JPS59197862A - Reagent for immunoassay to measure simultaneously multiple terms - Google Patents

Abstract

PURPOSE:To measure plural components in a sample liquid at one time by using one sample by color coding each of plural solid carriers according to measuring items, and bonding separate antigens or antibodies corresponding to the measuring items to the respective carriers of each color. CONSTITUTION:A sample to be measured is a sample contg. >=2 kinds of optional components which are previously measured by immunoassay. When an explanation is made by taking an example of a sandwich method, a specified amt. of the sample contg. the plural components to be measured is added to a reaction vessel and is caused to react for a specified time. The carriers are washed in some case after the reaction and thereafter a marked antibody or antigen is added and the sample is further caused to react. The carriers of respective colors are then separately drawn from the sample and the amt. of the marking agents bonded therewith are detected, by which the amts. of the measuring components for the respective carriers are known by one time of measuring operation. The material of the solid carriers may be any material like plastics such as polystyrene, and polycarbonate, cellulose, etc. The color coding of the carriers is accomplished preferably by adding coloring matters such as dyes and pigments to the materials of the respective carriers or color coding suitably the surfaces of the carriers with coloring materials, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical field of application] The present invention relates to a reagent for multi-item simultaneous measurement immunoassay that enables the simultaneous measurement of multiple test items using one sample.

[Prior art]

In recent years, immunoassays have been widely used as a quick and simple method in clinical testing and other biochemical fields. Immunoassay is a measurement method based on antigen-antibody reaction V'C, and is classified into competitive and non-competitive methods based on the reaction principle. Among these, the present invention particularly relates to measurement methods using an insoluble carrier to which an antigen or antibody is bound and a graphical labeling agent, representative examples of which are the competitive method and the Sand-Deutsch method.

In the sandwich method, an antibody is bound to a solid phase made of an inert insoluble solid carrier to form an insolubilized antibody, and a sample containing an antigen is reacted with this to form an insoluble antibody. This is the Fuji method, in which the antigen is bound to an antibody, and then an antibody labeled with a labeling agent such as oxygen is bound between the antigens, and the amount of the antigen in the sample is determined from the amount of bound labeling agent.

The competitive method involves making an insolubilized antibody bound to a solid phase competitively react with a known labeled antigen and an unknown amount of the antigen to be measured in the sample, and then determining the amount of labeling agent bound to the insolubilized antibody. This is a method for investigating the presence of antigens in a sample.

In the above competitive method and sandwich method, antibodies can also be measured by reversing the antigen and antibody.

The method of Sunta, Kotoriki, et al.
A), luminescence immunoassay (LIA), and other methods, but in the present invention, any labeling agent may be used.

[Purpose of the invention]

The present invention provides a multi-item simultaneous measurement immunoassay reagent that allows multiple components in a sample to be measured at once using one sample in clinical tests and other measurements.

[Structure of the invention]

In the immunoassay test sample for simultaneous multi-item measurement of the present invention, each of a plurality of solid carriers having a size that can accommodate two or more VCs in a reaction vessel is color-coded according to the measurement item, and each colored carrier corresponds to the measurement item. It is characterized by binding different antigens or antibodies and accommodating these carriers in one reaction container.

The solid support used in the present invention has a size that does not cause any trouble to the reaction when the solid support is input into a general immunoassay reaction container. Therefore, the shape of the carrier may be ball-shaped, plate-shaped, rod-shaped, angular, or any other shape, and is preferably a test tube (about 168 mm in diameter,
It has a shape and size, for example, a ball shape with a diameter of 4 to 5 mm, so that two or more pieces can be inserted at a time into a length of about 100 mm. Conventionally, such Ic/J% carriers have not been generally used, but according to the research of the present inventors, such Ic/J% carriers have not been used.
It has been found that measurements can be performed with sufficient sensitivity and accuracy even in small carriers.

The material of the solid carrier may be plastic such as polystyrene or polycarbonate, paper such as cellulose, or any other material conventionally used in immunoassays.

The carriers may be color-coded by adding dyes, pigments, or other coloring matter to the material of each carrier, or by appropriately color-coding the surface of the carrier with a coloring material or the like. This color classification may be a combination of several colors, or a combination of transparent and opaque colors or a combination of colorless and colored colors.

The plastic carrier is preferably melt-colored11. For example, a dye is added to polystyrene to color it, and then it is processed into a desired carrier shape. For yellow coloring, Chromofine Yellow 6G etc., orange percentage, ? 1 Chromofacourt Orange 2G'6 for color VC, Chromofacourt Bordeaux RN etc. for red coloring, Chromofine Blue 6 L "9 for blue coloring, Chromofine Green 2GO for green coloring For other colors, dyes can be similarly selected as appropriate.According to the research of the present inventors, polystyrene carrier-5 colored with these dyes can bind antigens or antibodies without any problems. It has been found that it is possible to do so, and that it does not have any undesirable effect on the measurement.

The reagent of the present invention can be obtained by binding each antigen or antibody to each of the carriers thus colored by a conventional method and storing each carrier in one reaction vessel. The reaction container is appropriately selected depending on the purpose, such as a small test tube or a vial.

The sample to be measured by the reagent of the present invention is a sample containing two or more arbitrary components conventionally measured by immunoassay. Such components include, for example, enzymes, hormones, serum proteins, urine proteins, glycoproteins, polysaccharides, lipids, haptens, and other antigenic substances, as well as antibodies thereof.

By using the reagent of the present invention, it is possible to simultaneously measure each component in the same sample containing a plurality of these components in any combination. For example, simultaneous measurement of IgG, IgM, and IgA in serum is also possible.

Next, a method for measuring antigen or antibody-sensitized color using the reagent of the present invention, which is comprised of a container containing a carrier, will be explained using the sandwich method as an example. A predetermined amount of a sample containing multiple components to be measured is added to a reaction container and allowed to react for a predetermined period of time. After the reaction, the carrier is optionally washed, then a labeled antibody or labeled antigen is added, and the carrier is further reacted for a certain period of time.

Next, by taking out the carriers of each color separately and detecting the t of the bound labeling agent, the amount of the component to be measured corresponding to each carrier can be determined in a single measurement operation.

The labeling agent may be any one such as an enzyme, fluorescence, luminescence, eintog, etc. as described above. When an enzyme is used as a labeling agent, it is convenient to detect the amount of the labeling agent bound to the carrier using a reaction system that generates a color due to its deep purple color.

Next, the present invention will be explained in more detail based on test examples and examples.

Test Example 1 Difference in measurement due to carrier size A polystyrene ball with a diameter of 44 mm and a polystyrene ball with a diameter of 6.5 mm, which has been conventionally generally used as a solid phase, were used to measure the size of the solid phase. We investigated the effects on

Each polystyrene ball was sensitized with the same anti-AFP antibody by a conventional method. Separately, an enzyme-labeled antibody was prepared by conjugating peroxidase to the same anti-AFP antibody using a conventional method. Using the obtained antibody-sensitized polystyrene balls and labeled antibodies, AFP was measured by the Sand-Germany method.

In a regular test tube with a diameter of 13 mm and a length of 100 mm, a reaction buffer (0.01M of pH 7.2 containing 0.1% BSA) was added.
0.3 volumes of IJ acid buffer (hereinafter referred to as PBS) were added, and 20μ of standard AFP antigen or serum to be measured was added. Next, the above-mentioned polystyrene balls (solid phase) with diameters of 44 μm and 6.5 μ were added to each test tube separately, and
Shake and incubate for hours. The solid phase was washed 3 times with 2 portions of physiological saline, 0.3% of the enzyme-labeled antibody was added, and further shaken at 37° C. for 1 hour for incubation. Wash the solid phase, transfer it to another test tube into which 0.3-units of IF prime coloring substrate solution (0-phenylenediamine-H2O,2) has been dispensed in advance, and shake at 57°C for 30 minutes. and incubated. Sulfuric acid (1,5N
) to 2ff17! In addition, after stopping the reaction, each enzyme activity was measured by absorbance measurement (OD492) to determine the concentration. The results are shown in Table 1.

As shown in Table 1 and above, even if the size of the polystyrene ball is reduced, a standard line that is almost the same as the conventional one can be drawn, and therefore, the antigen concentration in an unknown sample can be measured satisfactorily from this standard line.

Test Example 2 Effect of coloring of carrier on measurement Using polystyrene balls with a diameter of 4.4U, which can be placed in a general reaction container, one colorless (white) one and 1 liter of Chromofine Blue 6L were used. It was colored blue by mixing up to 2% or less, and EIA was performed using the Sanderuch method using AFP as the object. The operation was performed in the same manner as in Test Example 1. Antibody sensitization to polystyrene balls, both colorless ones and blue ones, was carried out by physical adsorption as follows. Wash polystyrene balls with cleaning solution (E, xtra
Wash with water (e.g., liquid solution) and rinse well with distilled water. Next, completely immerse these balls in the antibody solution (approximately 0.01 μm) and
Incubated for 4 hours at 7'c. After incubation, wash with distilled water and soak in PBS containing 2% BSA for 3
Incubate for 2 hours at 7°C. After washing with pBS, the cells were stored in PBS containing 0.1% BSA in the trench used.

Table 2 shows the relationship between each concentration of the standard AFP antigen and the absorbed absorbance in this test example.

Table 2 110 ' 0.1353 ' 0.1412
11 160 i 0.552710.5551 □
.

Ro20 1 [L9128 1 0.89
51 1640 1+, 2341 1 1.168
4.1 As is clear from these results, it was found that even if colored polystyrene balls were used, it was not possible to obtain a standard line that was substantially the same as that of a colorless ball.

Next, an example will be shown in which various measurement items were simultaneously measured using the reagent of the present invention.

[Example 1] Simultaneous measurement of IgG, IgM, and IgA in serum 1) Preparation of reagents Anti-IgG antibody, anti-■μ antibody, and anti-I
A sensitized carrier was prepared by physically adsorbing the gA antibody using a conventional method. The red coloring is Chromophata Lou Bordeaux RN.
The green coloring is Chromofine Green 2GO.
This was done by The reagent of the present invention was prepared by placing these balls in a test tube with a diameter of about 16 cm and a length of about 100 cm.

Separately, enzyme-labeled antibodies were prepared by binding peroxidase to each of the three types of antibodies in a conventional manner. These labeled antibodies can be mixed in predetermined amounts in advance, dispensed into ampoules, vials, etc., lyophilized, and attached to the reagent to immediately perform the sandwich method, further increasing the usefulness of the reagent of the present invention. I can do it. If a labeled antigen is attached in the same manner instead of a labeled antibody, a competitive assay can be carried out immediately.

Furthermore, the reagent of the present invention includes a measurement buffer, a reaction terminator,
If you also attach an enzyme coloring substrate solution, etc., it will be easier to use.

2) Simultaneous measurement Using the reagents and labeled antibodies prepared in 1) above, IgG and Ig in serum samples were determined by EIA using the sandwich method.
M and IgA were measured simultaneously. The operations were carried out in the same manner as in Experimental Examples 1 and 2, with the exception that:
0.4 ml of solution, standard antibodies (standard IgG, IgM,
IgA (mixed with IgA) or unknown samples were used as 01G, and the labeled antibodies were ``Veroxida'' labeled anti-IgG antibodies,
A mixture of a peroxidase-labeled anti-IgM antibody and a peroxidase-labeled anti-IgA antibody at a specific concentration was used. Measurement of enzyme activity bound to a solid phase carrier (ball) is performed using an enzyme substrate (0-phenylenediamine-H,O
,,) Each ball was placed in one dispensing test tube.

A calibration curve was drawn from the standard substance for each of the 611 constant substances (Ga, IgM, IgA) to determine the concentration in the unknown sample.

The results of the calibration curve using the standard substances are shown in Table 6 and the figure.

Table 3 As is clear from the results, simultaneous measurement is possible using three color-coded solid phases for the three items.

[Example 2] Multi-item measurement-2 In this example, AFP, a cancer-related substance in serum, was measured.
An example of simultaneous measurement of four types: , CEA, β2-microglobulin, and ferritin is shown. The method is based on the sandisothi method <E
IAVC, yes.

1) Preparation of reagents) Add anti-AFP antibody, anti-CEA antibody, anti-β2-microglobulin antibody, and anti-ferritin antibody to polystyrene balls of four colors (colorless, blue, yellow, and orange) with a diameter of 4° and 4 holes using a conventional method. A sensitized carrier is prepared by physically absorbing the water. The blue coloring was done with Chromofine Blue 6L, the yellow coloring was done with Chromofine Yellow 6G, and the elm coloring was done with Chromofatale Orange 2G. These balls were placed in a test tube in the same manner as in Example 1 to prepare a reagent.

2) Simultaneous measurement AFP, CEA, β in serum samples using the above reagents
2-Microglobulin and ferritin were measured simultaneously. The measurement operation was performed in the same manner as in Example 1. Table 4 shows the results of the calibration curve using standard substances.

As is clear from these results, simultaneous measurements could be carried out satisfactorily even when four color-coded solid phases were used for the four items.

In the above embodiments, <EI
Although the reagent of the present invention has been explained mainly using A, it has a wide range of applications and can also be used in competitive reactions, and can be applied to methods such as LIA, FIA, and RIA.

〔Effect of the invention〕

The reagent for immunoassay for simultaneous measurement of multiple items of the present invention is intended to be able to measure multiple items using one sample. Therefore, unlike the conventional method, it is not necessary to prepare a sample for each measurement item, and therefore, it is possible to measure a large number of test items using a sample such as rIn liquid, which is difficult to obtain in large quantities.

Furthermore, since the reagent of the present invention performs the immune reaction stage of each measurement in a single operation, it can save measurement time and effort, and is therefore suitable for rapid measurement of a large number of samples, which is especially required in clinical tests. This can make a major contribution.

[Brief explanation of the drawing]

The figure is a graph showing the standard heddle of Example 1. (1 other person)

Claims (1)

[Claims] Each of the multiple solid carriers is color-coded according to the measurement item,
A reagent for immunoassay for simultaneous measurement of multiple items, which is obtained by binding different antigens or antibodies corresponding to the measurement items to carriers of each color and then storing them in one reaction container.