-CPBA is preferred. US-A-5242842, US-A-5631364 and US-A-5739318, particularly with regard to the boronic acid conjugates described therein, are hereby incorporated herein by reference.

The detectable non-immobilised selective binding agent for glycated haemoglobin as described herein may be contacted with the blood derived sample by means of capillary flow through the absorbent substrate, particularly when the non-immobilised selective binding agent for glycated haemoglobin is disposed within the absorbent substrate. Alternatively, the detectable non-immobilised selective binding agent for glycated haemoglobin as described herein may be contacted with the blood derived sample by means of a pre-treatment step. The sample comprising bound glycated haemoglobin (detectable glycated haemoglobin) will then generally be applied to the absorbent substrate.

Lysing reagents as used in any aspect or embodiment of the present invention may be any surfactant capable of lysing erythrocytes, e.g. deoxycholates, saponins, Tritons and Tweens (polysorbates). In one embodiment, non-ionic surfactants such as Tritons, in particular Triton X-100 (polyethylene glycol p-(1 ,1 ,3,3-tetramethylbutyl)- phenyl ether) are preferred. Alternatively, saponins may be used very effectively for this purpose. The zinc or zinc compounds referred to in all aspects and embodiments of the present invention will typically be inorganic zinc salts. Zinc chloride is preferred. Such zinc salts may be immobilised to the absorbent substrate by known methods.

The washing reagent is conveniently an aqueous buffer, e.g. Hepes. A particularly suitable washing reagent comprises 50 mM morpholine, 200 mM NaCI, 0.5% w/v Triton-X-100, 0.1 % w/v glycerol, 0.05% w/v NaN3, pH 9.1.

Generally, the detection method for the first and second signals will comprise irradiation with at least one wavelength of light. In one embodiment, detection comprises irradiation with a first wavelength and measurement of a first reflectance as a first signal and irradiation with a second wavelength and measurement of a second reflectance as a second signal. The first wavelength may be about 630 ± 50 nm (e.g. 625 to 640nm). The second wavelength may be about 475 ± 50 nm (e.g. 460 to 490nm).

The fist and second detected signals will be related to the relative concentrations of glycated haemoglobin and total haemoglobin. Such a relation may be calculated by means of a simple ratio, accounting for the relative intensities of the signals at equal concentration. Alternatively, such signals may be related to the relative

concentrations of glycated haemoglobin and total haemoglobin by means of a standard table or curve. Such a standard table or curve may be pre-calculated or may be measured by means of standard solutions. The generation and use of standard curves of this type is described in detail in US 7,374,943, which is hereby incorporated by reference. Corresponding methods may evidently be used in the present invention.

The invention will now be farther illustrated by reference to the following non-limiting examples and the attached figures, in which:

Figure 1 shows a first embodiment of the device of the invention comprising application region A, washing region B, capture region Zn and sink region S.

Figure 2 shows a second embodiment of the device of the invention comprising an application region A, washing region B, binding region C, capture region Zn and sink region S. Figure 3 shows a third embodiment of the device of the invention comprising an application region A, washing region B, haemolysis region H, binding region C, capture region Zn and sink region S.

Examples: Materials:

Reagents used in the first embodiments of the invention: Zn- solution

50 mmol ZnCI₂ in purified water. HbA1 c buffer:

Glycinamide hydrochloride 53.8 mM/l

NaCI 215 mM/l.

Triton X 100 0.12 % w/w

Na-Azide 0.053 % w/w.

Albumin, bovine. 1.07 mg/ml.

pH = 7.9.

HbA1 c washing solution:

Morpholineum 50 mM 4,36 g/l

NaCI 200 mM 12 gram/I.

Triton X-100 0.1 % w/w = 1 gram per litre = 10 ml 10% l0sning/l.

Glycerol 0.1 % v/w = 1 ml/I.

Na-Azide 0.05 % = 500 mg/l.

5 M HCI till pH = 9.3.

Blue dye solution:

7.2 mg XC-DAPOL-CPBA is dissolved in 3.2 grams of DMSO, and 32 ml of HbA1 c buffer is added and mixed.

Preparation of filter materials:

Sartobind 94IDA-42-001 was used for binding of zinc ions. Sartobind membrane adsorbers are made from regenerated cellulose and have been stabilized for high chemical stability. They are derivatized with conventional chelating functional groups to a metal chelate adsorber for protein and peptide purification with high selectivity. The large pores minimize the size exclusion effect.

The supplier Sartorius Ltd. provides the following specifications:

Pore size 3-5 μηι

Reusable Yes

Delivery 1 sheet, 1 manual

Membrane type Metal chelate membrane

Ligand Iminodiacetic acid

Ligand density > 5 με /αη²

Dimensions 21 x 29,7 cm

Adsorption area 624 cm²

Membrane thickness 275 μιη

Binding capacity 100 μ§/αη²

Flow rate at 1 bar > 30 ml/min

Reinforcement

Polyester

material

pH stability 1 - 12 (short term), 3 - 9 (long term)

Chemical Stable in all common chromatography buffers exept peroxide compatibility and other oxidizing or reative reagents

Reference protein Polyhistidine tagged protein

Recommended

Ni²+, Co²+, Cu²+ or Zn²+

chelating ions

Membrane Stabilised reinforced cellulose

Immobilisation

Immobilisation of Zn on areas an aqueous solution of ZnCI₂ on the Sartobind 94IDA-42-001 membrane was performed by pipetting a solution of 50 mM ZnCI₂ in purified water on to the membrane and drying it at room temperature for 24 hours. Excess of Zn ions were removed by washing the Sartobind 94IDA-42-001 membrane by purified water, followed by a further period of drying at room temperature for 24 hours.

Porous containers for dried boronic acid dye conjugate XC-DAPOL-CPBA.

We have worked with conjugate pads from Pall, both polyester, cellulose and borosilicate binders. The polyester pads and boroslicate pads worked best, but all were able lo release XC-DAPOL-CPBA when HbA1c buffer was passed through the membrane.

Conjugate Base Material Typical Typical Typical Basis Tensile

Pad Type Thickness Thickness Weight (g/m²) Strength (lbs in

(mils (μιτι) MD)

6613 Spun bonded polyester 16.5 419.1 98.3 31.4

(binder free)

6615 Spun bonded polyester 20.0 508.0 135.6 105.0

(binder free)

8301 Cellulose and synthetic 14.0-17.5 355.6-444.5 50.0 10.3 blend with PVA binder

8964 Borosilicate glass fiber 14.0-20.0 355.6-508.0 75.1 0.9 with PVA binder

8975 Borosilicate glass fiber 9.0-13.0 228.6-330.2 49.1 24.5 ith PVA binder

Conjugate pads were prepared by soaking a pad in the blue dye solution (see above). Excess dye solution was removed by placing the conjugate pad an blotting paper, and drying at room temperature for 24 hours.

Absorbent pads "sink" for downstream absorbance of solutions.

We have used absorbent pads from Pall made from cellulose.

Part Number Description

S70006 Cellulose Absorbent Pad 1 1 1 , ί 3" x 10" sheet

S70007 Cellulose Absorbent Pad 113, ί 3" x 10" sheet

S70008 Cellulose Absorbent Pad 133, ί 3" x 10" sheet

S70009 Cellulose Absorbent Pad 165, ί 3" x 10" sheet

S70010 Cellulose Absorbent Pad 197, ί 3" x 10" sheet

The choice of material for absorbent pads is believed to be non-critical. Preparation of lateral flow strips.

We have worked with three different exemplifications of a lateral flow test strip. The strips are 10 mm wide and 60 mm long. All three have a sink at the downstream end, example no. 2 and 3 also have a conjugate pad. The rest of all three test strip examples are made from Sartobind 94IDA-42-001 membrane, with an area of Zn- ions immobilized, as described above.

Example Assays:

Assay Example 1 :

In a separate vial, a small drop (approximately 15 μΙ) of whole blood was mixed with 300 ul of blue dye solution, supplied in a pre-measured vial, typically an Eppendorf vial (composition of the blue dye solution as described above). During mixing of blood sample with reagents, erythrocytes were lysed to release haemoglobin, (both glycated and non-glycated). The blue dye XC-DAPOL-CPBA bound only to glycated haemoglobin.)

A small drop of the reaction mixture was transferred to area A on the strip of example 1 (See Figure 1). Thereafter 5 small drops of haemoglobin washing solution

(composition: See above) was slowly transferred to area B in the strip of example 1. The liquids migrated towards the absorbance pad /sink (S), and excess liquid was absorbed into the absorbance pad.

When the liquids passed through the area where Zn had been immobilised, the haemoglobin molecules (glycated and non-glycated) were captured on the Zn (with and without XC-DAPOL-CPBA bound), and all other constituents of the liquids passed through the Zn modified area.

Immediately thereafter, the result was read by reading with a NycoCard reader at the reflectance of light at wavelengths of 632 and 476 nm respectively. The ratio of the reflectance signal was correlated to a standard curve obtained by samples of whole blood (similar to what was described in US 7,374,943).

Assay Example 2:

In a separate vial, a small drop (approximately 15 μΙ) of whole blood was mixed with 300 ul of HbA1 c buffer, supplied in a pre-measured vial, typically an Eppendorf vial. (During the mixing, the erythrocytes of the blood samples were lysed, and haemoglobin (both glycated and non-glycated) was released from erythrocytes into the solution.

A small drop of the assay mixture is transferred to area A on the strip example 2 (see Figure 2).

Thereafter 5 small drops of HbA1c buffer (composition: See above) was slowly transferred to area B in the strip of example 2.

The liquids migrated through the conjugate pad, where dried XC-DAPOL-CPBA was liberated and allowed to react with the sample constituents. The sample mixture then migrated further towards absorbance pad (S), and any excess liquid was taken up in to the absorbance pad.

When the liquids passed through the area where Zn had been immobilised, haemoglobin molecules (glycated and non-glycated) were captured on the Zn (with and without XC-DAPOL-CPBA bound), all other constituents of the liquid sample passed through the Zn treated area.

Immediately thereafter, the result was determined by reading with a NycoCard reader to measure the reflectance of light having a wavelength 632 and 476

nmrespectiveely. The ratio of the reflectance signal was correlated to a standard curve obtained by samples of whole blood (similar to what was described in US 7,374,943).

Assay Example 3:

1 ul whole blood was transferred to area A on test trip example 3 (See Figure 2), using a glass capillary which was brought in contact with the test strip at this area. Thereafter 5 small drops of HbA1c buffer (composition: See above) was slowly transferred to area B in the said strip example 3. By passing through area A, the HbA1 c buffer dissolved and lysed the blood sample, and the liquids migrated through the conjugate pad where dried XC-DAPOL-CPBA was liberated and allowed to react with the sample constituents. Sample mixture thenmigratedfurther towards the absorbance pad (S), and any excess liquid was absorbed in to the absorbance pad. When the liquids passed through the area where Zn had been immobilised, haemoglobin molecules (glycated and non-glycated) were captured (with and without XC-DAPOL-CPBA bound), and other constituents of the liquid sample passed through the Zn modified area.

Immediately thereafter, the result was determined by reading with a NycoCard reader to measure the reflectance of light having a wavelength 632 and 476 nm

respectiveely. The ratio of the reflectance signal was correlated to a standard curve obtained by samples of whole blood (similar to what was described in US 7,374,943).

Claims

Claims:

1) A method for determination of a relative concentration of glycated

haemoglobin to total haemoglobin in a blood derived sample, said method comprising; i) contacting a said sample with a detectable non-immobilised selective binding agent for glycated haemoglobin, optionally within an absorbent substrate, whereby to generate a detectable glycated haemoglobin; ii) subsequently contacting said sample with an immobilised binding agent for both glycated and non-glycated haemoglobin within said absorbent substrate whereby to generate an immobilised haemoglobin sample, iii) exposing said immobilised haemoglobin sample to conditions allowing for the independent detection of a first signal corresponding to said total haemoglobin and second signal corresponding to said detectable glycated haemoglobin either sequentially or simultaneously; and iv) relating the said first signal and said second signal to the relative concentrations of glycated haemoglobin and total haemoglobin in said sample.

2) The method of claim 1 wherein said blood derived sample is an unmetered sample.

3) The method of any preceding claim wherein between steps ii) and iii) any excess detectable non-immobilised selective binding agent for glycated haemoglobin is at least partially removed from said immobilised sample.

4) The method of any preceding claim wherein said non-immobilised selective binding agent for glycated haemoglobin is in the form of a dry reagent.

5) The method of any preceding claim wherein said non-immobilised selective binding agent for glycated haemoglobin comprises a boronic acid derivative. 6) The method of claim 5 wherein said boronic acid derivative is photometrically detectable.

7) The method of any preceding claim wherein said immobilised binding agent for both glycated and non-glycated haemoglobin is in the form of a dry reagent.

8) The method of any preceding claim wherein said sample is contacted with said non-immobilised selective binding agent for glycated haemoglobin by means of a pre-treatment step prior to application to an absorbent substrate.

9) The method of any preceding claim wherein said sample is contacted with said non-immobilised selective binding agent for glycated haemoglobin by means of capillary flow of said sample through an absorbent substrate.

10) The method of any preceding claim wherein said sample is contacted with said immobilised binding agent for both glycated and non-glycated haemoglobin by means of capillary flow of said sample through an absorbent substrate.

1 1 ) The method of any of claims 3 to 10 wherein said excess detectable non- immobilised selective binding agent for glycated haemoglobin is at least partially removed from said immobilised sample by means of capillary flow of a fluid past said immobilised sample.

12) The method of any preceding claim wherein said immobilised binding agent for both glycated and non-glycated haemoglobin is immobilised on said absorbent substrate between a washing region and a sink region.

13) The method of any preceding claim wherein said immobilised binding agent for both glycated and non-glycated haemoglobin is zinc or a zinc compound.

14) The method of any preceding claim wherein said sample is haemolysed by contacting said sample with at least one haemolysing agent prior to step iii).

15) The method of claim 14 wherein said sample is contacted with said at least one haemolysing agent by means of capillary flow through an absorbent substrate. 16) The method of any preceding claim wherein said conditions allowing for the independent detection of a first signal corresponding to said total haemoglobin and second signal corresponding to said detectable glycated haemoglobin comprise irradiation with at least one wavelength of light.

17) The method of any preceding claim wherein said first and second signals are optical signals.

18) The method of any of claims 16 or 17 wherein said first and second signals are independently generated by absorption, reflection, scattering or fluorescence induced by said irradiation.

19) The method of any preceding claim wherein said first and second signals are optical signals at or corresponding to first and second wavelengths of light respectively wherein said first and second wavelengths are different.

20) A device for the assessment of the relative concentration of glycated haemoglobin to total haemoglobin in a blood derived sample, said device comprising: i) an absorbent substrate having a first major surface;

ii) a washing region at, or in fluid communication with, a proximal end of said absorbent substrate;

iii) a sink region at or in fluid communication with a distal end of said absorbent substrate;

iv) a sample application region on said first major surface disposed between said washing region and said sink region;

v) a capture region of said absorbent substrate disposed between said sample application region and said sink region;

vi) optionally a binding region of said absorbent substrate disposed between said sample application region and said capture region; wherein said capture region contains at least one binding agent for both glycated and non-glycated haemoglobin immobilised to said absorbent substrate.

21) A device as claimed in claim 20 wherein said immobilised binding agent for both glycated and non-glycated haemoglobin comprises zinc or at least one zinc compound. 22) A device as claimed in claim 20 or claim 21 wherein said binding region is present and comprises at least one non-immobilised specific binding ligand for glycated haemoglobin.

23) A device as claimed in claim 22 wherein said non-immobilised specific binding ligand for glycated haemoglobin comprises at least one detectable boronic acid derivative.

24) A device as claimed in any of claims 20 to 23 further comprising a haemolysis region of said absorbent substrate disposed between said sample application region and said binding region wherein said haemolysis region comprises at least one haemolysing agent.

25) A method as claimed in any of claims 1 to 19 comprising applying a blood derived sample to the sample application region of a device as claimed in any of claims 20 to 24.

26) A method as claimed in claim 25 further comprising applying at least one washing solution to the washing region of a device as claimed in any of claims 20 to 24.

27) A method as claimed in claim 25 further comprising detecting said first and second signals optically from the capture region of a device as claimed in any of claims 20 to 24.