WO1998058077A1

WO1998058077A1 - Reagent mixture

Info

Publication number: WO1998058077A1
Application number: PCT/GB1998/001744
Authority: WO
Inventors: Murdo Mcphee Black; Jeremy Leonard; Stephanie Ramsey
Original assignee: Hypoguard Limited
Priority date: 1997-06-17
Filing date: 1998-06-15
Publication date: 1998-12-23
Also published as: AU7927098A; GB2326476A; GB9712484D0

Abstract

A reagent mixture for testing for the presence of a component in a biological fluid sample applied thereto, comprises a polymeric binder material, a benzidine chromogen, a catalyst system which will interact with the component to be tested so as to cause a colour change in the chromogen, and a soluble or dispersible sulphonate buffer. Another aspect of the invention provides a reagent mixture which has a binder and a buffer in a weight ratio of from 1:5 to 5:1. The invention also provides coating or impregnating fluid compositions containing the reagent mixtures, a porous membrane which contains the reagent mixtures, a method of making the membrane, and a fluid test device which incorporates the membrane.

Description

REAGENT MIXTURE

BACKGROUND OF THE INVENTION

The present invention relates to a reagent mixture for testing blood or other biological fluids, to a support medium which carries the reagent mixture, and to a test strip which carries the reagent mixture. The invention also provides a method of producing the support membrane and a solution for use in the method.

In a known type of test strip, for testing blood for the presence of glucose or other materials, a blood sample is applied to one side of a translucent porous membrane carrying a reagent mixture within the pores so that plasma of the blood flows into the pores and reacts with the reagent to develop a colour which is then observed from the other side of the membrane as it develops . Such a technique will be referred to as a back reading technique.

European Patent 0 591 315 discloses a test strip for back reading which comprises a porous membrane mounted on a support . At least part of the pores of the membrane has been blinded by a material capable of forming a solid, gel, or matrix form, whereby fragments of the walls of cells in a fluid sample applied thereto are substantially prevented from passing through the pores of the membrane, whereas the component to be tested is allowed to pass therethrough. This development helps prevent cell debris from affecting the colour observed. A typical reagent formulation comprises a hydrolysed gelatine binder, a phosphate buffer, a benzidine chromogen and one or more enzymes which react with the analyte to be tested to produce a characteristic colour change in the chromogen. Benzidine chromogens, for example o-tolidine, are desirable because they produce strong colours, and different concentrations of analyte produce different gradations of colour so that analyte concentration can be assessed by comparing the generated colour with a calibrated colour chart.

A problem with known benzidine systems is that the colour produced is not stable, but changes with time, because of the transformation of the coloured species produced initially by the chromogen into a second species of a different colour. A finite time is also required for the initial colour change to reach its end point, and this time tends to be increased by the presence of a high level of binder which acts as the blinding material . The user therefore needs to check the colour change within a limited time period after the end point has been reached, and before the developed colour changes too much. If the concentration of analyte (for example, glucose) is too high, an end point may not be reached at all. It is therefore desirable to have a benzidine-containing reagent mixture which gives a stable end-point colour over several minutes. It is also desirable for an end point to be reached at a range of analyte concentrations.

Another problem with known benzidine systems is that solutions of these systems, which are used to impregnate or coat suitable membrane or other substrates, tend to be unstable. The coating formulations are typically aqueous solutions with a co-solvent such as methoxyethanol . Precipitation from solution occurs, often within a few minutes, so such solutions have a short shelf life. SUMMARY OF THE INVENTION

According to an aspect of the present invention there is provided a reagent mixture for testing for the presence of a component in a biological fluid sample applied thereto, the reagent mixture comprising a polymeric binder material, a benzidine chromogen, a catalyst system which will interact with the component to be tested so as to cause a colour change in the chromogen, and a sulphonate buffer.

We have surprisingly found that by using a sulphonate buffer instead of a phosphate or citrate buffer, the reagent mixture can reach an end point at a wide range of component concentrations, and the developed colour is stable for longer. In certain cases, the end point is also reached more quickly. Moreover, the reagent mixture forms a stable solution which can be used to impregnate a porous membrane for longer periods without precipitation.

The term "benzidine chromogen" is used herein to refer to benzidine and substituted benzidines, for example, o- tolidine, and salts thereof.

The term "sulphonate buffer" is used herein to refer to a sulphonic acid or salt thereof, which tends to maintain a constant pH in solution. The sulphonate buffer preferably tends to maintain an aqueous solution pH of from 5.5 to 8.

Preferably an amine, particularly a tertiary amine, is associated with the sulphonate buffer. In a particularly preferred embodiment the sulphonate buffer has amine functionality, particularly tertiary amine functionality. Preferred sulphonate buffers are 2- [N-morpholino] ethane sulphonic acid (MES) and 2- [bis (2- hydroxyethyl) amino] ethane sulphonic acid (BES). Both materials are sold by Sigma. MES has a useful pH range of 5.5 to 6.7; BES has a useful pH range of 6.4 to 7.8. MES is particularly preferred.

The sulphonate material preferably comprises from 10% to 75% by weight of the reagent mixture. It is particularly preferred that the sulphonate material comprises from 40% to 65% by weight of the reagent mixture.

Suitable binders include hydrolysed gelatine and carboxymethylcellulose (CMC) , hydroxyethyl cellulose, hydroxypropyl cellulose and cellulose acetate.

Any suitable catalyst system may be used. Examples of known suitable catalyst systems are glucose oxidase and a peroxidase, for testing for glucose; and cholesterol esterase, cholesterol oxidase and a peroxidase, for testing for cholesterol .

To make a membrane for a back-read test strip, the reagent mixture components are prepared in a fluid composition which is applied to an initially porous membrane, whereby the fluid penetrates the pores of the membrane; and allowing the composition to solidify so as to form a solid within the pores which substantially completely occupies at least part of the length of the bores of the pores.

A preferred porous membrane is poly (ether sulphone) (PES) . The pore size is preferably in the range 0.1 to 0.8 μ , notably 0.2 μ.

To increase the speed at which a reagent reaches its end point, it is desirable to reduce the level or concentration of binder material which blinds the pore membrane in a back-read test strip. However, reducing the binder level too much allows lysis of blood cells and this affects the perceived colour which develops.

We have surprisingly found that the binder concentration may be reduced without significant lysis of blood occurring, providing that the ionic strength of the reagent mixture is sufficiently high.

Accordingly, a second aspect of the invention provides a reagent mixture for testing for the presence of a component in a biological fluid sample applied thereto, the reagent mixture comprising a polymeric binder material, a chromogen, a catalyst system which will interact with the component to be tested so as to cause a colour change in the chromogen, and a buffer, characterised in that the weight ratio of buffer to binder is in the range 1:5 to 5:1. It is particularly preferred that the weight ratio is the range 1:1 to 2.5:1.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described, by way of example, with reference to the following drawings in which:

Figure 1 is a graph of % reflectance v time for a known reagent mixture with a phosphate buffer, with different concentrations of glucose;

Figure 2 is a graph of % reflectance v time for a known reagent mixture with phosphate buffer and a reagent mixture in accordance with an aspect of the present invention;

Figure 3 is a graph of % reflectance v time for another reagent mixture in accordance with an aspect of the present invention, with different blood glucose concentrations; and

Figure 4 is a graph of % reflectance v buffer mass for a test mixture for use in formulating a reagent mixture in accordance with another aspect of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All reagent mixtures tested were contained in the pores of a Supor 200 PES membrane supplied by Gelman. Supor 200 is a trade mark. The membrane is 145 μ thick, and has a pore diameter of 0.2 μ and a water flow rate of 15 to 25 seconds/100 ml/9.62 cm² at 24 in Hg pressure.

The initially porous membrane was impregnated by a solution of the reagent mixture and then allowed to dry for two to three minutes at 85°C and high air velocity.

The impregnated membrane was then cut or punched to form discs which were mounted over a circular aperture in a plastics support member, in a manner well known to those skilled in the art. The diameter of the aperture was 5.5 mm, and the void volume in a similar area of uncoated membrane is calculated to be about 2 μl .

Blood or other biological fluid to be tested was applied to the top surface of the test strip, over the aperture, and colour which developed in the membrane was observed using the back reading technique.

Measurement of % reflectance was carried out at 660 nm, using an LRE research reflectometer .

A % reflectance reading was taken every second after application of the fluid and, from the third reading onwards, the mean % reflectance was calculated for the latest three consecutive readings. The end point was defined as the time when the difference between successive mean values was less than 0.1%.

Figure 1 shows test results for a glucose reagent mixture with a known sodium phosphate buffer, with three blood samples with different glucose concentrations (118, 214 and 316 mg/dl) . The reagent mixture solution which was used to impregnate the PES membrane was made up as follows:

A solution was made by stirring together overnight, hydrolysed gelatine of mwt 2,000 to 4,000 (Byco O, from Croda Colloids) (300 g) , water (300 ml) and sodium phosphate (0.5 M, pH 7) (300 ml).

The following day EDTA (1.5 g) was added to complex out metal ions, together with o-tolidine hydrochloride (15 g) , glucose oxidase (60 ml of a stock solution from Biozyme) , horse radish peroxidase (1.27 g, from Biosynth) and methoxyethanol (300 ml) .

The mixture was applied to a PES membrane as described above and tested with whole blood. Precipitation from the coating solutions began to be noticeable after about 15 to 20 minutes.

The prior art reagent mixture reached end point with the lower (118 mg/dl) glucose concentration, after about 11 to 12 seconds, but did not reach an end point with the higher glucose concentration samples . The % reflectance values do not stay constant; after about 17 to 20 seconds the reflectance values for the lower glucose concentration dip and then rise. Conversely higher levels of glucose show further drops in the % reflectance values as the end point is not attained. If a blood glucose measurement is not taken within the window between the end point and the start of significant further change in reflectance values, a false reading may be obtained.

Coating solutions of reagent mixtures in accordance with a first aspect of the present invention were prepared from the following components:

Example 1 (Glucose System)

MES 345 ml (1.2 M solution, pH 6)

Gantrez AN139 1.5 g

EDTA 2.25 g o-tolidine.2HCl 15 g Glucose Oxidase 90 ml (ex Biozyme) Horse Radish Peroxidase 1.9 g

Methoxyethanol 300 ml

Hydrolysed Gelatine 50 g (Byco 0, mwt 2,000 - 4,000)

This formulation was used to dip coat a 0.2 μ pore size PES membrane in a one stage process .

Example 2 (Cholesterol System)

MES 600 ml (0.8 M solution, pH 6)

Gantrez AΝ139 1.5 g

EDTA 2.25 g o-tolidine.2HCl 15 g

Cholesterol Esterase 1 g (bovine pancreas)

Cholesterol Oxidase 1 g

(Nocardia erythropolis)

Horse Radish Peroxidase 0.4 g

Sodium Cholate 2.5 g Methoxyethanol 300 ml

Hydrolysed Gelatine 220 g (Byco A, mwt 4,000 - 6,000)

The formulation was used to dip coat a 0.2 μ pore size PES membrane in a one stage process. Example 3 (Glucose System)

Two Stage Coating Procedure

Bath 1 Methoxyethanol 300 ml

Water 225 ml o-tolidine.2HCl 15 g

4% 7L Carboxymethyl cellulose 75 ml (from AquaIon) Bath 2 4% 7 Carboxymethyl cellulose 150 ml BES (0.42 M, pH7) 300 ml

EDTA 2.25 g

Glucose Oxidase 52 ml

Horse Radish Peroxidase 1.45 g

The membrane was initially coated using the Bath 1 formulation. The membrane was then dried and wound on to a roll and passed through the Bath 2 formulation. The membrane was dried at 85°C for 2 to 3 minutes with high air flow velocity.

None of the coating mixtures produced any noticeable precipitate over two to three hours.

The ratio of dried components in test strips was calculated, and approximate quantities (in μg) for each test strip are given in Table 1 below.

TABLE 1

The graph in Figure 2 shows % reflectance against time for the prior art reagent mixture above (phosphate buffer) , and Example 3 (BES buffer) , in a test strip with whole blood applied, read using the back reading technique. The blood glucose concentration was 118 mg/dl. The reagent mixture in Example 3 reaches end point more quickly than the prior art reagent mixture, and the colour produced is substantially stable for the duration of the test.

Figure 3 shows results for a test strip with the reagent mixture of Example 1, tested with samples of whole blood of glucose concentrations ranging from 0 mg/dl to 660 mg/dl. In all cases, the end point is reached within about 30 seconds (less for low glucose concentrations) , and the colour which develops is substantially stable for the duration of the test.

To examine the effect of the buffer on stability of the colour which is generated, a number of different buffers, at pH 6, were used in reagent formulations in test strips. The % change in reflectance was measured 1 minute after end point was reached, for whole blood with a glucose concentration of 120 mg/dl. Results are summarised in Table 2 below.

ADA = N- [2 -acetamido] -2 - iminodiacetic acid

Bis-TRIS = 2 -bis [2 -hydroxyethyl] amino-2 - [hydroxymethyl] - 1-

3 -propanediol

TABLE 2 The buffers which gave best results are the two sulphonic acids, MES and BES, and citrate. The coating solutions which incorporate MES and BES were stable for hours, but the coating solution with the citrate buffer gave precipitation within 30 to 40 minutes.

Turning now to Figure 4, there are shown results for a test strip in which increasing quantities of sodium citrate buffer (pH 7 when in solution) are in a test strip with about 117 μg of binder. No chromogen or enzymes are present. Whole blood is applied to each test strip, and

% reflectance is measured using a back reading technique.

At low ionic strength (less than 10 mM) the reflectance obtained is similar to that obtained by Pre-haemolysing the blood. The degree of haemolysis is reduced with increasing buffer concentrations until at 200 μg per strip, where the reflectance was the same value as for plasma.

The relatively small concentration of binder, and relatively high concentration of buffer salts means that analyte in a fluid applied to the test strip can dissolve more rapidly and pass more readily along the pores in the membrane, thereby reaching the end point sooner. Without in any way limiting the invention, it is thought that a possible reason why the use of increased concentrations of salts may help prevent haemolysis could be due to water loss by osmosis from the erythrocytes .

The addition of high molecular weight dextran (mwt > 100,000) to the formulation aids in protecting red cells from haemolysis.

Claims

1. A reagent mixture for testing for the presence of a component in a biological fluid sample applied thereto,

> the reagent mixture comprising a polymeric binder material, a benzidine chromogen, a catalyst system which will interact with the component to be tested so as to cause a colour change in the chromogen, and a soluble or dispersible sulphonate buffer.

2. A reagent mixture as claimed in claim 1, wherein an amine is associated with the sulphonate buffer.

3. A reagent mixture as claimed in claim 1 or claim 2, wherein the sulphonate buffer has amine functionality.

4. A reagent mixture as claimed in claim 3, wherein the sulphonate buffer has tertiary amine functionality.

5. A reagent mixture as claimed in any one of the preceding claims, wherein the sulphonate buffer is selected from a group comprising 2- [N-morpholino] ethane sulphonic acid (MES) and 2- [bis (2- hydroxyethyl) amino] ethane sulphonic acid (BES). 5

6. A reagent mixture as claimed in claim 5, wherein the sulphonate buffer is MES.

7. A reagent mixture as claimed in any one of the 0 preceding claims, wherein the sulphonate buffer comprises from 10% to 75% by weight of the reagent mixture.

8. A reagent mixture as claimed in claim 7, wherein the sulphonate buffer comprises from 40% to 65% by weight of 5 the reagent mixture.

9. A reagent mixture as claimed in any one of the preceding claims, wherein the weight ratio of sulphonate buffer to binder is in the range 1:5 to 5:1.

10. A reagent mixture as claimed in claim 9, wherein the weight ratio of sulphonate buffer to binder is in the range 1:1 to 2.5:1.

11. A reagent mixture as claimed in any one of the preceding claims, further including high molecular weight dextran.

12. A fluid composition for coating or impregnating a membrane for use in testing for the presence of a component in a biological fluid sample applied thereto, the fluid composition comprising a reagent mixture according to any one of the preceding claims in a fluid carrier.

13. A porous membrane suitable for use as the support for testing for the presence of a component in a biological fluid sample applied thereto, at least part of the length of the bores of the pores therein being blinded with a material capable of forming a continuous solid, gel or matrix form when introduced therein, whereby fragments of the wall of cells in a fluid sample applied to the membrane are substantially prevented from passing through the pores of the membrane, whereas the component to be tested is allowed to pass therethrough, the blinding material comprising a reagent mixture in accordance with any one of claims 1 to 11.

14. A method of making a porous membrane as claimed in claim 13, comprising applying a fluid composition in accordance with claim 12 to an initially porous membrane whereby the fluid penetrates the bores of the pores of the membrane, and allowing the composition to solidify so as to form a solid within the pores which substantially occupies at least part of the length of the bores of the pores .

15 A fluid testing device incorporating a membrane as claimed in claim 13.

16. A reagent mixture for testing for the presence of a component in a biological fluid sample applied thereto, the reagent mixture comprising a polymeric binder material, a chromogen, a catalyst system which will interact with the component to be tested so as to cause a colour change in the chromogen, and buffer, characterised in that the weight ratio of buffer to binder is in the range 1:5 to 5:1.

17. A reagent mixture as claimed in claim 16, wherein the weight ratio of buffer to binder is in the range 1:1 to 2.5:1.

18. A reagent mixture as claimed in claim 16 or claim 17, wherein the binder is carboxymethyl cellulose.

19. A reagent mixture as claimed in claim 16 or claim 17, wherein the binder is a hydrolysed gelatine.

20. A reagent mixture as claimed in any one of claims 16 to 19, wherein the buffer is selected from the group comprising citrate, MES and BES.

21. A reagent mixture as claimed in any one of claims 16 to 19, further including high molecular weight dextran.

22. A fluid composition for coating or impregnating a membrane for use in testing for the presence of a component in a biological fluid sample applied thereto, the fluid composition comprising a reagent mixture according to any one of claims 16 to 21, in a fluid carrier.

23. A porous membrane suitable for use as the support for testing for the presence of a component in a biological fluid sample applied thereto, at least part of the length of the bores of the pores therein being blinded with a material capable of forming a continuous solid, gel or matrix form when introduced therein, whereby fragments of the wall of cells in a fluid sample applied to the membrane are substantially prevented from passing through the pores of the membrane, whereas the component to be tested is allowed to pass therethrough, the blinding material comprising a reagent mixture in accordance with any one of claims 16 to 21.

24. A method of making a porous membrane as claimed in claim 23, comprising applying a fluid composition in accordance with claim 22 to an initially porous membrane whereby the fluid penetrates the bores of the pores of the membrane, and allowing the composition to solidify so as to form a solid within the pores which substantially occupies at least part of the length of the bores of the pores .

25. A fluid testing device incorporating a membrane as claimed in claim 23.

26. A fluid testing device incorporating a reagent mixture according to any one of claims 16 to 21, which device contains 150 to 300 ╬╝g of buffer.

27. A fluid testing device as claimed in claim 26, which device contains 200 to 250 ╬╝g of buffer.