IMPROVED VISUAL READING OF COLOUR TESTS

Abstract: A new method for the visual detection of the colour of a liquid in a series of vessels containing different dyestuff concentrations is described, in which the detection is carried out in the presence of one or more additional dyestuffs, resulting in the observation of different colours or colour shades in the vessels containing different concentrations of the first dyestuff.

Description: IMPROVED VISUAL READING OF COLOUR TESTS. The invention relates to a method for the visual detection of the intensity of the colour of a liquid in a series of vessels with different dyestuff concentrations, and test kits to use in this method. Visual determination of the colour intensity of a liquid is, by virtue of its simplicity and rapidity, an attractive method for among others detection and/or concentration determination of substances in a test medium. Such visual determinations are carried out as a matter of routine, in very large numbers, in numerous laboratories. A substantial problem in this context is, however, that, depending on the dyestuff in the solution, the eye is relatively insensitive to the frequently only very slight colour intensity differences. This has an adverse effect on the reliability of such a determination. The object of the process according to the invention is to make such methods of determination substantially more reliable. The invention is characterised in that the detection is carried out in the presence of one or more additional dyestuffs, so that, in the vessels containg different concentrations of the first dyestuff a different colour or colour shade is produced. The process according to the invention is particularly suitable for visual detection after having carried out a chemical reaction which leads to a colouration or change of colour. Comparison of the resulting colour with others in the series of vessels, including blanks and standards, permits a qualitative detection of the substance to be determined. According to the invention it is possible to incorporate the extra dyestuff, or - if several are present one or more of the extra dyestuffs, in the material from which the vessels for carrying out the determination are produced. Another possibility is to add an extra dyestuff, or several of the extra dyestuffs, to the liquid in the vessels. The dyestuff(s) can be in true solution, or e.g. in dispersed form. It is also possible1 when using several extra dyestuffs, to add one of these to the liquid and another to the material from which the vessels for carrying out the determination are produced. The dyestuff or dyestuffs can also be incorporated in a transparent or translucent sheet through which light striking the vessels is transmitted. An extra dyestuff added in accordance with the invention can be complementary to the other dyestuff or dyestuffs present, resulting in extinction, or noncomplementary, resulting in colour differences when there are concentration differences of the first dyestuff. A chemical reaction which, in the method according to the invention, can change the colour intensity may comprise the addition of a compound which reacts with the substance to be determined, but can also comprise a plurality of addition steps, possible intermediate processing as well as heating, cooling, crystallisation and adsorption, as well as the addition of reactants which are later removed again. The process according to the invention can advantageously be employed for immunochemical determinations wherein a colour reaction occurs, as in the use of dye or metal sol particles, but in particular those determinations where detection takes place through the measurement of enzyme activity with the aid of a substrate, which upon conversion leads to a formation or change of colouration. The routine performing of immunochemical determinations frequently employs ready-to-use sets which comprise reagents, reaction vessels and auxiliaries, and which are referred to as test kits. 'For using the method according to the invention, special test kits can be employed. These test kits for example comprise, in addition to the conventional components for the immunochemical determination, a series of coloured vessels, a translucent or transparent coloured sheet or a loose added dyestuff or a plurality of dyestuffs having a colour different from that of the liquid which is formed as a result of the particular immunochemical determination. Depending on the type of immunochemical determination, a test kit for example includes, as particular conventional components, a labelled immuno component, an immuno component optionally bonded to a carrier, wash liquid and/or buffer. A test kit for enzyme immuno-assay according to invention comprises one or more enzyme-coupled immuno component, a substrate for the coupled enzyme and a chromogen, as well as a series of coloured vessels, a translucent or transparent coloured sheet or a loose added dyestuff or a plurality of dye stuffs having a colour different from that of the liquid which was formed as a result of the particular immunochemical determination. The invention is illustrated with the aid of the examples which follow. Example 1 The enzyme activity of samples containing horseradish peroxidase is determined by incubation of the samples in buffer (0.01 mol of sodium acetate/litre; adjusted to pH 6.0 with solid citric acid) to which substrate (H,O,; 20 mmoles/litre) and chromogen (3,3', 5,5'-tetramethylbenzidine, TMB"; 0.4 mmol/iitre) are added. After incubation (1 hour at room temperature) the reaction is stopped by adding sulphuric acid 2 mol/ litre). The enzyme activity present ultimately results in a yellow colour (absorption maximum: 450 nm), with differences in activity between samples manifesting themselves as differences in colour intensity. The differentiation between these in the case of a +J¯n reading, for example made visually, is accentuated by converting the intensity difference into a colour change. This is done by adding to the sulphuric acid solution (see above) the blue dyestuff Toluidine Blue 0; concentration corres ponding to A1 cm = 1.860. The series of "low to high A596 intensity of yellow" (or: light yellow to dark yellow) is thus changed to a series varying from blue via bluish green / greenish yellow to yellow. EMI5.1 <tb> <SEP> Without <SEP> activity <SEP> Without <SEP> Toluidine <SEP> With <SEP> Toluidine <tb> Enzyme <SEP> activity <SEP> Blue <SEP> Blue <tb> <SEP> - <SEP> colourless/light <SEP> blue <tb> <SEP> yellow <tb> + <SEP> ... <SEP> +++ <SEP> light <SEP> yellow/dark <SEP> bluish <SEP> green/ <tb> <SEP> yellow <SEP> greenish <SEP> yellow <tb> <SEP> dark <SEP> dark <SEP> <SEP> yellow <SEP> yellow <tb> Example 2 The determination according to example 1 was carried out, except that the reaction was in this case not stopped with sulphuric acid. Enzyme activity present now results in a blue colour (absorption maximum: 655 nm); activity differences between samples manifest themselves as different intensities of blue. The differences again become more clearly readable by converting the intensity difference into a colour change, namely by adding a solution of the yellow dyestuff PrimazinR Yellow GRL, BASF in distilled water; concentration corresponding to A1 cm = 1.820. 415 EMI5.2 <tb> <SEP> PrimazinR <SEP> Without <SEP> PrimazinR <SEP> <tb> Enzyme <SEP> activity <SEP> Yellow <SEP> Yellow <tb> <SEP> colourless/light <SEP> yellow <tb> <SEP> blue <tb> + <SEP> ... <SEP> <SEP> +++ <SEP> light <SEP> blue/dark <SEP> yellowish <SEP> green/ <tb> <SEP> blue <SEP> bluish <SEP> green <tb> <SEP> ++++ <SEP> dark <SEP> blue <SEP> blue <tb> Example 3 The determination according to example 2 was carried out, except that N,N-dimethyl-p-phenylenediamine dihydrochloride was used as a chromogen in place of TMB. Enzyme activity present now results in a red colour (absorption maximum: 520 nm). The conversion of the colour intensity difference into a colour change isachievedby adding a solution of the blue dyestuff Toluidine Blue O in distilled water (see example 1). EMI6.1 <tb> <SEP> Without <SEP> activity <SEP> Without <SEP> Toluidine <SEP> With <SEP> Toluidine <tb> Enzyme <SEP> activity <SEP> Blue <SEP> Blue <tb> <SEP> - <SEP> colourless/light <SEP> blue <tb> <SEP> red <tb> + <SEP> .... <SEP> +++ <SEP> light <SEP> red/dark <SEP> reddish <SEP> violet/ <tb> <SEP> red <SEP> purple <tb> <SEP> dark <SEP> red <SEP> red <tb> Example 4 The determination according to example 1 was carried out; however, in this case a blue and a red dyestuff were added to the sulphuric acid solution, namely Toluidine Blue 0, concentration corresponding to A1 ciii = 1.860 and 596 LevaflixR Brilliant Red E-4BA (Bayer AG), concentration corresponding to A 1 cm = 0.600). The dyestuff concen 510 trations are so chosen that in the case of the negative samples the background colour (light yellow) was "extinguished", to a bluish grey/grey, by subtractive mixing with the two other primary colours, blue and red. The positive samples are greenish yellow/yellow. Example 5 The determination according to example 2 was carried out; after the end of the reaction, subtractive colour mixing as in example 4 is again employed, but in this case using the two primary colours yellow (PrimazinR Yellow GRL, BASF, concentration corresponding to A41cm = 1.820) and red A415 (LevafixR Billiant Red E-4BA (Bayer AG), concentration corresponding to 1 cm = 0.720). The background colour A510 of the negative samples (light blue) is now "extinguished" to yellowish grey/grey. The positive samples are blue/bluish green. Example 6 In a Sandwich enzyme immuno-assay for Hepatitis B R surface antigen (Hepanostika HBsAg; Organon Teknika) use is made of an antibody labelled with horseradish peroxidase. The presence of the antigen in the blood sample to be investigated results in the bonding of a quantity of enzyme to the well in a polystyrene microtitration plate; the enzyme activity is rendered visible by. incubation with buffer/substrate (H202)/chromogen (TMB), followed by addition of sulphuric acid. The "positive/ negative" result is now determined by visual comparison of the colour of the wells containing sample with those of the wells with positive and negative control. The reading is greatly simplified by following the procedure described in example 1. The positive samples "stand out" as greenish yellow/yellow amongst the blue negative samples.

Claims: CLAIMS Method for the visual detection of the intensity of the colour of a liquid in a series of vessels containing different dyestuff concentrations, characterised in that the detection is carried out in the presence of one or more additional dyestuffs, so that, in the vessels containing different concentrations of the first dyestuff a different colour or colour shade is produced. Method according to claim 1, characterised in that the visual detection is employed after carrying out a chemical reaction in the series of vessels, which reaction gives rise to a colouration or colour change of the reaction medium. Method according to claim 1 or 2, characterised in that vessels produced from a material wherein at least one dyestuff is included are employed. 4. Method according to claim 1 or 2, characterised in that one or more extra dyestuff(s) is or are added to the liquid in the vessels. 5. Method according to claim 1 or 2, characterised in that at least two extra dyestuffs are employed, with at least one dyestuff being included in the material from which the vessels are produced and at least one other extra dyestuff being added to the liquid in the vessels. 6. Method according to one of the preceding claims, characterised in that an extra dywstuff with a colour which is non-complementary to that of the first dyestuff is employed. 7. Method according to claim 6, characterised in that yellow and cyan are employed as non-complementary colours0 8. Method according to claim 2, characterised in that the chemical reaction is an immunochemical determination. 9. Method according to claim 8, characterised in that the determination is an enzyme immunochemical determina tion. 10. Test kit for use in the method according to claims 8 or 9 for the visual detection of the intensity of the colour of a liquid in a series of vessels containing different dyestuff concentrations, characterised in that the test kit contains, in addition to the conventional components for immunochemical determination, a series of coloured vessels having a colour different from the colour which is formed as a result of the particular immunochemical determination. 11. Test kit for use in the method according to claims 8 or 9 for the visual detection of the intensity of the colour of a liquid in a series of vessels contain ing different dyestuff concentrations, charRcterised in that the test kit contains, in addition to the con ventional components for immunochemical determinations, a dyestuff having a colour different from the colour which is formed as a result of the particular immuno chemical determination. 12. Test kit for use in the method according to claims 8 or 9 for the visual detection of the intensity of the colour of a liquid in a series of vessels containing different dyestuff concentrations, characterized in that the test kit contains, in addition to the conven tional components for immunochemical determinations, one or more translucent or transparent sheets having a colour different from the colour which is formed as a result of the particular immunochemical determination.

Inventors: GRIBNAU, THOMAS CORNELIS JOSEPHUS, KUYPERS, LEONARDUS PAULUS CLEMENT
Applicants: AKZO N.V.
Application year: 1983
Main classification: C12Q1
IPC codes: G01N33, G01N31