JPS607229B2 - Immunochemical measurement method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for immunochemically measuring physiologically active substances.

insulin, ciliary gonadotrobin, growth hormone, Q
- Antigenic substances such as fetobrotin and immunoglobulin (hereinafter referred to as antigenic substances) have very specific and sensitive binding reactions with antibodies.
Many methods for measuring antigenic substances or antibodies to antigenic substances using this reaction have been developed, and many immunochemical measurement methods are already in practical use. For example, in-gel precipitation method in which antigen and antibody are reacted in agar gel, agglutination reaction and agglutination inhibition reaction in which microparticles such as blood cells or polystyrene latex are used as antigen or antibody carriers, and radioactive isotope, enzyme, or fluorescent substance Radioimmunoassay (Radio-jm) used as a labeling agent for antigens or antibodies
m skin oassay is hereinafter abbreviated as RIA. ), enzyme immunoassay (abbreviated as EIA).

), and fluorescence immunoassay. On the other hand, a substance to be measured instead of an antibody in an immunochemical measurement method (hereinafter referred to as a measurement substance).

), ie, receptors or binding proteins, can also be measured by competitive protein assays. Each of these methods has its own characteristics and is widely used to take advantage of these characteristics, but RIA and E
Compared to other methods, IA has extremely high measurement sensitivity and excellent quantitative performance, so it has a wide range of applications, and the substances that can be measured range from polymeric substances such as protein hormones, viruses, and immunoglobulins to bebutides. , steroids, synthetic drugs, and other low-molecular substances.

The measurement principle of RIA and EIA is common, and conventionally two methods called competitive method and sandwich method have been used, and the measurement principle is as follows.

Hereinafter, in this specification, an antigen is used as a measurement substance, and a substance that specifically binds to this substance (hereinafter referred to as a binding substance) is used.

), but the present invention is not limited to this, and the present invention is not limited to this, and may also be applied to cases where the substance to be measured is an antibody and the binding substance of this substance is an antigen, or the case where an antigen-antibody system is used. However, the present invention is not limited to this, and a physiologically active substance-receptor protein system can also be implemented in the same manner. Next, an outline of the competitive method and sandwich method in RIA and EIA will be explained using drawings.

The measurement principle of the competitive method is as shown in the schematic diagram of Fig. 1. An unknown amount of unlabeled antigen 1 to be measured and a fixed amount of labeled antigen 2 labeled with a labeling agent are combined with a fixed amount of the corresponding antibody 3. Make a competitive reaction between the amounts.

In this case, unlabeled antigen 1 and labeled antigen 2 bind to antibody 3 in proportion to their respective amounts, and the amount of labeled antigen 2 that binds to antibody 3 increases or decreases in inverse proportion to the increase or decrease in the amount of unlabeled antigen 1. do. Next, labeled antigen 2a that bound to the antibody and labeled antigen 2 that did not bind are separated by an appropriate method, and the activity of the labeling agent in either fraction is measured. At the same time, a standard curve is prepared in the same manner using a standard substance of known concentration, and the amount of unknown antigen 1 is calculated using this standard curve. As shown in the schematic diagram of FIG. 2, the measurement principle of the sandwich method is to react a specific amount of unlabeled antigen 1 to be measured with insolubilized antibody 4, which is an insolubilized antibody for the antigen.

Both combine to form an antigen-antibody complex 5 (first reaction). This complex 5 is once separated from the reaction mixture, and is reacted with a certain amount of labeled antibody 6, which is an antibody of the measurement antigen bound to a labeling agent (second reaction). The labeled antibody 6 binds to the complex 5, but the labeled antibody 6 in excess of the binding capacity of the complex 5 does not bind and exists in a free state. Next, labeled antibody 6a that bound to the complex and labeled antibody 6 that did not bind are separated, and the activity of the labeling agent in either fraction is measured. At the same time, a standard curve is prepared in the same manner using a standard substance of known concentration, and the amount of unknown antigen 1 is calculated using this standard curve. In the competitive method, the amount of change in the activity of the labeling agent is smaller than the change in the amount of antigen to be measured, so the slope of the standard curve is small and the range of the amount of antigen that can be measured is narrowed. In this method, changes in the amount of antigen to be measured directly correspond to changes in the activity of the labeling agent, so the slope of the standard curve is large and the presence of antigen over a wide range can be measured.

However, with the conventional sandwich method, the upper limit of the amount of antigen that can be satisfactorily measured is 1 to 1 times the lower limit, whereas the upper limit of the amount of the substance to be measured can range from 1 to 1 times the lower limit. It's finally here. For example, the blood concentration of Q-fetoprotein is less than 1 × 1 billion per day in normal people, but
Concentrations of 1×1 ping/' can be reached in patients with primary liver cancer. In such a case, there is the inconvenience of having to prepare a sample diluted in several stages so as to fall within the measurable range of the assay method. In addition, ``As the number of dilutions increases, there is a risk that measurement accuracy will decrease.As a result of repeated research by the present inventors to develop a measurement method that does not have the above-mentioned disadvantages and has a wide measurement range, we have found that In this book, we discovered that in the sandwich method, it is possible to measure a much wider range than conventional methods by actively utilizing the antigen-rich region, which was thought to be impossible to measure due to zone phenomena. Completed the invention.

That is, the measurement method of the present invention involves reacting a analyte with a binding substance and then reacting a labeling substance without separating the reaction product from the reaction solution to form a binding substance-analyte-labeling substance complex. In an immunochemical assay method in which the analyte is measured by forming a compound and measuring the activity of a labeling agent in the complex, the ratio of the analyte to the binding substance varies from an excessive amount (for example, excess antibody) to an excessive amount (e.g., excess antibody). For example, antigen excess), a standard curve is obtained in which the curve representing the relationship between the amount of the substance to be measured and the activity of the labeling agent gradually rises and then falls. This method measures the amount of the substance to be measured by measuring at least two points.

To carry out the present invention, the following steps are usually performed: A sample containing a large amount of antigen (hereinafter referred to as a large amount) and a sample containing a small amount of antigen (hereinafter referred to as a small amount) are separated from one specimen solution.
One sample is prepared, and an immobilized antibody suspension is added to it to allow the antigen and antibody to react.

Next, a labeled antibody solution is added to this reaction mixture to react, and then the solid phase is separated by centrifugation, and the solid phase is thoroughly pre-liquid, and the activity of the labeling agent contained therein is measured. For example, when using an enzyme as a labeling agent, a well-refined solid phase
Add an appropriate substrate solution, react, and measure enzyme activity. Compare the two measured values, and if the measured value for a large amount > the measured value for a small amount, then the standard curve will be on the rising side (left side), and if the measured value for a large amount < the measured value for a small amount, it will be on the downward side (right side). If the measured value of a large amount is equal to the measured value of a small amount, the measured value of the large amount is applied to the decreasing side and the measured value of the small amount is applied to the increasing side to determine the amount of antigen in the sample solution. Note that different amounts of antigens are usually collected by collecting different volumes of specimens with the same concentration, but it is of course possible to collect the same volume of specimens with different concentrations. Next, the principle of the measuring method of the present invention will be explained as shown in the schematic diagrams of FIGS. 2 and 3.

When a certain amount of antigen 1 to be measured and its insolubilized antibody 4 are reacted, if the amount of antigen 1 is smaller than the amount of insolubilized antibody 4, the principle of the sandwich method shown in FIG. 2 is activated. (However, do not separate the antigen-antibody complex from the reaction mixture.)

), the amount of unknown amount of antigen 1 is measured. Next, if the amount of antigen 1 to be measured is larger than the amount of insolubilized antibody 4, as shown in FIG. When a certain amount of labeled antigen 6 is reacted with the solution, the antigen-antibody complex 5 and the free antigen 1 cause a competitive reaction with the labeled antigen 6, and the more free antigen 1 is, the more it binds to the antigen-antibody complex 5. The amount of labeled antibody 6 decreases.

Therefore, the activity of the labeling agent bound to the antigen-antibody complex decreases in inverse proportion to the increase in the amount of antigen. That is, in the present invention, when the amount of antigen to be measured is small compared to the amount of insolubilized antibody, the principle of the sandwich method operates, and when the amount of the antigen is greater than the amount of the antibody, the principle of the competition method operates. , the operating principle is automatically selected depending on the quantitative relationship between antigen and antibody, and the standard curve is , a mountain-shaped curve that gradually rises and then falls as the amount of antigen to be measured increases, and a valley-shaped curve that gradually falls and then rises when the activity of the unbound liquid phase labeling agent is measured. draw

As is clear from the standard curve of the measurement method of the present invention, quantities that give the same measurement value exist on the rising side and the falling side of the standard curve. Therefore, from a single measured value, it cannot be determined whether the measured value is applied to the rising side or the falling side. Therefore, measure at least two quantities, and compare the magnitudes of the obtained measured values. If the measured value for a large quantity is larger than the measured value for a small quantity, then the standard curve is on the rising side. In the case of , the descending side is used, and in the case of large measured value = small measured value, both sides are used. To separate labeled antibodies that have bound to antigen-antibody complexes from labeled antibodies that have not bound to antigen-antibody complexes, the immobilized antibodies that are immobilized by binding the antibodies to a carrier such as a cellulose compound, polystyrene, or dextran are usually used. However, other separation methods such as the two-antibody method can also be used.

Labeling agents include radioactive isotopes (e.g., Yama 51, Skin 1, 3 Day, etc.), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, 3-D galactosidase, glycose oxidase, etc.), or fluorescent substances (e.g., , fluorescens isothiocyanate, rhodamine, etc.), each of which has its own characteristics, but it is most advantageous to use enzymes in consideration of measurement sensitivity, accuracy, and simplicity.

To carry out the present invention, the following steps are generally performed.

An insolubilized antibody suspension is added to a sample solution at an appropriate concentration to allow the antigen and antibody to react.

Next, a labeled antibody solution is added to this reaction mixture to react, and the solid phase is separated by centrifugation, thoroughly refined, and the activity of the labeling agent contained therein is measured. For example, when using an enzyme as a labeling agent, a well-refined solid phase
Add an appropriate substrate solution, react, and measure enzyme activity. The amount of antigen in the sample solution is measured by applying the obtained measurement value to a standard curve obtained by similarly operating a standard substance of known concentration. When performing measurements, conditions such as the amount of sample, concentration and amount of reagent used, reaction temperature and time, etc. vary depending on the type of substance to be measured, the titer of the antibody used, the type of labeling agent and its activity, etc. Therefore, the most appropriate conditions for each measurement are determined experimentally.

The activity of the labeling agent can be measured in either the solid phase or the liquid phase after centrifugation, but in the present invention, after the reaction between the sample solution and the insolubilized antibody, the antigen-antibody complex is reacted and mixed. Since it is not separated from the liquid, when measuring the activity of the liquid phase, the activity measurement may be interfered with by impurities present in the sample solution.

It is therefore advantageous to measure the activity of the solid phase, especially when enzymes are used as labeling agents. The present invention has the following features compared to conventional measurement methods.

As described above, the present invention includes the principles of the sandwich method and the competitive method in one measurement method, and as a result, a very wide concentration range can be measured.

This eliminates the need to prepare multiple dilution solutions to ensure that the measurement range is not exceeded, unlike in the past, even when the amount of the substance to be measured is wide and it is difficult to predict the amount present in the sample solution. , the operation is simple, there are few errors due to dilution, and the measurement accuracy can be improved. Also,
In the conventional sandwich method, it was necessary to perform the reaction product separation operation twice, but with the present invention, it is only necessary once, which simplifies the operation and reduces loss of sample due to the separation operation, improving measurement accuracy. can be improved. moreover,
In the present invention, after the antigen and antibody are reacted, the reaction proceeds without separating the antigen that did not bind to the antibody, so that even the antigen that did not react sufficiently with the antibody in a short period of time can be reacted with the antibody. Because the binding is promoted and the amount of antigen that binds to the antibody increases, ``the measurement sensitivity can be increased compared to conventional measurement methods.

The present invention will be specifically explained with reference to Examples.

Example 1 Measurement of Q-fetoproteina Preparation of Q-fetoprotein standard solution Nishi et al.
cer Res-, 30, 2507-2513, (19
Q-fetoprotein (hereinafter abbreviated as AFP) was extracted and purified from ascites of patients with liver disease by the method described in 70)).

) in phosphate buffered saline (hereinafter abbreviated as BSA) containing 0.5% Vienna 2.0 and 1% bovine serum albumin (BSA).
Hereinafter abbreviated as P Satoshi. ), he was relegated to the concentration of 1bi, 1pi, 1pi, 1pi, 1bi, 1pi and 107n Yu/gu. b Production of anti-AFP antibody A that was extracted and purified from ascites of patients with liver disease by the method of Nishi et al.
FP was dissolved in physiological saline to a concentration of 2:9/secretion, 0.5' of it was mixed with Freund's complete adjuvant, and rabbits were immunized five or more times to obtain anti-AFP serum.

This antiserum was salted out with sodium sulfate to obtain anti-AFP antibody globulin. c. Preparation of anti-AFP antibody/cellulose conjugate Add cellulose powder (manufactured by Merck & Co., Ltd.) to 2.5% bromic cyanide solution 320°C for 8 minutes, adjust the pH to 10-11 with IN sodium hydroxide solution, and heat under light for 2 minutes. After reacting, the mixture was filtered through a glass filter and washed with a 0.1M sodium carbonate hydroxide solution to obtain activated cellulose.

Next, this activated cellulose is suspended on a 0.1M sodium bicarbonate solution 32, and the anti-AF is added to this suspension.
P antibodies 8 and 9 were added and allowed to react at 4°C for 2 hours. After the reaction, wash with PBS and mother M urea/0.2 M glycine mixture (pH 7.0), and add 0.5% Tween 2.
．． They were suspended in PBS containing 0 and 1% MoA at lo% concentrations. d Production of anti-AFP antibody/enzyme conjugate Horseradish peroxidase (Horserraddjsh Peroxidase, hereinafter abbreviated as HRP).

) was dissolved in 0.9M sodium hydroxide solution of 1%, 1% 2,4-dinitrofluorobenzene of 0.1% was added, and the mixture was incubated at room temperature for 1 hour. 0.0 in this solution
After adding 1 part of 8M sodium periodate solution and mixing at room temperature for 30 minutes, 1.0 part of 0.18M ethylene glycol solution was added and mixed for 1 hour at room temperature. After overnight dialysis against 0.01 M carbonate buffer (pH 9.5),
The anti-AFP antibody prepared in b) above was dissolved in 0.01 M carbonate buffer (pH 9.5) to a concentration of 5:9, and an anti-AFP antibody solution of 1.0' was added thereto, and the mixture was heated at room temperature. After reacting for 3 hours, add sodium borohydride from 5 to 9.
The reaction was continued for an additional 3 hours at °C.

After the reaction, it was dialyzed against P plaques (PH7.2) overnight, and then fractionated and purified using Cephadex C200 to obtain anti-AFP.
An antibody/HRP conjugate (hereinafter abbreviated as antiAFP/HRP) was obtained. e. Creation of standard curve 0.1' of the standard AFP solution of each concentration prepared in a above.
into a test tube, and add the anti-AFP antibody produced in step c above.

Add 0.4 ml of cellulose conjugate suspension to 60 ml at room temperature.
Allowed to react for minutes. After the reaction is complete, add the above d to this reaction mixture.
antiAFP/HRPO. Add ' of 1,
The reaction was allowed to proceed for 12 minutes at room temperature.

After the reaction is completed, centrifuge and remove the solid phase with 0.005% Vienna 2.
Wash thoroughly twice with physiological saline containing 0, and add 5-aminosalicylic acid 60% 9/d' and 0.3% hydrogen peroxide solution 1/2 times.
Substrate solution 3′ containing

After stopping the reaction, it was briefly centrifuged and the absorbance of the supernatant was measured (5
0m). The standard curve at this time is shown in FIG. Next, a measurement example using this standard curve will be described.

f. Measurement of AFP in the serum of normal subjects and patients The amount of AFP in the serum of normal subjects, pregnant women, and subjects with liver disease was measured according to the method for preparing the standard curve in e. above.

Place 0.05 and 0.1 samples of the measurement sample in test tubes, and add 0.0 and 0.1 samples of the anti-AFP antibody/cellulose conjugate suspension prepared in step c above to the test tubes. The mixture was added and reacted for 60 minutes at room temperature.

Thereafter, the absorbance in 50 plates was measured in the same manner as in e. ．． Absorbance of sample amount 0.05 (ODo
．． .

5) and the absorbance (ODo.,) of a sample amount of 0.1 secretion. Hawk SOD.

．． , then the rising side of the standard curve, ODo. o5>ODo. ．．
If so, the amount of AFP in the serum was calculated using the descending side. The results are shown in Table 1.

Table 1 Example 2 Measurement of hCG a Preparation of standard hCG solution Dissolve the hCG standard product listed in the Japanese Pharmacopoeia in PBS containing 0.1% BSA, 1bi,1pi,1pi,1pi,I04
hCG solutions with concentrations of , 1 and 1 pi/miu were prepared.

b Production of anti-hCG antibody Commercially available hCG was purified using a gel furnace method using an anion exchange resin and Cephadex, and this was used as an antigen in Example 1.
A domestic rabbit was immunized using method -b.

Normal human serum and urine components were added to the obtained antiserum to absorb and remove antibodies against the serum and urine components, followed by salting out with sodium sulfate to obtain anti-hCG antibodies. c.
Production of anti-hCG antibody-sensitized polyethylene twill Anti-hC in b.
G in glycine buffer (pH 8.2) to a concentration of 10 μg/secretion.
), dissolve in this antibody solution 10' diameter 3 skin, length 8
100 rib polyethylene twills were placed and incubated at 37q0 for 2 hours.

After washing this with physiological saline, P plaques containing 1% normal rabbit serum were added and left at 4° C. overnight to produce anti-hCG sensitized polyethylene strips. d Production of anti-hCG antibody/HRP conjugate According to the method of Example 11d, HRP of 5-9 and the above c
Anti-hCG antibody/HRP
A conjugate was obtained.

e. Creation of standard curve P spot 0.4' containing 0.1% MoA, standard hC of above a.
0.1 of the G solution and three anti-hCG antibody-sensitized polyethylene sticks prepared in c above were placed in a test tube and incubated at 37°C for 60 minutes.

Next, add 0.1 of the anti-hCG antibody/HRP conjugate solution of d above.
and incubated at 3700 for 180 minutes.
After the reaction is complete, add 0.005% Tween 20 to the polyethylene rod.
Wash with physiological saline containing 0.5 oz of substrate solution (o
-Phenylenediamine: 5 of 9/enemy and P spots, PH6.4
> added. After reacting overnight at room temperature, two IN day CIs were added and the optical intensity at 49 km was measured. The obtained standard curve is shown in FIG. Next, an example of measurement using this standard curve will be described. f Measurement of hCG in the serum of normal subjects and patients The amount of hCO in the serum of non-pregnant women, pregnant women, and patients with hydatidiform mole was measured according to the method for creating the standard curve in e. above.

Place 0.05 and 0.1' of the measurement sample in a test tube, and add 0.4' of P containing 0.1% A.
Then, the anti-hCG sensitized polyethylene resin prepared in step c above was added, and the mixture was allowed to react at room temperature for 6 minutes.

Thereafter, the absorbance at 49 m was measured in the same manner as in e. Absorbance (ODo.) with sample amount 0.05.

5) and the absorbance (OD) above the sample amount of 0.1.

．． ), and ODo. If <ODM, then on the rising side of the standard curve, ODo. o5>ODo. , then the amount of hCG in the serum was calculated using the descending side. The results are shown in Table 2. Table 2

[Brief explanation of drawings]

Figure 1 is a model diagram to explain the measurement principle of the competitive method, Figure 2 is a model diagram to explain the measurement principle of the sandwich method, and Figure 3 is a model diagram to explain the measurement principle of the measurement method of the present invention. 4
The figure is a graph showing a standard curve for AFP measurement in Example 1, and FIG. 5 is a graph showing a standard curve for hCG measurement in Example 2. In the figure, 1...unlabeled antigen, 2...labeled antigen, 3...antibody, 4...insolubilized antibody, 5...
...antigen-antibody complex, 6...represents a labeled antibody. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Claims (1)

[Claims] 1. After a substance to be measured and a substance that specifically binds to this substance (hereinafter referred to as a binding substance) are reacted, the reaction product is further bonded without separating the reaction product from the reaction solution. By reacting a labeling substance with a labeling agent bound to a binding substance,
A method for immunochemically measuring a target substance by forming a target substance-labeling substance complex and measuring the activity (hereinafter referred to as activity value) of the labeling agent in this complex. A standard curve is obtained in which the curve representing the relationship between the amount of the substance to be measured and the activity value gradually rises and then falls by reacting at various proportions of the substance ranging from too little to too much. At least two quantities of the substance to be measured, one of which contains more of the substance to be measured than the other (hereinafter referred to as a large amount and a small amount, respectively), are collected from the test solution, and the measurement operation is performed on each of them, and the thus obtained Examine the magnitude relationship between the two activity values, a. If the activity value for the large amount is greater than the activity value for the small amount, apply each measurement value to the rising side of the standard curve, b. If the activity value for the large amount is smaller than the activity value for the small amount, Fit each activity value to the falling side of the standard curve, and c If the large and small activity values are equal, fit the large activity value to the falling side of the standard curve and the small activity value to the rising side to determine the amount of substance to be measured. An immunochemical measurement method characterized by: 2. Collect a large amount and a small amount of the same test liquid, one with a large volume,
The method according to claim 1, wherein the other method is carried out by collecting a small amount. 3 Collect a large amount and a small amount of the same test solution, one undiluted,
The method according to claim 1, wherein the other one is undiluted and the same volume is collected from each other. 4. The method according to claim 1, wherein a large amount and a small amount of the same test liquid are collected by concentrating one sample liquid and collecting the same volume of the same sample liquid from the other sample without concentrating the other liquid.