JPH06308125A - Improved method for immunoreaction of enzyme - Google Patents

Abstract

PURPOSE:To provide a reagent which can keep the detecting sensitivity in a predetermined range in an immunoassay for an enzyme and a measuring method using the reagent. CONSTITUTION:This is a measuring reagnet wherein an enzyme to be used in an immunoassay is combined with a coupling reagent. The reagent is characterized in that the activity of the enzyme included in the reagent and/or the titer of the coupling reagent is regulated. Moreover, the reagent is featured in that, at an immunoassay for an enzyme, the activity of the enzyme in the measuring reagent wherein the enzyme is coupled with the coupling reagent and/or the titer of the coupling reagent is regulated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved enzyme immunoreaction method, and more particularly, to an improvement for keeping a detected amount within a certain range when detecting a labeled enzyme in the enzyme immunoassay. It is a thing.

[0002]

2. Description of the Related Art The content of trace substances, such as proteins, contained in biological samples such as serum and urine can be measured by immunoassay using antibodies and antigens. The immunoassay is a method for quantitatively measuring the concentration of an antigen or antibody by utilizing the fact that the reaction of binding of an antigen and an antibody thereto is extremely specific and occurs even at an extremely low concentration, and is generally a sandwich. There are two methods, a competitive method and a competitive method, but in most cases, the measuring reagent is composed of a solid-phased antibody or antigen and a labeled antibody or antigen.

Originally, the reaction rate of an antigen and an antibody is extremely high, and the binding constant of an antigen-antibody complex is extremely high, so that the detection sensitivity (lower limit of detection) of immunoassay is high, and the antigen or antibody to be measured is Even if the concentration of is low, rapid and quantitative measurement is possible.

[0004]

It is necessary to keep the sensitivity in detecting an antigen-antibody reaction within a certain range in order to facilitate quality control in the production of a measurement reagent related to an immune reaction,
This is important in considering the ease of control on the machine side when providing a measurement reagent to an automatic immunoassay system.

On the other hand, the actual detection sensitivity of the immunoassay reagent is
It usually varies slightly depending on the production lot. This is because even if antibodies or antigen materials of the same origin are used, the amount of solid phase immobilized at the manufacturing stage, the heterogeneity of the solid phase manipulation, etc.
It occurs because it is impossible to remove almost all the fluctuation factors, but in order to keep this within the certain range, quality control is performed for each manufacturing batch of the material used for the measurement reagent, etc. In practice, extremely difficult work is required.

[0006]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventors have found that, in particular, the enzyme in the assay reagent used in the assay in which the enzyme and a binding reagent such as an antigen or an antibody are bound to each other. It has been found that variations in the activity or binding reagent titer can be a major obstacle to keeping the detection sensitivity within a certain range.

Therefore, in the present invention, the detection sensitivity can be kept within a certain range by adjusting the activity of the enzyme used for labeling and / or the titer of the binding reagent to be constant when the enzyme immunoassay is performed. It is intended to provide an enzyme immunoassay method capable of performing the above. That is, the present invention is the enzyme immunoassay method characterized by adjusting the enzyme activity and / or the titer of the binding reagent in the assay reagent in which the enzyme and the binding reagent are bound, when performing the enzyme immunoassay. The present invention is also a measurement reagent in which an enzyme and a binding reagent used for enzyme immunoassay are bound, and the reagent is characterized in that the enzyme activity and / or the titer of the binding reagent contained therein are adjusted. The above-mentioned reagent attached. Hereinafter, the present invention will be described in detail.

[0008] When an enzyme having a constant specific activity is used, and this is combined with an antigen or an antibody, for example, protein A capable of binding to these, or an avidin-binding antibody in place of a usual labeled antibody in the sandwich method, etc. When the measurement reagent in the present invention is prepared by binding it with a binding reagent such as biotin in the above, a good reagent can be provided for keeping the detection sensitivity within a certain range. However, it is generally very difficult to obtain an enzyme having a certain activity due to the problem that the activity of the enzyme is due to the repetitiveness of the purification operation, the stability of the enzyme itself and the like. In the present invention, by using at least two kinds of enzymes having different specific activities, for example, an enzyme having a high specific activity and an enzyme having a low specific activity, in combination, the detection sensitivity can be kept within a certain range.

[0009] If the antigenic substance to be measured changes, the antigen, antibody, etc. used in the measurement also change accordingly. Therefore, it is difficult to obtain a detection amount that can be commonly used for different measurement objects because of the different detection sensitivity for each measurement object. However, according to the present invention, this problem can also be solved by adjusting the specific activity of the enzyme used for labeling to a value such that a detected amount can be obtained that can accurately respond to the measurement target and measurement concentration range.

In the present invention including the above-mentioned regulation, any enzyme having a different specific activity can be used as the enzyme, and it is suitable for other conditions in the original enzyme immunoassay, such as optical detection. It may be appropriately selected in consideration of the presence or absence of the substrate. For example, in the case of alkaline phosphatase, some purified products have different activities of the enzyme itself. In addition, in general, those with different specific activities due to differences in purity, those with the same activity but with different origins due to different origins, and those with artificial mutations during production by genetic engineering techniques There are those whose activity has been changed.

The binding reagent which is bound to the enzyme to form the measuring reagent is, for example, an antigen or an antibody which itself directly binds to the antigenic substance to be measured in the sandwich method. Further, for example, in the competitive method, it binds to the immobilized antibody or antigen in competition with the antigenic substance to be measured,
The antigenic substance or an analog thereof. That is, the binding substance in the present invention is directly or indirectly bound to the enzyme,
It means all that is used for the purpose of introducing a label into the immune complex formed on the solid phase during the reaction. When an antibody is used as a binding reagent and the titer of the binding reagent is not adjusted as described below, it is particularly preferable to use a monoclonal antibody.

As described above, if two or more kinds of enzymes having different specific activities are combined with the binding reagent, at least two kinds of measurement reagents can be prepared. Therefore, in the actual measurement, by mixing this measurement reagent in an appropriate ratio, the enzyme immunoassay with any detection sensitivity can be carried out, or a plurality of measurements can be carried out within a certain detection sensitivity range. . On the other hand, with respect to other elements in the measurement reagent, that is, the binding reagent, if the measurement reagent is adjusted to have a constant titer, that is, a binding force (affinity) with the partner to which it binds, the detection sensitivity is kept constant A good reagent can be provided. However, it is very difficult to obtain a constant titer of these binding reagents due to problems such as the repetitiveness of the purification procedure and the stability of the binding reagent as well as the activity of the enzyme. Another aspect of the present invention is to solve this problem by mixing at least two types of binding reagents having different titers, for example, a binding reagent having a high titer and a binding reagent having a low titer, to thereby obtain a detection sensitivity within a certain range. Can fit in.

As the binding reagent, any one having a different titer can be used. As described above, the binding reagent itself does not always bind directly to the measurement target, so the titer of the binding reagent means the titer of the binding partner.

For example, in the case of directly binding to an object to be measured, an enzyme-labeled antibody in the sandwich method can be exemplified. In this case, for example, two or more kinds of monoclonal antibodies obtained from hybridomas of different clones, two or more kinds of monoclonal antibodies having different classes or subclasses, and further different titers as a whole. A group of two or more polyclonal antibodies shown can be exemplified.

In the present invention, the effect of keeping the detection sensitivity within a certain range is achieved even when only one of the above-mentioned adjustment of the specific activity of the enzyme and the adjustment of the titer of the binding reagent is carried out. It is more preferable to carry out these adjustments simultaneously. In this case, the above effect can be achieved by mixing at least four kinds of measurement reagents in an arbitrary ratio.

[0016]

As described above, according to the present invention, it is possible to easily manufacture a measurement reagent having a constant quality, that is, a detection sensitivity within a certain range. Therefore, from the standpoint of reagent manufacturing, it is possible to improve the production amount, from the standpoint of quality control, it is possible to provide more uniform reagents, and from the standpoint of use, more accurate measurement results can be obtained. Obtainable. Particularly, according to the measuring method of the present invention, the effect of eliminating the variation in the detection sensitivity of the immunoassay reagent containing the binding reagent, and being able to obtain the results irrelevant to the concentration of the measurement target is important.

On the other hand, when an automatic measuring device is used for carrying out the immunoassay, it is extremely important to prevent the device from malfunctioning, deteriorating, and being erroneously operated by the operator. If the reagent provided at this time has a detection sensitivity within a certain range, it is easy to detect such malfunctions, deteriorations, and malfunctions.

[0018]

EXAMPLES In order to explain the present invention in more detail, the following examples are described, but these examples do not limit the present invention.

Example 1 Japanese Patent Application No. 61-038279 of ethylene-vinyl acetate copolymer (EVA) pellets (manufactured by Tosoh Corporation) having an average diameter of 1.4 mm and an average length of 1.5 mm obtained by the water strand method.
No. 1 was spheroidized by the method described in No. 1, was heat-sealed with ferrite (manufactured by Tosoh Corporation), and was further polymer-coated with glycidyl methacrylate (GMA). The obtained polymer-coated beads were treated with a caustic soda / methanol solution to open the epoxy group of the surface layer to form a diol.

Example 2 Using the beads obtained in Example 1, mouse anti-human PA (prostate specific antigen) monoclonal antibody (antibody 1) was immobilized on the beads as shown below.

Beads 10000 obtained according to Example 1
500 pieces by the method of Japanese Patent Application No. 61-038279.
mg N, N'-carbonyldiimidazole (CDI,
It was vigorously stirred for 30 minutes at room temperature under a nitrogen atmosphere with 25 ml of dry acetone containing Tokyo Chemical Industry Co., Ltd.). After washing the activated beads, 2.5 mg / 20 ml of mouse anti-human PA monoclonal antibody was added and shaken at room temperature for 4 hours to bind the antibody to the particles. After washing this, a phosphate buffer (pH 7.0) containing 1.0% bovine serum albumin (BSA) was added for blocking treatment.

Example 3 As an enzyme used for labeling, alkaline phosphatase derived from bovine small intestine was used. As its specific activity, 3005
U / mg protein (enzyme 1) and 6485 U / mg protein (enzyme 2) were used to produce a labeled antibody conjugated with a mouse anti-human PA monoclonal antibody (antibody 2).

Example 4 Using the antibody-immobilized beads prepared in Example 2, enzyme immunoassay of human PA was carried out. The reaction conditions are
Twelve antibody-immobilized beads were placed in a plastic cup, and 100 μl of the labeled antibody produced in Example 3 was added to the cup to prepare a beads. This was set in an automatic enzyme immunoassay device AIA-1200 (manufactured by Tosoh Corporation).
Further, a 0.55 ng / ml human PA solution was set as the antigen solution. 20 μl of human PA solution was dispensed into a plastic cup containing antibody-immobilized beads,
Incubated at 37 ° C. for 40 minutes. At this time,
The beads were shaken and incubated. afterwards,
After washing the reaction vessel with a washing solution (B / F separation), 4-methylumbelliferone phosphate (4MUP), which is a substrate of alkaline phosphatase, was dispensed, and 4-methylumbelliferone (4MU) obtained by the enzymatic reaction was added. Increase speed (n
M / sec) was measured. The results are shown in Table 1.

[0024]

[Table 1]

As is clear from Table 1, the amount detected by the labeled antibody prepared using the enzymes 1 and 2 varies depending on the specific activity of the enzyme. In other words, it is clear that by controlling the specific activity of the enzyme used for labeling, an almost constant detection amount can be controlled.

Example 5 Using the antibody-immobilized beads prepared according to Example 2,
An enzyme immunoassay of human PA was performed. The reaction conditions were such that 12 antibody-immobilized beads were placed in a plastic cup, and 100 μl of the labeled antibody produced in Example 3 was added to the portion having a low titer (F
L) and the high portion (FH) were mixed in the ratio shown in Table 2 to prepare a mixture. In the same manner as in Example 4, the increasing rate (nM / sec) of 4-methylumbelliferone (4MU) obtained by enzymatic reaction with an automatic enzyme immunoassay device was used.
Was measured. The results are shown in Table 2.

[0027]

[Table 2]

From Table 2, it was shown that when the labeled antibodies having different titers were mixed and measured, the detection amount varied depending on the mixing ratio. Therefore, it has been clarified that the detection amount can be controlled to a substantially constant level by mixing the labeled antibodies.

From the above, when used in an immunoassay according to the present invention, the amount of detection can be adjusted by treatment such as specific activity of the enzyme and proper mixing of the labeled antibody, so that a high-quality immunoreaction can be prepared. It will be possible to provide reagents.

Claims (4)

[Claims] 1. An enzyme immunoassay method, which comprises adjusting the enzyme activity and / or the titer of the binding reagent in the assay reagent in which the enzyme and the binding reagent are bound, when performing the enzyme immunoassay. 2. A measurement reagent comprising a binding reagent bound to an enzyme and at least one assay reagent comprising a binding reagent bound to an enzyme having a different specific activity from said enzyme and / or having a different potency. The enzyme immunoassay method according to claim 1, wherein the enzyme activity / and / or the titer of the binding reagent in the measurement reagent is adjusted thereby. 3. A measuring reagent in which an enzyme used in enzyme immunoassay and a binding reagent are bound to each other, wherein the reagent has an enzyme activity and / or a titer of the binding reagent adjusted. The reagent attached. 4. A measurement reagent comprising a binding reagent bound to an enzyme and at least one assay reagent comprising a binding reagent bound to an enzyme having a different specific activity from said enzyme and / or having a different potency. The enzyme immunoassay method according to claim 3, wherein the enzyme activity and / or the titer of the binding reagent are adjusted by the above.