JP6040595B2 - Hapten standard solution and hapten quantitative reagent containing the same - Google Patents

Description

  The present invention relates to a hapten standard solution used when quantifying a hapten contained in a sample, and a hapten quantitative reagent containing the standard solution.

  When measuring a specific hapten using an antigen-antibody reaction, the high specificity of the antibody that recognizes the hapten affects the accuracy of the measurement system.

  Usually, in order to obtain an antibody having high specificity for a hapten, an antibody that does not show cross-reactivity with a similar structural substance of the hapten is selected. For example, when the hapten is a steroid hormone, the cross-reaction with a substance having a sterol skeleton or a substance having a structure similar to the sterol skeleton present in a sample (for example, a body fluid such as blood or serum) is investigated. In addition, an antibody having the lowest possible cross-reactivity with the substance is selected. However, in the case of steroid hormones, there are many types of similar structural substances of the steroid hormone present in the sample, and even with the technology of a monoclonal antibody having a single specificity, an antibody that recognizes only the steroid hormone can be detected. It is difficult to get. Therefore, generally, if the cross-reaction rate with respect to the said similar structural substance is 5% or less, it will be said that it has the favorable specificity as an antibody with respect to the said steroid hormone. However, even in the case of an antibody having a good specificity, among the similar structural substances, there is a substance present in a sample at a concentration 10 times or more that of the steroid hormone, in which case the antibody The slight cross-reactivity that has a significant impact on steroid hormone quantification. In addition, when the hapten in a sample (for example, body fluid such as blood or serum) is actually measured by using an antigen-antibody reaction, there is a problem that the influence of the cross reaction differs for each sample.

JP-A-11-352126

As a method for solving the above-mentioned problem, there is a method for measuring the hapten by an antigen-antibody reaction using a standard solution in which a hapten as a measurement target substance and a substance having cross-reactivity with the hapten are used (patent) Reference 1). In this method, when a standard solution is measured, a cross-reaction according to the concentration of the substance to be measured (hapten) occurs, so a calibration curve that takes into account the cross-reaction can be created. Therefore, by applying the measurement result of the sample containing hapten to the calibration curve, a quantitative value can be obtained in a form that suppresses the influence of the cross-reactive substance (similar structure substance of the hapten) contained in the sample. be able to. However, if the hapten is a steroid hormone,
(1) The steroid hormone-like structural substance added to the standard solution is expensive,
(2) The concentration of the similar structure substance added to the standard solution is very low from the order of pg / mL to the order of ng / mL,
(3) Since similar structural substances may act as endocrine disrupting substances (environmental hormones), the risk of manufacturing, using or discarding standard solutions cannot be denied.
The method could not be easily applied.

  Therefore, an object of the present invention is a hapten standard solution used for quantifying a hapten contained in a sample, and even if the hapten concentration is in a low concentration range, the hapten is not affected by the hapten-like structural substance. The object is to provide a standard solution capable of accurately quantifying the concentration.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have prepared a calibration curve using a standard solution in which a saccharide is allowed to coexist, and quantified the hapten concentration in the sample using the standard curve. The present inventors have found that the influence of the hapten contained in the hapten by the similar structural substance can be suppressed, and as a result, the hapten concentration in the sample can be accurately determined.

  That is, the first aspect of the present invention is a hapten standard solution in which saccharides are allowed to coexist with the hapten, which is used when the hapten contained in a sample is quantified.

  A second aspect of the present invention is the standard solution according to the first aspect, wherein the hapten is a steroid hormone.

  A third aspect of the present invention is the standard solution according to the second aspect, wherein the sample is a serum sample and the saccharide is 4% (w / v) to 6% (w / v) sucrose. is there.

  Furthermore, the fourth aspect of the present invention is a hapten quantitative reagent comprising a hapten measurement reagent containing an antibody that recognizes a hapten and the labeled hapten, and a standard solution in which a saccharide is allowed to coexist with the hapten.

Furthermore, the fifth aspect of the present invention provides
Using a hapten-measuring reagent containing an antibody that recognizes a hapten and the labeled hapten, a hapten standard solution in which a saccharide coexists with the hapten is measured,
Create a calibration curve from the measured value for the hapten concentration in the standard solution,
From the measurement value obtained by measuring the sample using the hapten measurement reagent, the amount of the hapten in the sample is quantified using the calibration curve,
This is a method for quantifying a hapten.

  Hereinafter, the present invention will be described in detail.

  The hapten in the present invention is not particularly limited as long as it is a low molecular weight substance usually measured by a competition method. Examples thereof include triiodothyronine (T3), thyroxine (T4), 3,5-diiodo-L- Thyroid hormones such as thyronine (T2), estrone (E1), estradiol (E2), estriol (E3), progesterone (corgestol), testosterone, dehydroepiandrosterone sulfate (DHEA-S) ) And other steroid hormones. In particular, a steroid hormone represented by estradiol (E2) is preferable as a hapten to be measured with the reagent of the present invention.

  The hapten standard solution of the present invention is characterized in that a saccharide is allowed to coexist in a hapten having a constant concentration. The standard solution may be in a liquid state at least during use, and may be in a liquid state before use or may be in a lyophilized state. The carbohydrate to be coexisted in the hapten standard solution of the present invention is not particularly limited as long as it is water-soluble, and examples include monosaccharides such as glucose, fructose, galactose, disaccharides such as sucrose, lactose, maltose, trehalose, β- Examples thereof include polysaccharides such as cyclodextrin and methyl-β-cyclodextrin, and sugar alcohols such as mannitol, xylitol, inositol and cyclitol. Note that the type and concentration of a substance that has a cross-reactivity with the hapten (a similar structural substance of the hapten) that affects hapten measurement differs depending on the sample to be measured. For this reason, the type and concentration of carbohydrates to coexist in the hapten standard solution of the present invention may be appropriately selected according to the sample to be measured.

An example of a method for screening (selecting) saccharides to coexist in the hapten standard solution of the present invention is shown below. In addition, when screening the concentration of the saccharide coexisting in the hapten standard solution of the present invention, the same method as shown below can be used.
(1) Prepare a hapten standard solution in which saccharides as candidates coexist with haptens at various concentrations.
(2) The standard solution prepared in (1) is added to a hapten measurement reagent containing an antibody that recognizes the hapten and the labeled hapten, and measurement is performed by a competition method.
(3) A calibration curve for the hapten concentration in the standard solution prepared in (1) is prepared from the measured values (for example, fluorescence intensity and luminescence intensity) obtained by the measurement in (2).
(4) For a plurality of samples to be measured (for example, body fluids such as blood and serum), the same measurement as in (2) is performed, and the hapten concentration (quantitative value) in the sample based on each calibration curve created in (3) Are calculated respectively.
(5) The hapten concentration (quantitative value) obtained by measuring the same sample as that measured in (4) with a measurement method (for example, chromatographic mass spectrometry) without the influence of cross-reaction, and (4) The calculated quantitative value is compared, and the saccharide having the best match between the two values is adopted as the saccharide to be included in the hapten standard solution of the present invention.

  In addition, if the influence of coexisting carbohydrates is strong, the response when measuring the hapten standard solution is lower than the response when measuring the sample, and the hapten concentration (quantitative value) in the sample calculated from the calibration curve is negative. May be a value. Therefore, when a saccharide is allowed to coexist in the hapten standard solution of the present invention, it is necessary to examine a saccharide whose quantitative value is not a negative value and its concentration. Specifically, when the hapten is estradiol (E2), which is one of steroid hormones, and the sample to be measured is serum, 4% (w / V) to 6% (w / v) sucrose.

  The hapten standard solution of the present invention is a combination of a hapten measurement reagent containing an antibody that recognizes the hapten and a labeled hapten, whereby the hapten concentration contained in the sample is influenced by the substance that is cross-reactive with the hapten. Can be accurately quantified without exposure. The sample to be measured is not particularly limited, and examples include blood samples such as serum, plasma, and whole blood, and body fluids such as urine, lymph, amniotic fluid, saliva, and milk. The substance to be labeled on the hapten contained in the hapten measurement reagent may be any substance that can detect the presence or absence of the substance with a detector. Examples thereof include enzymes, radioisotopes, fluorescent substances, and luminescent substances. The substance may be directly labeled on the hapten, or may be indirectly labeled using a substance that specifically binds to the hapten (for example, an anti-hapten antibody).

  The hapten standard solution of the present invention can also be used as a control in measurement using a hapten measuring reagent containing an antibody that recognizes the hapten and the labeled hapten.

  The present invention is a hapten standard solution used for quantifying a hapten contained in a sample, and is characterized in that a saccharide is allowed to coexist. By combining the hapten standard solution of the present invention with a hapten-measuring reagent containing a labeled hapten and an antibody that recognizes the hapten, a sample containing a large amount of a substance that exhibits cross-reactivity to the hapten (a substance having a similar structure to the hapten). In contrast, the influence of the cross reaction can be suppressed, and as a result, the hapten concentration in the sample can be accurately quantified.

It is a figure which shows the result of having examined the density | concentration of the sucrose made to coexist in an estradiol standard solution. A calibration curve was prepared using a standard solution in which sucrose coexists with 5% (w / v) or a standard solution in which sucrose does not coexist, and the results of quantifying the estradiol concentration in each sample from the prepared calibration curves are shown. FIG.

  Hereinafter, the present invention will be described in more detail by taking the measurement of the amount of estradiol (E2) contained in a serum sample as an example, but the present invention is not limited to this example.

Example 1 Examination of saccharide concentration coexisting in standard solution The optimum saccharide concentration coexisting in the E2 standard solution was examined.
(1) For serum from which steroid hormones have been removed (manufactured by TRINA BIOREACTIVE AG), sucrose is 0% (w / v), 1% (w / v), 2% (w / v), 3% (w / V) 4% (w / v), 5% (w / v), 6% (w / v) and 7% (w / v) coexisting base serum (E2 non-added E2 standard solution) Each was produced.
(2) Obtained by labeling a solid-phase antibody obtained by binding an antibody recognizing E2 to magnetic beads with E2 derivative by bovine small intestine-derived alkaline phosphatase (ALP) (manufactured by Oriental Yeast Co., Ltd.) by chemical bonding. After adding 50 mM Tris buffer (pH 7.5) containing enzyme-labeled antigen and cowhide-derived peptide (Nippi), the reagent for E2 measurement was prepared by lyophilization.
(3) Using the E2 measurement reagent prepared in (2) and a commercially available fully automatic enzyme immunoassay device (AIA-2000, manufactured by Tosoh Corporation), a sample was measured by the method described below. As the measurement sample, the base serum prepared in (1) or the human serum sample (reference sample) whose E2 concentration was marked as zero by the isotope dilution gas chromatograph mass spectrometry (ID-GC / MS method) was used. .
(3-1) The measurement sample was added to the E2 measurement reagent prepared in (2) and reacted at 37 ° C. for 10 minutes.
(3-2) After the B / F separation operation, 4-methylumbelliferyl phosphate (4MUP), which is an ALP substrate, was added.
(3-3) The rate of increase in 4MU (nmol / sec) (FU) was calculated from the fluorescence intensity of 4-methylumbelliferone (4MU) produced by the decomposition of 4MUP by ALP.

  The results are shown in Table 1 and FIG. As the coexistence amount of sucrose increases, the FU value decreases. When sucrose coexists with 4% (w / v) to 6% (w / v), it is almost the same as the FU value of the reference sample (within 100 ± 2%). The value of was shown. That is, by allowing sucrose to coexist in the E2 standard solution at 4% (w / v) to 6% (w / v), a substance (E2 similar structure substance) that is cross-reactive with E2 contained in the serum sample is contained. The impact can be reflected.

実施例２ ヒト血清検体の測定
スクロースが共存しないＥ２標準液、またはスクロースを共存させたＥ２標準液で検量線を作成し、当該作成した検量線を基に、ヒト血清試料を測定した。
（１）（１）で作製した各ベース血清のうち、スクロースが共存しないベース血清（０％（ｗ／ｖ））、またはスクロースを５％（ｗ／ｖ）共存させたベース血清に対し、エタノールに溶解したＥ２をそれぞれ添加し、６点濃度のＥ２標準液をそれぞれ作製した。
（２）実施例１（３）に記載の方法により、Ｅ２標準液を測定し、ＦＵ値を算出した。ＦＵ値は試料中のＥ２濃度に比例するため、標準液中のＥ２濃度に対するＦＵ値をプロットすることで検量線を作成した。
（３）実施例１（３）に記載の方法により、ヒト血清試料３８試料（あらかじめ同位体希釈ガスクロマトグラフ質量分析計法（ＩＤ−ＧＣ／ＭＳ法）でＥ２濃度の値付けを行なった試料）を測定し、（２）で作成した検量線を用いて、各試料中に含まれるＥ２濃度を定量した。

Example 2 Measurement of Human Serum Sample A calibration curve was prepared with an E2 standard solution in which sucrose did not coexist or an E2 standard solution in which sucrose coexisted, and a human serum sample was measured based on the prepared calibration curve.
(1) Of the base sera prepared in (1), ethanol is used for the base serum in which sucrose does not coexist (0% (w / v)) or in the presence of 5% (w / v) sucrose. E2 dissolved in each was added to prepare a 6-point E2 standard solution.
(2) The E2 standard solution was measured by the method described in Example 1 (3), and the FU value was calculated. Since the FU value is proportional to the E2 concentration in the sample, a calibration curve was created by plotting the FU value against the E2 concentration in the standard solution.
(3) According to the method described in Example 1 (3), 38 human serum samples (samples for which E2 concentration was priced in advance by an isotope dilution gas chromatograph mass spectrometer method (ID-GC / MS method)) Was measured, and the E2 concentration contained in each sample was quantified using the calibration curve prepared in (2).

  The results are shown in Table 2 and FIG. When a calibration curve is prepared with an E2 standard solution in which sucrose, which is a saccharide, does not coexist, especially in a human serum sample with a low E2 concentration, the difference from the E2 concentration determined by the ID-GC / MS method is large, and the E2 concentration However, in human serum samples of 50 pg / mL or less, the value was more than twice as high. That is, it can be seen that when a calibration curve is prepared with an E2 standard solution in which sucrose does not coexist, it is influenced by a substance that cross-reacts with E2 contained in a human serum sample, particularly in a low concentration range. On the other hand, when a calibration curve is prepared with an E2 standard solution in which 5% (w / v) sucrose coexists, even if it is a human serum sample with an E2 concentration of 50 pg / mL or less, the value is obtained by the ID-GC / MS method. The deviation from the attached E2 concentration could be suppressed to about 30% at the maximum. In other words, by making a certain amount of sucrose, a saccharide, coexist in the E2 standard solution used when preparing a calibration curve, it is possible to reduce the influence of substances that cross-react with E2 contained in the sample (specimen). As a result, it can be seen that E2 can be accurately quantified even in a sample containing a low concentration of E2, which is easily affected by the substance.

Claims (3)

A hapten standard solution in which saccharides are allowed to coexist in the hapten for preparing a calibration curve for use in quantifying the hapten contained in a sample using a hapten-measuring reagent containing the labeled hapten and an antibody that recognizes the hapten . The standard solution, wherein the hapten is estradiol and the saccharide is 4% (w / v) to 6% (w / v) sucrose . A hapten determination reagent comprising a hapten-measuring reagent containing an antibody recognizing a hapten and the labeled hapten, and a standard solution in which a saccharide is allowed to coexist with the hapten, wherein the hapten is estradiol and the saccharide is 4% (w / V) to 6% (w / v) sucrose . Using a hapten-measuring reagent containing an antibody that recognizes a hapten and the labeled hapten, a hapten standard solution in which a saccharide coexists with the hapten is measured,
Create a calibration curve from the measured value for the hapten concentration in the standard solution,
From the measurement value obtained by measuring the sample using the hapten measurement reagent, the amount of the hapten in the sample is quantified using the calibration curve,
A method for quantifying a hapten ,
The quantification method, wherein the hapten is estradiol and the saccharide is 4% (w / v) to 6% (w / v) sucrose .

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