JPS63500962A - Immunoassay method using enzyme labels - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Immunoassay method by using enzyme label The present invention uses a measurement container as a solid phase. Concerning immunoassay methods of fluorescence or photometry using the bottom or wall of the instrument. Ru.

In the prior art, antibodies on antigen or antibodies pre-positioned on a solid surface. Alternatively, immobilization of antigen and use of antibody or antigen labeled with tracer A paced method is known. Such methods include, for example, RIA (radioimmunology). (chemical assay) and 5P-FIA (in-phase fluorescence immunoassay), the latter method should be dealt with in the present invention. In all these methods, immunological reactions are The solid surface where the reaction occurs and the reaction solution are separated by the excess trainer present in the reaction solution. Covers the trainer signal present in the antibody-based antigen immobilized on the top. The tracer signals must be separated from each other before being measured to avoid No. Signals that correspond to nuclei include, for example, radioactivity (RIA), fluorescence (FIA), etc. or even enzyme activity (RIA).

Separation of the solid phase from the reaction solution generally involves washing of the solid phase & i currently manual or or requires operations that are difficult to realize.

Both antibodies and antigens can be measured by the methods of the invention. of the present invention The basic idea is applicable to both photometric and fluorescent measurements. both places In some cases, the separation liquid is heavier than the reaction solution, while the bottom or wall of the vessel is After the antigen or antibody has been immobilized, put the separated solution into the reaction container and use the other side for measurement. The target antibody or antigen and the trainer-labeled antibody or antigen are combined with each other. Make it react.

In the photometric method, the separating liquid is preferably added as two layers and present as the bottom layer. Only the layer that should be present contains the 8-element substrate used as a trainer, and its color depends on the enzyme. It changes depending on the element. Related substances may include, for example, alkali or acid phosphants as enzymes. atase, paranitrophenyl phosphate as substrate and colored final product. The product is para-nitrophenol. In the fluorescence measurement method, excitation light or strongly absorbs emitted light (or both) and is used as a trainer The most suitable separation liquid is a substance that contains an enzyme substrate and the substrate emits fluorescence due to the enzyme. It is.

Specific examples of fluorescence measurement and photometric measurement of the present invention are shown in the attached drawings below. I explain. The so-called competitive method is described here, but other methods may also be used. stomach.

Brief description of the drawing Figure 1 shows a measurement container, the inner surface of which is coated with antigen (1); It also acts as a graystone container. Add the sample into the measurement container, and the sample containing the corresponding antibody (2) labeled with the GC# dye and containing the corresponding antibody (5) nothing. During immunological reactions, labeled and unlabeled antibodies are mixed in the reaction solution. is present on the bottom and wall of the Sj constant vessel due to immunological binding in proportion to the proportion present in the attached to existing antigens.

In Figure 2, the substrate for an enzyme tracer that emits fluorescence due to an enzyme is illustrated. The separated solution (4), which further contains (5) and is heavier than the test solution, has a fixed volume of 63 as shown in Figure 1. Add to 6.

Possible enzymes in this connection may be, for example, alkaline and acid phosphatases. and the substrate is methylumbelliferyl phosphate, which is also fluorescent. Releases methylumbelliferone as a substance. The separated liquid can be replaced with the test liquid. , only molecules that are attached by immunological binding remain there. FB1 as appropriate ．． 1, for example, with a 20 to 60% SilI sugar bath.

Fluorescence is -? Fireflies present at the top of the bottom of the bed By directing the luminescent light (6) as well as the GL light (7) emitted from the fluorescent layer Measuring K is measured from below. The stronger the fluorescence, the easier it is to measure in the sample. There is less antibody to be detected. This is because an antibody that has been labeled with a large amount of enzyme is attached. Because I arrived. Fluorescent substances present at the top of the separation liquid are not observed during measurement, This is because the separating liquid acts as an optical shutter.

The absorption factor of the separation liquid at the excitation and emission wavelengths of fluorescence is determined by the substrate of the cuvette. or form a substantial hindrance to the measurement of the fluorescent agent in the layer in direct contact with the wood. However, when measured through the entire separating layer, the excitation is measured through the entire thickness of the separating liquid. Such high light that substantially prevents the access of the luminous or emitted light (or both) selected to form the academic density. When measuring the fluorescence of methylumbelliferone, for example, chromate salt is used in the separation solution. or methyl orange (or a mixture thereof) as a coloring agent. It is possible.

Furthermore, in the specific examples shown in FIGS. 5 and 4, the antigen (1) is placed on the septum in the measurement container. Immobilized antibody (2) and labeled antibody (3) are added to the container. It will be done. After the immunological reaction, the sample is passed through the dosing end (8) near the bottom of the container. Separate the substrate without stirring, and the sample layer is also heavy and colorless. Solution (9) is first added, then preferably without removing the intermediate dosing end, 57 Attach a colorless substrate 0υ that is colored by the element and converted into an optically measurable substance. Additionally, a heavier, colorless separating liquid OQ is added. Absorption of colored substances is measured by the beam of light 0z that passes through it reasonably.

As enzymes, for example alkaline or acid phosphatases can be used. , as well as paranitrophenyl phosphate can be used as a substrate, In this case, paranitrophenyl is liberated as a colored compound. .

The specific example shown in Figures 3 and 4 also shows that in the final stage of addition of the same separation liquid, Preferably from the Th section duct opening proximate to the dispensing end into the liquid duct into the separating liquid. You may also try applying pressure to the substrate by adding 60% of the substrate.

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Claims (6)

[Claims] 1. The test solution to be tested and the corresponding enzyme-labeled antibody are placed in a container equipped with a transparent window. The antigen (1) of the antibody to be measured is immobilized on the inner surface of the window. In addition, in immunoassay methods using enzyme tracers, immunoreactivity is After that, a separated liquid (4, 10) heavier than the test liquid is added into the measurement container, and the separated liquid is fermented. a substrate for an enzyme that can be exchanged for a substance that can be measured fluorometrically or photometrically (5,11) and is present in the separation layer. An immunological measurement method characterized by measuring the amount of fluorometrically or photometrically. Law. 2. Contains a substrate that becomes fluorometrically detectable by the enzyme after the immunoreaction By adding the separation liquid (4), the excitation light (6) passes through the separation layer and the emission light (7) The amount of detectable compounds can be determined by fluorescence by passing through the measurement window and outside the separation layer. 2. A method according to claim 1, characterized in that the measurement is carried out photometrically. 3. Adding a separation liquid (4) that strongly absorbs at the excitation wavelength or emission wavelength The method according to claim 2, characterized in that: 4. Contains a substrate (11) that becomes photometrically measurable by the enzyme after the immune reaction Adding the separating liquid (10), the measurement bead is passed vertically through the amount of measurable compound. 2. A method according to claim 1, characterized in that the measurement is carried out by means of a system (12). 5. Before the separation solution (10), first prepare a sample that is heavier than the test solution and does not contain the enzyme substrate (11). The method according to claim 4, characterized in that a separately isolated liquid (9) is added. Law. 6. Special feature is the addition of an isolated separation liquid (9) that is lighter than the original separation liquid (10). The method according to claim 5, characterized in that