JP2521074B2 - How to regulate the immune response - Google Patents

Description

Description: TECHNICAL FIELD The present invention regulates the measurement sensitivity when a substance is quantified using an immunochemical reaction, and more specifically, an analyte and a labeled ligand are used. In the reaction of, by using a factor that inhibits this reaction, the activity of the analyte is partially reduced and the measurable concentration range of the analyte is adjusted, and thus a very diluted sample is used. The present invention relates to a method that enables accurate and accurate measurement of substances.

INDUSTRIAL APPLICABILITY Since the present invention is effective for analysis and measurement of various substances, especially for trace analysis of biological components, it is importantly applied in each technical field such as medical treatment, diagnosis, clinical examination, analysis and quantification.

(Prior Art) With the development of medicine in recent years, the physiological significance of many trace components contained in biological fluids has become clear. Accompanied by this, in the field of clinical examinations, an accurate method for quantifying these trace components in biological fluids has become increasingly important. One of the methods for quantifying these trace components is labeled antibody,
A labeled antibody method using an insoluble carrier in which an antibody is insolubilized is known. In this labeled antibody method, the antigen is bound to an antibody-insolubilized carrier, and then the labeled antibody is bound to the antigen bound to the carrier, and then the amount of labeled antibody bound to the carrier is measured to determine the value. The amount of antigen is obtained more. In addition, a method of measuring the amount of antibody using an antigen-immobilized carrier and a labeled second antibody; a method of labeling the antigen, competing with the antigen, and measuring the amount of antigen by binding with the labeled antibody; Has been.

On the other hand, various kinds of various components are contained in biological fluid at various concentrations, and even if the same component is present, there are individual differences. The concentration range that must be achieved (hereinafter referred to as the "required range") is different for each component. However, even in the labeled antibody method, depending on the performance of the antibody used, the labeling performance of the enzyme radioactive substance, the fluorescent substance, etc., the reaction conditions, etc. There is a concentration region of the sample that can be accurately measured (hereinafter referred to as “measurement range”).

Therefore, the most important thing in each kit is that the necessary range is included in the measurement range.

In order to solve this problem, only a few methods are currently used in the labeled antibody method, particularly for the following assay components, in which the sample is diluted very thinly: β ₂ -microglobulin (hereinafter referred to as “β
MG ”), α ₁ -microglobulin, C-reactive protein, immunoglobulin (IgG, IgA, IgE, etc.), complement component (C
₃ , C ₄ etc.), transferrin, protein C (protein S), various hormones or hormone-like substances (triiodothyronine, thyroxine-binding globulin, cortisol-binding protein, sex hormone-binding globulin, hPL (hum)
an Placental Lactogen) etc.).

As one example, BMG is a measurement component that is currently being actively measured in the field of clinical examination as one index in the determination of diagnostic and therapeutic effects on smart dysfunction and various malignant tumors. When measuring BMG by an enzyme immunoassay (hereinafter also referred to as "FIA"), which is one of the currently widely used labeled antibody methods, a buffer solution should be used so that the measurement range characteristics of each kit are met. The sample diluted to 100 to 1000 times is used for 10 to 100μ measurement. This enormous dilution operation is not only very complicated, but also in the development of a fully automatic measuring machine aiming at rapid processing of a large number of specimens, the complexity of the machine is increased and the cost is increased. In addition, it may cause a bad influence on the measurement accuracy. As a matter of course, it is theoretically possible to measure the sample without diluting in such a large amount, and in that case, the amount of the sample to be used may be 10 to 1000 nl. Since the amount of the sample liquid is too small in this case, the sample cannot be dispensed accurately, and accordingly, the measurement accuracy is unavoidably deteriorated and is not put into practical use.

Therefore, although it is complicated at present, this method that requires a large amount of dilution operation is unavoidably put to practical use.

(Problems to be Solved by the Invention) The present invention provides a method that uses a reaction between an analyte and a corresponding labeled ligand, such as the labeled antibody method described above. It was made for the purpose of newly developing an analytical method that does not require a special dilution operation.

(Means for Solving Problems) As a result of conducting research from various fields to achieve the above-mentioned object, it is quite unexpected that, in the labeled antibody method, the analyte to be measured and the corresponding label When a factor that inhibits this reaction is applied during the reaction with the prepared ligand, the measurement range can be moved to a higher concentration range, in other words, the sample can be accurately diluted without extensive dilution. It was found that the light can be measured.

As a result of further studies, it was also confirmed that the same excellent effect can be obtained even if the reaction inhibitor is allowed to react with the analyte in advance, in which the analyte and the labeled ligand are all reacted.

The present invention is based on these new findings, further studies,
As a result of the detection, it was completed.

In the present invention, the analyte is an object to be measured, and the ligand is a substance showing a biological correlation with the analyte, and these are relative and when one is the analyte, the other is It becomes a ligand. A wide variety of substances are widely used as these analytes (ligands). For example, the following biological components can be enumerated: antigen (antibody), antibody (second antibody), antibody (anti-idiotype). Antibody), hormone (receptor), DNA (m
RNA), enzyme (substrate, reaction product, coenzyme, competitive inhibitor).

As the analyte, avidin, biotin, protein A, etc. may be bound to the analyte to modify it so as to further enhance the reactivity with the ligand. On the contrary, it has a direct relationship with the reaction with the ligand. Remove the part you don't
It is also possible to modify it by making it a subunit.

The ligand is labeled so as to facilitate the measurement after the reaction, and this labeled ligand (hereinafter, also referred to as “labeled ligand”) is used in the present invention. The labeling treatment may be performed according to a conventional method, and as the label, an enzyme, a fluorescent substance, a radioactive substance, ferritin or the like is appropriately used according to a conventional method.

In the present invention, a factor that inhibits this reaction is allowed to act on the reaction between the analyte (and / or its modified product) and the labeled ligand, whereby the analyte can be accurately measured without significantly diluting the sample. It is possible to analyze and measure (modified products). Therefore, as the factor, any substance can be used as long as it inhibits the above reaction, and for example, it can react with an analyte (specifically, an antigen to be measured: an antibody, etc.) or a modified product thereof. In addition to an antibody, an antigen, or a modified fragment thereof that can be used, a substance containing these (blood, serum, body fluid, etc.) can also be used in some cases. Further, for example, a substance having an action of weakening an immune reaction such as an eluent such as ethylene glycol can be used as the factor, and a buffer, a lysate, a substrate and a reaction inhibitor can also be used depending on the case. . It should be noted that these substances are given as examples only and any substance can be used as long as it is a substance that inhibits the above reaction.
The factor is the amount of analyte, the ability to bind the labeled ligand,
Adjust the concentration so that the concentration is appropriate while taking into consideration the type of sample and each condition.

This reaction-inhibitory factor may act on the above reaction,
The time of the addition treatment does not matter. Therefore, the factor may be allowed to coexist in the reaction solution during the reaction between the analyte and the labeled ligand, or the labeled ligand may be allowed to act after allowing the analyte and the factor to act in advance.

 Next, examples of the present invention will be described.

Example 1. An antibody against BMG obtained from a rabbit was immobilized on the inner surface of a polystyrene test tube by a conventional method (hereinafter referred to as "immobilized" tube). A glycol oxidase-labeled antibody was obtained from a rabbit. Using the obtained anti-MBG antibody and glucose oxidase, it was prepared according to the method described in "Enzyme-linked immunosorbent assay" 2nd edition (Medical Shoin) P.105-P.108. As the inhibitory factor, the same antibody as that used for immobilization was used, and the antibody concentration was adjusted to 10 ug / ml with 0.1% albumin and 100 mM sodium chloride.
It was prepared using mM phosphate buffer (hereinafter referred to as "dissolution").

On the other hand, peroxidase 10 mg was added to 300 ml of 0.1 M phosphate buffer (pH 7.5) containing 0.1 M glucose and 15 mM phenol.
50 mM phosphate buffer (pH 7.5) containing 50% glycerol 1
10 ml of a 15 mM 4-aminoantipyrine solution was sequentially added to prepare a substrate solution.

Then, first, dissolve the solution into each of the nine solid-phase tubes.
0.5 ml was added, and 0.1 ml of a solution of the inhibiting factor was further added. Next, set BMG to 0.01, 0.03, 0.1, 0.3, 1.0, 3.
The solution prepared to each concentration of 0, 10, 30, 100 μg / ml was dispensed by 10 μm and stirred. After reacting at 37 ℃ for 2 hours, remove the reaction solution, wash with phosphate buffer, and add 0.6 ml of Grillchol oxidase-labeled antibody dissolved in diluent to 0.1 u / ml and stir. did. After standing at 37 ° C for 2 hours, 1 ml of the substrate solution was added and stirred. 9 at 37 ° C
After standing for 0 minute, the absorption of the reaction solution at 505 nm was measured with a spectrophotometer. The relationship between the BMG concentration and the absorbance of each standard solution obtained was plotted on a graph on a logarithmic scale to obtain a first result.

Example 2. The solid-phase immobilized tube and the enzyme-labeled antibody used were those prepared by the same method as in Example 1. The factor that inhibits is different from the rabbit from which the antibody used for immobilization was obtained.An antibody against BMG obtained from another rabbit was used, and its concentration was 0,10,
100,500ug / ml was prepared.

First, 0.4 ml of the diluent was placed in each of the 9 solid-phase tubes, and then 0.1 ml of the inhibitor having a concentration of 0 ug / ml was added.

In the same manner as in Example 1, the absorbance was determined for each of the 9 BMG standard solutions. Then, the solution of the inhibiting factor was changed to a concentration of 10,100,500 ug / ml and the same operation was repeated. The relationship between the concentration of the standard solution and the absorbance with respect to the concentration of each inhibiting factor obtained was plotted on a graph on a logarithmic scale to obtain the results shown in FIG.

Example 3 As the solid-phased tube and the oxygen-labeled antibody, those produced by the same method as in Example 1 were used. As the inhibiting factor, an antibody against BMG obtained from goat was used, and the concentration was 50 ug /
Prepared to ml. Then, the absorbance at 505 nm for each standard solution was determined in the same manner as in Example 1. The obtained results are plotted in a graph on a logarithmic scale and shown in FIG.

Example 4. An antibody against BMG obtained from goat was immobilized on the inner surface of a polystyrene test tube by a conventional method. As an inhibitory factor, an antibody against BMG obtained from goat was used, and the concentration was 0.1%.
It was adjusted to be mg / ml. First, 0.5 ml of the dissolution liquid was dispensed into each of the nine solid-phase tubes described above, and further, Example 1
The same standard solution was dispensed in 10 μm aliquots. Next, the inhibitory factor having a concentration of 0 mg / ml was added by 100 μm and stirred. The following operations were performed in the same manner as in Example 1.
Next, the same operation as described above was performed for the case where the concentration of the inhibiting factor was 1 mg / ml. The obtained results are plotted on a graph on a logarithmic scale and shown in FIG.

(Effects of the Invention) As shown in these examples, the use of the method of the present invention using this inhibiting factor enables measurement without performing a large amount of diluting the sample, which has been complicated until now, and maintains the measurement accuracy. Since it is dripping, it is an optimum means for rapid processing of a large amount of samples. Further, even when the fully automated measurement is carried out by a machine, a pretreatment operation of enormously diluting the sample is not required, which allows a simpler design, which is convenient.

Further, in the method of the present invention, the measurement range can be finely adjusted by changing the amount of the inhibiting factor to act. That is, as the amount of the inhibiting factor to act increases, the measurement range also shifts to the high concentration range, so that it is possible to optimally respond to a slightly different necessary range for each biological fluid, which is convenient.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a BM of the standard solution obtained from Example 1.
The relationship between G concentration and absorbance is plotted on a graph on a logarithmic scale and each point is connected by a curve (hereinafter referred to as "calibration curve"). FIG. 2 shows a calibration curve based on the results obtained in Example 2 for each concentration of the inhibitory factor added.
(If the concentration of the inhibiting factor is 0 ug / ml, it is-●-
10 ug / ml is-▲-, 100 ug / ml is-○-
The values of 500 ug / ml are indicated by-△-. ) FIG. 3 shows the results obtained from Example 3 as a calibration curve. (Note that the calibration curve obtained when the inhibitory factor was not added (see Example 2) is also indicated by ... so that the effect of the inhibitory factor can be easily determined.) FIG. The obtained results are drawn as a calibration curve. (In addition, the calibration curve for the inhibitory factor concentration of 0 mg / ml is-○-, and the calibration curve for 1 mg / ml is-●.
It is indicated by-. )

Claims (6)

(57) [Claims] 1. In the reaction of an analyte or a modified product thereof with a corresponding labeled ligand, a factor inhibiting this reaction is allowed to coexist in the reaction solution; or a factor inhibiting this reaction and the analyte or After a predetermined action with the modified product, a labeled ligand is allowed to act on the analyte or the modified product so that the activity of the analyte or the modified product is reduced to a measurable concentration range, and A method for accurately measuring an analyte or a modified product thereof without excessively diluting a sample, which is characterized by measuring the activity of the modified product. 2. The method according to claim 1, wherein the factor that inhibits the reaction is an antibody corresponding to the analyte. 3. The method according to claim 1, wherein the analyte is an antigen and the ligand is an antibody corresponding to the antigen. 4. The method according to claim 1, wherein the analyte is an antibody and the ligand is a second antibody against the antibody. 5. The method according to claim 1, wherein the analyte is an antibody and the ligand is an anti-idiotype antibody against the antibody. 6. The method according to claim 1, wherein the analyte is an antibody and the ligand is an antigen that reacts with the antibody.