IE68446B1

IE68446B1 - A colour stable chromogen composition

Info

Publication number: IE68446B1
Application number: IE233489A
Authority: IE
Inventors: James Walsh; Brendan Kevin Farrell
Original assignee: Noctech Technology Limited
Priority date: 1989-07-19
Filing date: 1989-07-19
Publication date: 1996-06-12
Also published as: BE1001417A6; GB2269231A; IE892334A1; GB9216075D0

Abstract

A method for preparing a colour stable chromogen composition for use in an enzyme immunoassay kit comprises preparing a first solution by heating a solvent, preferably glycerol, adding tetramethylbenzidine (preferably the dihydrochloride salt) to the solvent, preferably below atmospheric pressure, and mixing the solution. A second solution is prepared by mixing tetramethylbenzidine perchlorate and solvent, preferably ethanol. When the first solution has been mixed, the vacuum is slowly released and the second solution is added to the first solution in the flask. The temperature of the mixture in the flask is continuously maintained and the flask is again evacuated to a pressure below atmospheric, typically 250 mBar, and the mixture is mixed.

Description

The present invention relates to a colour stable chromogen composition for use in an enzyme immunoassay.

The invention also relates to a method for preparing the colour stable chromogen composition and to a kit including the colour stable chromogen composition.

Typical enzyme immunoassays include competitive or sandwich enzyme immunoassays which are suitable for assaying for antigens, antibodies and the like, and an enzyme linked immunosorbent assay which include direct and indirect enzyme linked immunosorbent assay methods.

These assays will be well known to those skilled in the art and may be qualitative, quantitative or semiquantitative. In these tests an enzyme label is provided and one of the most commonly used methods of enzyme activity estimation is by the use of specific enzyme substrates. A typical enzyme used is the enzyme horseradish peroxidase, and the substrate may typically be composed of a solution of hydrogen peroxide and a chromogenic material to donate the required protons for detection of the horseradish peroxidase. Several chromogenic materials are available for horseradish «I peroxidase detection, and a particularly commonly used chromogenic material is o-Phenylenediamine, however it * has been found that this chromogenic material has carcinogenic properties, and it has recently become less popular as a chromogenic material. Another chromogenic material is tetramethylbenzidine dihydrochloride. Where tetramethylbenzidine dihydrochloride is used it is generally prepared as a solution in dimethylsulphoxide. Another chromogen composition is 2,2'-Azino-(3 Ethylbenzthiazolihe-6-Sulphonic Acid) (ABTS). However, while these materials give reasonable results, they have been found to be relatively colour unstable and do not store well. Furthermore, and more importantly, the rate of change of colour, in other words, the rate of colour development and the degree of colour change is relatively low.

There is therefore a need for a chromogen composition for use in an enzyme immunoassay test which overcomes the problems of known chromogen compositions, and in particular which has a relatively good rate of colour change and a relatively good degree of colour change. There is also a need for a method for preparing such a chromogen composition. Further, there is a need for a kit comprising the chromogen composition as well as a method for preparing a solution from the kit for carrying out an enzyme immunoassay test.

The present invention is directed towards providing a method for preparing such a chromogen composition and also is directed towards the chromogen composition. Further, the present invention is directed towards providing a kit comprising the chromogen composition and a method for preparing a solution from the kit for use in an enzyme immunoassay test.

According to the invention, there is provided a method for preparing a colour stable chromogen composition for use in an enzyme immunoassay, the method comprising preparing a first solution by dissolving tetramethylbenzidine or a derivative thereof in a solubilising liquid, preparing a second solution comprising tetramethylbenzidine perchlorate and mixing the second solution into the first solution at a pressure less than atmospheric pressure.

Preferably, the second solution is mixed into the first solution at a temperature , at a pressure P^ and for a time T^. In one embodiment of the invention, Q.j may be any temperature in the range of 22°C to 90°C, P.j may be any pressure in the range of 150 mBar to 350 mBar, and T1 may be any time in the range of 30 to 90 minutes. Preferably, may be any temperature in the range of 50°C to 70°C, P^ may be any pressure in the range of 200 mBar to 300 mBar, and T1 may be any time in the range of 45 to 75 minutes. Advantageously, is 60°C, P^ is 250 mBar, and is 60 minutes.

* In another embodiment of the invention, the solubilising liquid of the first solution is glycerol, and the tetramethylbenzidine of the first solution is 3,3’,5,5' tetramethylbenzidine dihydrochloride. In another embodiment of the invention, the 3,3',5,5’ tetramethylbenzidine dihydrochloride is dissolved in the glycerol at a temperature Q2, at a pressure Pg and for a time T2· Preferably, Q2 may be any temperature in the range of 22°C to 90°C, Pg may be 10 any pressure in the range of 150 mBar to 350 mBar, and T2 may be any time in the range of 90 to 150 minutes. Advantageously, Qg may be any temperature in the range of 50°C to 70°C, Pg may be any pressure in the range of 200 mBar to 300 mBar, and Tg may be any time in the range of 105 to 135 minutes, and preferably, Qg is 60°C, Pg is 250 mBar, and Tg is 120 minutes.

In one embodiment of the invention, the 3,3’,5,5' tetramethylbenzidine dihydrochloride is dissolved in the glycerol of the first solution in a proportion in the range of 2 g to 18 g of 3,3’,5,5' tetramethylbenzidine ♦ dihydrochloride per litre of glycerol.

In another embodiment of the invention, the tetramethylbenzidine perchlorate of the second solution is 3,3’,5,5' tetramethylbenzidine perchlorate.

Advantageously, the 3,3',5,5' tetramethylbenzidine perchlorate of the second solution is mixed with the ethanol in a proportion in the range of 0.025 mg to 0.175 mg of tetramethylbenzidine perchlorate per 1 ml of ethanol.

Additionally, the invention provides a colour stable chromogen composition for use in an enzyme immunoassay, the chromogen composition being prepared according to the method of the invention and comprising a first solution of tetramethylbenzidine or a derivative thereof dissolved in a solubilising liquid, and a second solution comprising tetramethylbenzidine chlorate.

Further, the invention provides a kit for use in an enzyme immunoassay, the kit comprising the colour stable chromogen composition according to the invention and a hydrogen peroxide solution.

Additionally, the invention provides a method for preparing an enzyme substrate solution for carrying out an enzyme immunoassay, the method comprising the step of mixing the colour stable chromogen composition and the hydrogen peroxide solution of the kit according to the invention to form the enzyme substrate solution.

The invention will be more clearly understood from the e following description of some non-limiting examples of preferred embodiments of the invention.

Two examples of kits for carrying out enzyme immunoassays according to the invention will now be described. In each example, a solution comprising hydrogen peroxide is used. The hydrogen peroxide solution used in the kit of each example are similar; each is prepared according to the following method. After describing the two examples of the kits, examples of uses of the kit will be described.

Hydrogen peroxide solution The hydrogen peroxide solution is prepared as follows. 14.19 g of disodium hydrogen phosphate is dissolved by stirring in 1 litre of distilled deionised water, which is already preheated to 50°C, .6 g of citric acid is added and dissolved in the solution, 0.1 g of thimerosal is added and dissolved in the solution, 300 microL of an aqueous solution of 30% .4 weight/volume hydrogen peroxide is then added to the solution and stirred, the resulting solution is poured into glass bottles and may be stored indefinitely without its activity being impaired.

Example 1 Chromogen Composition 1 A colour stable chromogen composition is prepared according to the following method, two solutions, namely a first solution and a second solution are prepared as follows and subsequently mixed together as described below.

First Solution a first solution is prepared as follows: litre of a solubilising liquid in this case glycerol is placed in a 2.5 litre, stout walled, round bottomed flask and is heated to a temperature of 60°C. The heating is achieved by partly immersing the flask in a thermostatically controlled water bath. g of 3,3',5,5' tetramethylbenzidine dihydrochloride is added to the glycerol in the flask, the flask is attached to a vacuum pump and the pressure within the flask is reduced to 250 mBar, the flask is rotated in the thermostatically controlled water bath for two hours, during which * time the temperature and pressure of the first solution in the flask is maintained at 60°C and 250 mBar, respectively, rotation of the flask for the two hour period causes the 3,3',5,5’ tetramethylbenzidine dihydrochloride to fully dissolve in the glycerol.

Second Solution a second solution comprising 3,3',5,5' tetramethylbenzidine perchlorate and ethanol is prepared as follows: grm of 3,3',5,5' tetramethylbenzidine perchlorate is added to and mixed with 1 litre of ethanol.

After the two hours, when the first solution is ready, the vacuum is slowly released from the flask. The second solution is then added to the first solution in the flask. The flask is still in the thermostatically controlled water bath and thereby the temperature of the mixture in the flask is maintained at 60°C.

The pressure is again reduced in the flask to 250 mBar and the flask is rotated in the thermostatically controlled water bath for a further sixty minutes. The composition in the flask is then cooled and is ready as a colour stable chromogen composition which is chromogen 4 composition 1. The chromogen composition 1 is then poured from the flask into glass or plastic bottles and can be stored indefinitely without loss in activity or precipitation.

A kit for carrying out enzyme immunoassays is then prepared which comprises one vial containing one part by volume of the chromogen composition 1 and one vial containing 25 parts by volume of the hydrogen peroxide solution.

To prepare the enzyme immunoassay substrate solution according to the invention, the contents of the two vials are mixed together.

Example 2 Chromogen composition 2 A colour stable chromogen composition according to another embodiment of the invention is prepared using a method identical to the method of Example 1 and using the same ingredients in the same proportions with the exception that instead of using 10 grm of 3,3’,5.,5' tetramethylbenzidine dihydrochloride, 5 grm of 3,3’,5,5’ tetramethylbenzidine dihydrochloride are used in the preparation of the first solution.

A kit is then prepared which comprises one vial containing one part by volume of the chromogen » composition 2 and one vial comprising 20 parts by volume of the hydrogen peroxide solution.

An enzyme substrate solution for carrying out an enzyme immunoassay according to this embodiment of the invention is prepared by mixing the contents of the two vials together.

The following examples illustrate tests carried out using the enzyme substrate solutions of Examples 1 and 2. In each case, visual comparative tests have been carried out with other enzyme substrate solutions to ascertain whether an improvement in the rate of colour development is achieved by using the enzyme substrate solutions of the present invention. The two enzyme substrate solutions against which the present invention has been compared are an enzyme substrate solution using ABTS as a chromogenic composition and tetramethylbenzidine dihydrochloride prepared in dimethylsulphoxide as a chromogenic composition. The tests using substrates for these latter two chromogen compositions were standard tests which will be known to those skilled in the art.

Example 3 In this example, the kit of Example 2 is used to detect Hepatitis Delta Virus Antigen (HDV) in serum using a sandwich enzyme linked immunosorbent assay (ELISA).

Micro ELISA flat well polystyrene microtitration plates are coated with a dilution of anti-delta antibodies.

Such a procedure will be well known to those skilled in the art. The anti-delta antibodies used in the sandwich are prepared by separating the immunoglobulin fraction of serum with 40% ammonium sulphate. The wells are allowed stand overnight at room temperature, and are washed three times and blocked by filling them with a quantity of 1% bovine serum albumin. microL of serum samples to be tested for delta antigen are placed in the wells prepared above. 25 microL of an extraction buffer, namely, a phospate buffered solution of pH 6.5 containing tween 20 detergent and potassium thiocyanate is added to each well and the wells are incubated overnight at room temperature. The wells are then washed and 75 microL of a dilution of horseradish peroxidase labelled anti-delta antibody is added. After incubation at 37°C for two hours, the wells are washed and incubated with 75 microL of the enzyme substrate solution prepared in Example 2. After 30 minutes incubation, the substrate reaction is stopped with 50 microL of 4N HgSO^. The optical density of each well is then measured at 650 nM.

The rate of colour development using the enzyme substrate solution prepared in Example 1 is quantified by measuring the rate of change of optical density at 650 nM. It has been found that the rate of colour development is three times greater than that achieved with ABTS and twice as much as would be achieved using a formulation of tetramethylbenzidine dihydrochloride prepared in a solution of dimethylsulphoxide.

Example 4 Detection of Hepatitis Delta Antibody (Anti HDV) in Serum by Enzyme Immunoassay Micro ELISA flat-well polystyrene microtitration plates are coated with an appropriate dilution of anti-delta antibodies. Such a procedure will be well known to those skilled in the art. The anti-delta antibody is prepared by separating the immunoglobulin fraction of serum with 40% ammonium sulphate. The solid phase plate wells are incubated overnight at room temperature with 75 microL of delta antigen diluted in phosphate buffered saline solution. After incubation the wells are emptied and washed. A 25 microL sample of neat serum to be tested for anti-delta antibodies is added to each well followed by 50 microL of phosphate buffered saline solution and the wells are incubated for 2 hours at 37°C. The wells are then washed four times with phosphate buffered saline solution Tween 20 solution. Thereupon 75 microL antibody-enzyme conjugate is added to each well and the well incubated at 37°C for 2 hours.

The wells are again washed and incubated with 75 microL of the enzyme substrate solution prepared in Example 2. After 30 minutes incubation the substrate reading is stopped with 50 microL of 4N HgSO^ and the optical density of each well is measured at 650 nM.

It has been found that the rate of colour development in the case of this example is three times greater than that achieved with ABTS and twice as great as that achieved using tetramethylbenzidine dihydrochloride prepared in dimethylsulphoxide.

Example 5 Detection of IgM Class Antibodies to Hepatitis Delta Antigen by Enzyme Immunoassay Samples to be tested for anti-delta IgM antibodies are diluted in phosphate buffered solution Tween. Duplicate 50 microL volumes of the diluted sera are incubated with a solid phase consisting of micro ELISA plates coated with rabbit anti-human IgM specific antiserum. The wells are then washed and incubated overnight at room temperature with delta antigen in one member of each pair of duplicate wells, and normal human serum in the other member. Washing, conjugate addition, substrate addition, stopping and colour development evaluation are carried out as in Examples 3 and 4. In this case, the enzyme substrate solution is that of Example 2.

A sample is considered positive for IgM class of anti-delta antibodies when it fits the following criteria: (a) if the optical density is greater than twice the optical density of the mean negative control, (b) if the sample control well, with normal human serum in place of delta antigen, is negative.

Colour developed in the delta IgM assay is improved by factors of approximately 2 and 3 over that achieved with ABTS and tetramethylbenzidine dihydrochloride prepared in dimethylsulphoxide respectively.

Example 6 Detection and Quantization of Progesterone in Body Fluids It has been found that the enzyme substrate solution of Example 1 can be successfully used in a procedure for determining haptens in body fluids. In particular, the enzyme substrate solution has been found particularly suitable for the quantitative assays of progesterone in bovine milk by competitive enzyme immunoassay.

Presently known enzyme immunoassay tests for steroids are based on the principle of competitive binding between endogenous steroid and a small fixed amount of endogenous horseradish perxidase labelled steroid for sites in a limited quantity of a specific antisteroid serum. The amount of horseradish peroxidase labelled steroid (the tracer) bound to the antibody is inversely proportional to the amount of endogenous steroid, that is, the dose present. The bound labelled steroid is measured by allowing the horseradish peroxidase label to act on its substrate, hydrogen peroxide, in the presence of the chromogen tetramethylbenzidine dihydrochloride (Example 1). The colour produced, namely the response, may be quantified spectrophotometrically.

By carrying out the assay for known amounts of endogenous steroid, namely standards, a dose-response curve may be constructed. The response for an unknown amount of endogenous steroid can then be quantified in terms of the dose initially present.

To make possible the assay of the antibody bound horseradish peroxidase-labelled steroid it must first be separated from the unbound tracer present in the reaction mixture at the end of the appropriate incubation period. The kit of Example 1 makes use of solid phase technology to achieve this separation. Components other than those bound to the solid phase are simply washed away. The solid phase chosen is the well surface of a microtitration plate. The well surface is first coated with a donkey anti-rabbit IgG, which then binds the specific steroid antiserum. The end result is a highly sensitive, easy-touse enzyme immunoassay for progesterone with good reproductibility.

Prior to commencing the assay, the dilution, distribution and identification plan is carefully established.

The following procedure is used.

Select required number of strips and place in holder.

Transfer 10 microL from each milk solution (standards, controls and samples) followed by 200 microL of diluted tracer to the appropriate well. Cover wells with adhesive strips provided.

Gently mix the contents of the wells. Cover the wells and incubate at 37°C for 1 hour.

Aspirate contents of wells and wash three times with diluted washing solution.

Add 150 microL of the enzyme substrate solution of Example 1 into each well and incubate at room temperature. Colour development should be allowed to proceed for 30 minutes, and then stopped by adding 50 microL of stopping solution to each well. Mix.

Read optical density at 650nM.

It has been found that using the enzyme substrate solution of Example 1, the rate of colour development is increased by a factor of 2 over chromogens such as o-phenylenediamine, ABTS and tetramethylbenzidine dihydrochloride, dissolved in dimethylsulphoxide.

This ELISA protocol employing the enzyme substrate solution of Example 1 may also be adapted to progesterone testing using antibody coated dipstick technology.

It is believed the reason that the degree of colour change and rate of colour change achieved using the chromogen compositions of the invention is so much improved on prior art chromogen compositions is that precipitation of tetramethylbenzidine dihydrochloride out of solution of the chromogen compositions according to the invention is avoided. Precipitation has been a serious problem in chromogen compositions known heretofore. It is believed that the addition of tetramethylbenzidine perchlorate to the composition helps substantially in avoiding precipitation. It is also believed that the fact that the tetramethylbenzidine is dissolved in the glycerol at a temperature below atmospheric pressure further helps in avoiding precipitation. It is believed that precipitation which occurs in most prior art chromogen 5 compositions is a major factor in inhibiting the rate of colour change and the degree of colour change. Why this should be is not fully understood. It has also been found that precipitation is more commonly found in chromogen compositions where the compositions have been prepared in a dusty environment. However, by preparing the chromogen compositions according to the present invention, it has been found tha.t precipitation is avoided irrespective of the amount of dust in the environment in which the composition is being prepared.

The exact reason why the addition of tetramethylbenzidine perchlorate should reduce precipitation is not fully understood. However, the following explanation is offered as a possible reason.

By virtue of the fact that the tetramethylbenzidine perchlorate is an ionic solution, its addition to the chromogen composition keeps the composition charged. This it is believed may assist in keeping the tetramethylbenzidine dihydrochloride in solution, thus avoiding precipitation. However, it is emphasised that this theory is advanced purely as a possible reason.

Why dissolving of the tetramethylbenzidine dihydrochloride in the glycerol at a reduced pressure should further facilitate avoiding precipitation is hot known.

While specific examples of the invention and assays using the enzyme substrate solution of the present invention have been described, it will be readily apparent to those skilled in the art that the enzyme substrate solution of the invention is suitable for all enzyme immunoassay methods including direct and indirect, competitive and sandwich and inhibition assays. While it is preferable to carry out the procedure in the solid phase, namely using a plastic, glass or other plate or wells, other procedures using membrane or paper supports in all formats, such as, for example, dipstick, bead and microtitre plate could be used. Further, the enzyme substrate solution of the invention may also be used to determine enzyme activity in horseradish peroxidase labelled gene probes.

Needless to say, the enzyme substrate solution may be used for detecting antibodies or antigens of natural or artificial origin. Clinical use may include, for example, the detection and/or determination of blood group substances, of diseases of various microbiological origins, for example diseases caused by viruses, bacteria or fungi, or parasitic disease, of hormones, of substances that may be present under certain conditions, for example, during pregnancy, for example, pregnancy , specific proteins and foetal proteins in foetal or maternal material, for example blood, or in association with certain malignant states, of antibodies associated with autoimmune disease and certain cancers and drugs.

It has been found that the enzyme substrate solution according to the invention is particularly useful in forensic investigation, drug determination or detection, both in forensic and associated investigations.

The enzyme substrate solution of the invention may be 10 used to detect any of the above substances, and also any other substance that can be detected and/or determined by enzyme immunoassay or enzyme labelled gene probes.

While the chromogen compositions have been described in the examples as being prepared at a temperature of 60°C, while this is preferable, it is not essential. It is believed that good results could be achieved once the temperature is retained within the range of 50°C to 70°C, and adequate results are achieved once the temperature is retained within the range of 22°C to 90°C. Further, while the first solution of 3,3',5,5’ ,/ tetramethylbenzidine dihydrochloride is dissolved in glycerol at a pressure of 250 mBar, it is believed that good results could be achieved with a pressure in the range of 200 mBar to 300 mBar. Adequate results are achieved once the pressure is retained in the range of 150 mBar to 350 mBar.

Further, it is believed that is not essential that the pressure should be retained below atmospheric pressure during the mixing of the second solution of ethanol and 3,3’,5,5' tetramethylbenzidine perchlorate with the first solution, although it is believed that better results may be achieved with a pressure lower than atmospheric pressure and preferably a pressure in the range of 150 mBar and 350 mBar and advantageously a pressure in the range of 200 mBar and 300 mBar.

Needless to say, the times quoted in the examples for dissolving the tetramethylbenzidine dihydrochloride in the glycerol could be varied without departing from the scope of the invention. In fact, it is believed that adequate results would be achieved if the solution is mixed for a period in the range of 90 to 150 minutes and preferably for a period in the range of 105 to 135 minutes.

Needless to say, the mixing times for the mixing of the second solution with the first solution could also be varied and it is believed that adequate results would be achieved with a mixing time in the range of 30 to 90 minutes and preferably in the range of 45 to 75 minutes. r Further, while the solubilising liquid has been described as being glycerol, other suitable solubilising solutions could be used. Indeed, it will be readily apparent to those skilled in the art that while 3,3',5,5’ tetramethylbenzidine dihydrochloride has been described as being dissolved in glycerol any other tetramethylbenzidine dihydrochloride or a derivative thereof could be used without departing from the scope of the invention.

While specific ratios of the compounds used have been described in the examples, other ratios could be used without departing from the scope of the invention. For example, it is envisaged that the first solution could be prepared by dissolving tetramethylbenzidine dihydrochloride in the range of 2 g to 18 g in 1 litre of glycerol, although preferred results would be achieved by dissolving 5 g to 15 g in 1 litre of glycerol.

Further, it is envisaged that adequate results could be < 20 achieved provided the quantity of 3,3’,5,5’ tetramethylbenzidine perchlorate added to the ethanol is it in the range of 0.025 to 0.175 g per litre of ethanol.

Claims

1. A method for preparing a colour stable chromogen .composition for use in an enzyme immunoassay, the method comprising preparing a first solution by dissolving 5 tetramethylbenzidine or a derivative thereof in a solubilising liquid, preparing a second solution comprising tetramethylbenzidine perchlorate and mixing the second solution into the first solution at a pressure less than atmospheric pressure. 10

2. A method as claimed in Claim 1 in which the second solution is mixed into the first solution at a temperature Q 1 , at a pressure Ρη and for a time T r

3. A method as claimed in Claim 2 in which may be 15 any temperature in the range of 22°C to 90°C, Ρη may be any pressure in the range of 150 mBar to 350 mBar, and may be any time in the range of 30 to 90 minutes.

4. A method as claimed in Claim 3 in which Q 1 may be 20 any temperature in the range of 50 Q C to 70°C, Ρη may be any pressure in the range of 200 mBar to 300 mBar, and Τη may be any time in the range of 45 to 75 minutes.

5. A method as claimed in Claim 4 in which Q^ is 60°C, P.j is 250 mBar, and is 60 minutes.

6. A method as claimed in any preceding claim in which the solubilising liquid of the first solution is 5 glycerol.

7. A method as claimed in Claim 6 in which the tetramethylbenzidine of the first solution is 3,3',5,5' tetramethylbenzidine dihydrochloride.

8. A method as claimed in Claim 7 in which the 10 3,3',5,5' tetramethylbenzidine dihydrochloride is dissolved in the glycerol at a temperature Q 2 , at a pressure Pg and for a time Tg.

9. A method as claimed in Claim 8 in which Qg may be any temperature in the range of 22°C to 90°C, Pg may 15 be any pressure in the range of 150 mBar to 350 mBar, and Tg may be any time in the range of 90 to 150 minutes.

10. A method as claimed in Claim 9 in which Qg may be any temperature in the range of 50°C to 70°C, Pg may 20 be any pressure in the range of 200 mBar to 300 mBar, and T o may be any time in the range of 105 to 135 & & minutes.

11. A method as claimed in Claim 10 in which Q 2 is 60°C, P 2 is 250 mBar, and Tg is 120 minutes.

12. A method as claimed in any of Claims 7 to 11 in which the 3,3',5,5' tetramethylbenzidine dihydrochloride is dissolved in the glycerol of the first solution in a proportion in the range of 2 g to 18 g of 3,3',5,5’ tetramethylbenzidine dihydrochloride per litre of glycerol.

13. A method as claimed in Claim 12 in which the 3,3',5,5' tetramethylbenzidine dihydrochloride is dissolved in the glycerol in a proportion in the range of 5 g to 15 g of 3,3',5,5' tetramethylbenzidine dihydrochloride per litre of glycerol.

14. A method as claimed in Claim 13 in which the 3,3',5,5' tetramethylbenzidine dihydrochloride is dissolved in the glycerol in the proportion of 10 g of 3,3',5,5' tetramethylbenzidine dihydrochloride per litre of glycerol. ·

15. A method as claimed in Claim 13 in which the 3,3’,5,5’ tetramethylbenzidine dihydrochloride is mixed with the glycerol in the proportion of 5 grin 3,3',5,5' tetramethylbenzidine dihydrochloride per litre of glycerol.

16. A method as claimed in any preceding claim in which 5 the tetramethylbenzidine perchlorate of the second solution is 3,3',5,5' tetramethylbenzidine perchlorate.

17. A method as claimed in Claim 16 in which the 3,3',5,5' tetramethylbenzidine perchlorate of the second solution is mixed with the ethanol in a proportion in 10 the range of 0.025 mg to 0.175 mg of tetramethylbenzidine perchlorate per 1 ml of ethanol.

18. A method as claimed in Claim 17 in which the 3,3',5,5’ tetramethylbenzidine perchlorate is mixed with the ethanol in a proportion in the range of 0.075 mg to 15 0.125 mg of tetramethylbenzidine perchlorate per 1 ml of ethanol.

19. A method as claimed in Claim 18 in which the 3,3',5,5' tetramethylbenzidine perchlorate is mixed with the ethanol in the proportion of 0.1 mg of 20 tetramethylbenzidine perchlorate to 1 ml of ethanol.

20. A method as claimed in any preceding claim in which the first solution is heated to a temperature in the range of 50°C to 70°C prior to the second solution being mixed into the first solution.

21. A method as claimed in Claim 20 in which the first solution is heated to a temperature of 60°C prior to the 5 second solution being mixed into the first solution.

22. A method as claimed in any preceding claim in which the second solution is mixed with the first solution in a round bottomed flask.

23. A method as claimed in Claim 22 in which mixing of 10 the first solution and the second solution is achieved by rotating the round bottom flask.

24. A method as claimed in Claim 22 or 23 in which the first and second solutions are maintained at the temperature and Tg by partly immersing the round 15 bottom flask in a thermostatically controlled water bath.

25. A method for preparing a colour stable chromogen composition, the method being substantially as described herein with reference to the examples. 20

26. A colour stable chromogen composition for use in an enzyme immunoassay, the chromogen composition being prepared according to the method of any of Claims 1 to 25 and comprising a first solution of tetramethylbenzidine or a derivative thereof dissolved in a solubilising liquid, and a second solution 5 comprising tetramethylbenzidine chlorate.

27. A chromogen composition as claimed in Claim 26 in which the tetramethylbenzidine of the first solution is 3,3',5,5' tetramethylbenzidine dihydrochloride.

28. A chromogen composition as claimed in Claim 26 or 10 27 in which the tetramethylbenzidine perchlorate of the second solution is 3,3',5,5' tetramethylbenzidine perchlorate.

29. A chromogen composition as claimed in any of Claims 26 to 28 in which the solubilising liquid is glycerol. 15

30. A colour stable chromogen composition substantially as described herein with reference to the examples.

31. A kit for use in an enzyme immunoassay, the kit comprising the colour stable chromogen composition of any of Claims 26 to 30 and a hydrogen peroxide solution. 20

32. A kit as claimed in Claim 31 in which the hydrogen peroxide solution comprises disodium hydrogen phosphate, distilled deionised water, citric acid and thimerosal. Λ

33. A kit as claimed in Claim 32 in which the hydrogen —6 peroxide constitutes up to 10 % of the hydrogen peroxide solution. 5

34. A kit substantially as described herein with reference to the examples.

35. A method for preparing an enzyme substrate solution for carrying out an enzyme immunoassay, the method comprising the step of mixing the colour stable 10 chromogen composition and the hydrogen peroxide solution of the kit of any of Claims 30 to 32 to form the enzyme substrate solution.

36. A method as claimed in Claim 33 in which the colour stable chromogen composition constitutes in the range of 15 1% to 10% by volume of the enzyme substrate solution.

37. A method as claimed in Claim 34 in which the colour stable chromogen composition constitutes in the range of v 2. % to 7% by volume of the enzyme substrate solution.

38. A method as claimed in Claim 35 in which the colour 20 stable chromogen composition constitutes about 4% by volume of the enzyme substrate solution. <*

39. A method as claimed in Claim 36 in which the colour stable chromogen composition constitutes about 5% by volume of the enzyme substrate solution.

40. A method for preparing an enzyme substrate solution 5 for use in an enzyme immunoassay, the method being substantially as described herein.