WO1987001810A1

WO1987001810A1 - Method for carrying out immunoassays by using enzyme labels

Info

Publication number: WO1987001810A1
Application number: PCT/FI1986/000096
Authority: WO
Inventors: Hannu Harjunmaa; Juhani Luotola
Original assignee: Labsystems Oy
Current assignee: Thermo Fisher Scientific Oy
Priority date: 1985-09-13
Filing date: 1986-09-10
Publication date: 1987-03-26
Anticipated expiration: 1988-03-13
Also published as: FI872078L; FI853523A0; FI872078A0; EP0235224A1; JPS63500962A; FI872078A7

Abstract

A method for carrying out immunoassays by using enzyme tracer in a vessel onto whose inside face an antigen of the antobody to be determined has been immobilized. The sample solution to be studied as well as a corresponding enzyme-labelled antibody are added into the vessel. After the immuno-logical reaction, a separation liquid (4) is added, which is heavier than the sample solution and which contains such a substrate (5) of the enzyme as is converted by the enzyme so that it becomes fluorometrically or photometrically detectable.

Description

Method for carrying .out im unoassays by using enzyme labels

The present invention concerns a fluorometric or photometric immunoassay method in which the bottom or the walls of the measurement vessel are used as the solid phase.

In prior art, methods are known which are based on immobilization of an antibody or antigen on an antigen or antibody in advance placed on a solid face as well as on the use of an antibody or antigen labelled with a tracer. Such methods are, e.g., RIA (Radioimmuno- assay) and SP-FIA (Solid Phase Fluoroimmunoassay) , which latter one is to be dealt with in the present invention. In all of these methods, the solid face on which the immunological reaction has taken place and the reaction solution must be separated from each other before the signal of the tracer is measured in order that the excess tracer present in the reaction solution should not cover the signal of the tracer present in the antibody or antigen immobilized on the solid phase. The signal con¬ cerned may be, e.g., radioactivity (RIA), fluorescence (FIA) , or even enzyme activity (EIA) .

As a rule, the separation of the solid phase from the reaction solution includes washing of the solid phase, which at present requires manual operations or operations whose automation is difficult.

By means of the method of the present invention both antibodies and antigens can be determined. The basic idea of the invention can be applied both to photometric measurement and to fluorometric measurement. In both cases a separation liquid heavier than the reaction solution is used, which said separation liquid is administered into the reaction vessel after the antigen or antibody immobilized on the bottom or walls of the vessel, on one hand, and the antibody or antigen to be determined and also the antibody or antigen labelled with the tracer, on the other hand, have been allowed to react with each other. In a photometric method, the separation liquid is administered preferably as two layers, of which only the layer to be placed as the bottom layer contains a substrate of the enzyme used as the tracer, whose colour is changed by the enzyme. Substances that may be concerned are, e.g., alkaline or acid phosphatase as the enzyme, paranitrophenylphosphate as the substrate, and paranitrophenol as the coloured final product. In a fluorometric method, the separation liquid is most appropriately a substance which absorbs the excitation light or the emission light (or both) strongly and contains a substrate of the enzyme used as the tracer, which said substrate is made fluorescent by the enzyme.

A fluorometric embodiment and a photometric embodiment of the invention will be illustrated in the following by means of the accompanying figures. Herein a so-called competitive method is described, but other methods may also be used.

Figure 1 shows a measurement vessel, whose inside face is covered with an antigen 1 and which also acts as the reaction vessel. Into the measurement vessel, the sample has been administered, which includes the antibody 2 to be determined and, moreover, the cor¬ responding antibody 3 labelled with an enzyme. In the immunological reaction, labelled antibody and unlabelled antibody have adhered to the antigen present on the bottom and walls of the measurement vessel by means of an immunological bond in the proportion in which they are present in the reaction solution.

In Figure 2, a separation liquid 4 heavier than the sample solution has been added into the mea¬ surement vessel shown in Fig. 1, which said separation liquid 4 further includes such a substrate 5 of the enzyme tracer as is made fluorescent by the enzyme. A possible enzyme concerned may be, e.g., alkaline or acid phosphatase, and the substrate may be methylum- belliferylphosphate, whereby methylumbelliferone is liberated as the fluorescent substance. The separation liquid has displaced the sample solution and allowed only the molecules adhering by means of the immunological bond to remain therein. A suitable separation liquid is, e.g., a 20...60-% saccharose solution.

The fluorescence is measured from below by directing an excitation light 6 of suitable colour into the fluorescent layer present above the bottom of the cuvette and by measuring the amount of fluorescent light 7 emitted from same. The stronger the fluores¬ cence is, the less has there been of the antibody to be determined in the sample, because, then, a large quantity of the antibody labelled with the enzyme has adhered. The fluorescent substance present in the top portion of the separation liquid is not seen in the measurement, because the separation liquid acts as an optical shutter. The absorption factor of the separation liquid at the excitation and emission wavelengths of the fluorescence is chosen so that it does not form a substantial - obstacle for the measurement of the fluorescent agent in the layer in direct contact with the bottom or wall of the cuvette but that, when measured through the whole separation layer, it forms such a high optical density that access of the excitation light or of the emission light (or both) through the whole thickness of the separation liquid is substantially prevented.

When the fluorescence of methylumbelliferone is measured, in the separation liquid it is possible to use, e.g., potassium chromate or methyl orange (or a mixture of same) as the colouring agent.

Also, in the embodiment shown in Figures 3 and 4, antigen 1 has been immobilized onto the partition wall in the measurement vessel, and antibody 2 and enzyme-labelled antibody 3 have been administered into the vessel. After the immunological reaction, into the vessel, to the proximity of the bottom, through a dosage tip 8 and without stirring the sample, first a colourless isolating separation liquid 9 with no substrate and heavier than the sample layer has been administered, and then, preferably without removing the dosage tip in between, a still heavier colourless separation liquid 10 has been administered, which additionally contains such a colourless substrate 11 as is converted by the enzyme to a coloured, photometrica ly measurable substance. The absorbance of the coloured substance is measured by means of a beam of light 12 that passes vertically.

As the enzyme it is possible to use, e.g., alkaline or acid phosphatase, and as the substrate paranitrophenylphosphate, in which case paranitrophenyl is liberated as the coloured compound.

The embodiment shown in Figures 3 and 4 may also be accomplished so that, at the final stage of the administration of one and the same separation liquid, the substrate is added to the separation liquid, preferably into the liquid duct out of a side duct opening close to the dosage tip.

Claims

WHAT IS CLAIMED IS:

1. Method for carrying out immunoassays by using an enzyme tracer, wherein a sample solution to be

-5 studied as well as a corresponding enzyme-labelled antibody are added into a measurement vessel provided with a transparent window, onto whose inside face the antigen (1) of the antibody to be determined has been immobilized, c h a r a c t e r i z e d in that, aftur 0 the immunological reaction, a separation liquid (4, 10) heavier than the sample solution is added into the measurement vessel, which said separation liquid con¬ tains such a substrate (5, 11) of an enzyme as is con¬ verted by the enzyme so that it becomes fluorometrically 5 or photometrically detectable, and that the quantity of the detectable compound present in the separation layer is measured fluorometrically or photometrically.

2. Method as claimed in claim 1 , c h a r ¬ a c t e r i z e d in that, after the immunological 0 reaction, a separation liquid (4) is added which con¬ tains a substrate which is made by the enzyme fluoro¬ metrically detectable and that the quantity of the detectable compound is measured fluorometrically so that both the excitation radiation (6) is passed into the 5 separation layer and the emission radiation (7) is passed out of the separation layer through the measure¬ ment window.

3. Method as claimed in claim 2, c h a r ¬ a c t e r i z e d in that a separation liquid (4) is 0 added which absorbs strongly at the excitation wave¬ length or at the emission wavelength.

4. Method as claimed in claim 1, c h a r ¬ a c t e r i z e d in that, after the immunological reaction, a separation liquid (10) is added which con- 5 tains a substrate (11) that is made by the enzyme photometrically detectable and that the amount of the detectable compound is measured by means of a beam of measurement (12) passing vertically.

5. Method as claimed in claim 4, c h a r ¬ a c t e r i z e d in that, before the separation liquid (10) , first an isolating separation liquid (9) is added which is heavier than the sample solution and which does not contain substrate (11) of the enzyme.

6. Method as claimed in claim 5, c h a r ¬ a c t e r i z e d in that an isolating separation liquid (9) is added which is less heavy than the separation liquid (10) proper.