JP3451942B2 - Standard solution and immunoassay method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hapten antigen standard solution in the presence of a cross-reactive substance, a hapten antigen immunoassay method and an immunoassay reagent using the same.

[0002]

2. Description of the Related Art When a specific hapten antigen is measured by an immunoassay using an antigen-antibody reaction, the high specificity of the antibody that recognizes the hapten antigen to be measured affects the accuracy of the measurement system. To do.

Usually, in order to obtain a specific antibody against a specific hapten antigen, an antibody that does not cross-react with a substance having a similar structure is selected. For example, when the hapten antigen is a steroid hormone, cross-reactivity with various similar structural substances having a sterol skeleton or similar to a sterol skeleton present in body fluid is investigated, and an antibody showing the lowest cross-reactivity is selected. select. However, in the case of steroid hormones, there are many types of similar structural substances that are present in the body fluid that is the sample, and even with the technology of a monoclonal antibody having a single specificity, an antibody that recognizes only the target steroid hormone can be detected. It was hard to get. Therefore, in general, if the cross-reactivity rate is 5% or less, it was considered to have good specificity as an antibody against a specific target steroid hormone.

However, among these substances that show cross-reactivity, there are substances that are present in body fluid at a concentration 10 times or more that of the substance to be measured, and the slight cross-reactivity of the antibody causes a slight cross-reactivity in the actual measurement of body fluid. Was causing a big impact.

Further, when actually measuring the hapten antigen in body fluid, the influence of cross-reaction differs for each sample. Generally, the following two methods are used to determine the cross-reactivity of antibodies. That is, the response generated by the reaction between the antibody and the cross-reacting substance, a method of calculating as a measurement value converted from the standard curve of the measurement target substance, to create a standard curve for both the measurement target substance and the cross-reacting substance,
The concentration at which the standard curve has the maximum slope, that is, B / B0 is 5
It is a method of calculating the discriminating ability of an antibody from each concentration showing 0%. The cross-reactivity determined by these methods is an index showing the discriminating ability of an antibody for a cross-reacting substance under certain specific conditions. Even if there is any, the influence of the cross-reaction is different for each sample. That is, when measuring a high-concentration substance to be measured, the concentration of the substance to be measured is relatively high with respect to the concentration of the cross-reacting substance, so the influence of the cross-reaction on the measured value is small, and the substance to be measured with a low concentration is measured. In this case, since the concentration of the substance to be measured is relatively low with respect to the concentration of the cross-reacting substance, the influence of the cross-reaction on the measured value becomes large.

Due to these problems, in the measurement of a body fluid in which a substance exhibiting a cross reaction coexists, the substance to be measured may not be accurately measured.

[0007]

Therefore, an object of the present invention is to provide a standard solution for use in an immunoassay method for a hapten antigen, particularly a steroid hormone, and perform an immunoassay by a competitive method using the standard solution. Another object of the present invention is to provide a hapten antigen immunoassay method that suppresses the influence of cross-reactive substances.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the conventional problems, the present inventors have conducted immunoassay using a standard solution in which a hapten antigen as a substance to be measured and a cross-reactive substance coexist. When the method is performed, it is possible to cause a cross-reaction depending on the concentration of the target substance in the standard solution,
The present invention has been completed by finding that the sample measurement value in which the influence of the cross-reacting substance in the sample is suppressed can be obtained by converting the sample measurement value from the standard curve including the cross reaction.

That is, the present invention provides a standard solution of a hapten antigen in the presence of a cross-reactive substance, more specifically, a standard solution of a hapten antigen in the presence of a cross-reactive substance in an immunoassay method by the competition method, and the standard. An immunoassay method and an immunoassay reagent using a liquid are provided.

By using the standard solution of the present invention, the effect of cross-reaction can be suppressed and the target hapten antigen can be accurately identified even in a sample in which a substance exhibiting cross-reactivity is present in a large amount as compared with the substance to be measured. It is possible to obtain various measured values.

The present invention will be described in detail below.

The immunoassay method according to the competition method of the present invention is based on the competition between the hapten antigen to be measured in the sample and the hapten antigen used as a reagent for the antibody that recognizes the hapten antigen as the substance to be measured. This is the principle immunoassay method. For example, a solid phase-bound hapten antigen, a labeled antibody and a sample are competitively reacted, and a response based on the amount of labeled antibody bound to a solid phase is measured, or a solid phase-bound antibody, a labeled hapten antigen and a sample are competitively reacted, It means a measuring method such as measuring a response based on the amount of labeled antigen bound to the solid phase. In this case, the competitive reaction may be a simultaneous reaction or a delayed reaction, and the labeling substance may be a substance which directly binds to the antibody or antigen or which binds to the antibody or antigen, for example, an anti-immunoglobulin antibody, It may be bound to an anti-immune complex antibody or the like.

In the present invention, the hapten antigen is a low-molecular-weight antigen, which means an antigen in which a plurality of antibodies cannot react at the same time, that is, a sandwich reaction is not established.
Examples thereof include steroid hormones such as progesterone, cortisol, testosterone, estradiol (hereinafter sometimes referred to as E ₂ ) and estriol (hereinafter sometimes referred to as E ₃ ). Such a hapten antigen is suitable for measuring in a competitive reaction.

The standard solution of the present invention means a reagent showing a response which is a standard for measuring the concentration of a substance to be measured in a sample in an immunoassay method, and a substance to be measured and a cross-reactive substance having a desired concentration. And coexist. The standard solution may be a liquid at the time of use, and may be in a liquid or freeze-dried state before use.

The cross-reactive substance that is allowed to coexist in the standard solution is
It means a substance that has a structure similar to that of the substance to be measured and shows cross-reactivity with a specific antibody used in the immunoassay method. The cross-reactive substance is a substance that is present in a large amount in the body fluid among substances that show a cross-reactivity with the specific antibody used in the immunoassay by the competition method, and is appropriately selected from, for example, physiological substances and drugs. be able to. As a selection method, when serum is used as the sample to be measured, first, an immunoassay method by a competition method is performed using a substance to be measured and a desired specific antibody, and then it is present in serum and exhibits cross-reactivity. It is advisable to measure a substance that is expected to be the same as above, and select a substance that exhibits a strong cross-reactivity in the measurement system as a cross-reactive substance that coexists in the standard solution. Specifically, when the substance to be measured is progesterone or cortisol, corticosterone, pregnenolone, 17α-hydroxyprogesterone, deoxycorticosterone, pregnanediol, cholesterol, danazol, etc. are used as the cross-reactive substances, and the substance to be measured is testosterone. And E ₂ are cross-reactive substances, 5α-dihydrotestosterone, 11-ketotestosterone, E _2-3 -sulfate, cholesterol, danazol, etc., and E ₂ and E ₃ are cross-reactive substances. E _2-3 -sulfate, estrone (E ₁ ), estetrol (E ₄ ), E
_3-3-sulfate, E _{3-3-glucuronide,} E ₃
-16-Glucuronide, cholesterol, danazol, etc. can be mentioned.

The concentration of the cross-reactive substance that is allowed to coexist in the standard solution is preferably the concentration that actually exists in the body fluid used as the sample, and is a concentration that minimizes fluctuations in the measured values of the substance to be measured due to cross-reaction. Can be appropriately selected. For example, when the hapten antigen to be measured is progesterone, the cross-reactive substance is corticosterone, and the sample to be measured is serum, first, the physiological concentration range of corticosterone in serum is 100-200 ng / ml. A corticosterone-added standard solution is prepared, and healthy serum is measured using this added standard solution. If the cross-reaction occurring in the measurement of the standard solution is stronger than the cross-reaction occurring in the measurement of the healthy serum, the response when measuring the standard solution becomes lower than the response when measuring the sample, and the sample calculated from the standard curve The measured value becomes lower than 0. Therefore, the concentration of corticosterone as a cross-reactive substance coexisting in the standard solution is selected so that the sample measurement value does not fall below 0. The present invention is to obtain a cross-reactive substance equivalent to the sample in the standard solution, thereby obtaining a sample measurement value that is not affected by the cross-reactive substance. It is preferable that the concentration is comprehensively judged from the distribution of the measured values obtained by measuring a large number of actual samples. Specifically, when the substance to be measured is progesterone, the cross-reactive substance is 50 to 200.
When corticosterone of ng / ml and the substance to be measured is testosterone, the cross-reactive substance is 0.05 to 10 ng.
/ Ml dihydrotestosterone is preferred.

The sample to be measured in the present invention is not limited, and various body fluids such as serum, plasma, whole blood, urine, lymph, amniotic fluid, saliva and milk can be measured. Whatever body fluid is used as the sample, the cross-reactive substance to be coexistent with the standard solution can be appropriately selected by the method as described above. However, since the type and concentration of cross-reacting substances that affect the measurement of hapten antigens differ depending on the type of body fluid to be measured, the type and concentration of cross-reactive substances to be present in the standard solution should be selected according to the type of body fluid to be measured. There must be.

The labeling substance used in the immunoassay of the present invention is a substance that gives a response to detect the labeling substance, and examples thereof include an enzyme, a radioisotope, a fluorescent substance, and a luminescent substance. The law is not limited to these.

The standard solution of the present invention in which the substance to be measured and the cross-reactive substance are allowed to coexist can be applied to a control sample for immunoassay.

[0020]

The present invention will be described in more detail with reference to Reference Examples and Examples.

Example 1 Effect of crossing substance on measurement system When determining the concentration of progesterone in serum using this detection system, it was confirmed by the following method which steroid affects the measurement value. . Various steroids in an amount of 0.001 to 100 times the physiologically equivalent concentration were added to steroid-depleted serum (manufactured by AB Corporation) and used as a sample. Magnetic particles (manufactured by Nippon Paint Co., Ltd.) are used as the solid phase carrier, and a progesterone activated ester (manufactured by Boehringer Mannheim) is bound to mouse IgG as a support by the succinimide method, and an antigen is bound through the support. The prepared magnetic particles were used as a suspension (hereinafter referred to as a particle liquid in the present specification) for the reaction. Bovine serum albumin (manufactured by Intergen) is used as a particle diluent for diluting particles.
A 50 mM Tris-HCl buffer containing pH 7.2 was used. The anti-progesterone antibody (manufactured by Dako) was combined with bovine small intestine-derived alkaline phosphatase (manufactured by Oriental Yeast) by the method of Yoshitake et al. To give an enzyme-labeled antibody. As a dilution buffer for dissolving the enzyme-labeled antibody, 50 mM female buffer containing bovine serum albumin (pH 6.8) was used. The standard antigen was prepared by adding a solution obtained by dissolving progesterone in ethanol to steroid-free serum. Particle liquid 250μ
l, labeled antibody 50 μl, standard antigen or specimen 20 μl
The immunoassay of the one-step competition method, in which the enzyme reaction is carried out at 37 ° C for 5 minutes using a luminescent substrate after the immunoreaction at 37 ° C for 20 minutes, the fully automatic chemiluminescence immunoassay system Lumipulse (manufactured by FUJIREBIO) is used. It was done using.

The results are shown in FIG. The response of the samples to which corticosterone and deoxycorticosterone showing strong cross-reactivity were added decreased in a dose-dependent manner. Cortisol, which showed low cross-reactivity,
No change was observed in the response of the samples to which estradiol, estriol and testosterone were added. Corticosterone has a higher serum concentration than progesterone and is 0.2 times higher than progesterone.
Since the cross-reactivity was 5% or more, the influence of the cross-reactivity was predicted in the low concentration region of protesterone.

Example 2 Based on the results of Example 1, a standard solution of progesterone 0.2 to 40 ng / ml was prepared by using the added serum prepared by adding 0 to 500 ng / ml of corticosterone to the steroid-depleted serum. The measurement method described in Example 1 was performed, and the standard curve created from the response of each standard solution is shown in FIG. Standard solution 5
In the region of ng / ml or less, the response of the standard solution was obviously decreased by the addition of corticosterone.

Example 3 Progesterone standard solution containing 200 ng / ml corticosterone (hereinafter referred to as additive standard solution) was used to obtain a measurement value when a healthy human sample was measured to obtain a progesterone standard without corticosterone. The measured value was compared with that obtained using a solution (hereinafter referred to as a non-added standard solution). The measurement method was the same as the method described in Example 1. The results are shown in Fig. 3. In FIG. 3, the vertical axis represents the sample measurement value obtained using the added standard solution and the horizontal axis represents the sample measurement value obtained using the non-added standard solution, and the variation of the sample measurement value due to the difference in the standard solution is shown. As shown in FIG. 3, the divergence of the sample measured values depending on the presence or absence of the cross reaction of corticosterone tended to be small in the high sample measured value region and large in the low sample measured value region.

When the variation of the measured value of less than ± 20% was set to be equivalent to the measured value obtained by the standard solution without addition, it was confirmed that the samples having progesterone of 8.0 ng / ml or more had the presence or absence of corticosterone. However, the measured values were equivalent, and the measured values were not equivalent for the samples with progesterone less than 8.0 ng / ml. The lower the measured value, the greater the effect of corticosterone cross-reactivity.

Example 4 Since the physiological corticosterone concentration in normal adult serum is 100 to 200 ng / ml, a corticosterone-added standard solution of 50 to 500 ng / ml including the physiological concentration range was prepared, 300 healthy adult sera were measured and the amount of corticosterone added to the standard solution was examined. The measurement method was the same as the method described in Example 1. The results are shown in Table 1. As shown in Table 1, corticosterone 200
The response of the sample is 0 of that of the standard solution under the addition condition of ng / ml or more.
More than ng / ml appeared. The frequency is 150
0 cases up to ng / ml, 1 case at 200 ng / ml, 30
9 cases at 0 ng / ml, 15 cases at 400 ng / ml, 50
There were 20 cases at 0 ng / ml, and the frequency of appearance of specimens with measured values of 0 ng / ml or less increased with increasing addition concentration.

[0027]

[Table 1]

Example 5 In order to investigate the fluctuation of the measured value of a low-concentration sample depending on the added concentration of corticosterone, a commercially available progesterone measuring reagent (DPC progesterone kit manufactured by Japan DPC Co.) and a standard solution without corticosterone added, 150 ng / Using the ml addition standard solution and the 400 ng / ml addition standard solution, the measurement values of 50 progesterone low-concentration serums were compared by the measurement method described in Example 1. The results are shown in Fig. 4. As shown in FIG. 4, the measured value of the sample fluctuates depending on the amount of corticosterone added to the standard solution, and the measurement system using the standard solution containing 150 ng / ml of corticosterone gives results equivalent to those of the commercially available progesterone measuring reagent. Obtained.

[0029]

Industrial Applicability According to the present invention, a hapten antigen standard solution coexisting with a cross-reactive substance, a hapten antigen immunoassay method and an immunoassay reagent using the hapten antigen standard solution, and a hapten antigen in which the influence of cross reaction is suppressed are provided. An accurate measurement value of can be obtained.

[Brief description of drawings]

FIG. 1 is a diagram showing the cross-reactivity of various steroids in the method for measuring progesterone.

FIG. 2 is a diagram showing the cross-reactivity of corticosterone in the method for measuring progesterone.

FIG. 3 is a diagram showing the difference in sample measurement values between a standard solution with added corticosterone and a standard solution without added corticosterone.

FIG. 4 is a diagram showing a difference in a measured value of a sample due to a difference in a coexisting concentration of corticosterone in a standard solution.

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Claims (8)

(57) [Claims] 1. A standard solution of a hapten antigen, wherein a cross-reactive substance coexists. 2. The standard solution according to claim 1, wherein the hapten antigen is a substance having a sterol skeleton. 3. The standard solution according to claim 1, wherein the cross-reactive substance is a steroid hormone. 4. The standard solution according to claim 3, wherein the steroid hormone is selected from progesterone, cortisol, testosterone, estradiol and estriol. 5. The standard solution according to claim 2, wherein the cross-reactive substance is selected from corticosterone, 5α-dihydrotestosterone and E _2-3 -sulfate. 6. The steroid hormone is progesterone and the cross-reactive substance is corticosterone 50.
The standard solution according to claim 4 or 5, wherein the standard solution is about 200 ng / ml. 7. A method for immunoassaying a hapten antigen by the competitive method using the standard solution according to claim 1. 8. An immunoassay reagent using the standard solution according to claim 1.