JPH08292192A - Immunoassary - Google Patents

Abstract

PURPOSE: To provide an immunoassay capable of simply and accurately measuring the amt. of an antigen or antibody in an antigen or antibody excessive region and capable of easily obtaining proper dilution factor even when it is necessary to dilute a specimen. CONSTITUTION: In a concn. curve, two calibration curves are formed with respect to a region before the generation of the so-called hook phenomenon and a region on and after the generation thereof and either one of the calibration curves is used to obtain an amt. of an object. The degree A of agglutination generated by the reaction of the object with the corresponding antigen or antibody is measured to form a calibration curve I showing the relation between the degree A and the amt. of the object. The object and the corresponding antigen or antibody are mixed in a more excessive ratio and the degree B of agglutination generated by antigen-antibody reaction is measured and B/A is calculated from the degrees A, B to form a calibration curve II showing the relation between B/A and the amt. of the object. A, B and B/A are calculated with respect to the specimen containing the object and the amt. of the object in the specimen is obtained from the calibration curves I, II.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method used in the field of clinical examination and utilizing the agglutination reaction of an antigen or an antibody against a substance to be measured.

[0002]

2. Description of the Related Art An antigen-antibody reaction is used to detect or quantify a specific substance from various biological components. Generally, an antigen to be measured or an antibody corresponding to the antibody or a method of reacting the antigen is used. To be As the measuring method, there are a method of using an insoluble carrier and a method of directly detecting agglutination without using a carrier.The former method carries an antibody against the antigen to be measured as a carrier such as latex, and its immune reaction. The antigen in the sample is detected or quantified by measuring the degree of aggregation generated by.

As a method for detecting the degree of agglutination, there are a method of visually judging the presence or absence of agglutination, and a method of irradiating the reaction solution with light to measure scattered light or transmitted light. It is used as a quantitative method, and the optical measurement method is used as a quantitative method of antigens.

Since the reactivity of the above-mentioned antigen-antibody reaction varies depending on the ratio of the antigen to the antibody, even if the degree of aggregation is measured by the above method, the value is in the antibody excess region or in the antigen excess region. It is necessary to judge whether it is a thing.

For example, in Japanese Unexamined Patent Publication No. 4-204378, the absorbance of a mixed solution of a sample and a reaction reagent is measured at two wavelengths, and after a certain period of time, the same two wavelengths are measured.
It is disclosed that the ratio of the difference in absorbance between different times at both wavelengths is compared with a preset reference value to determine whether the region is an antigen or antibody excess region. However, it is possible to determine whether it is an excess region of an antigen or an antibody, but it is not possible to obtain an accurate concentration.

Further, in Japanese Patent Laid-Open No. 62-293163, after the first measurement, the second measurement is carried out by changing the amount of the antigen or antibody in the sample or reagent, and the change in the measured value It is disclosed that the excess of antigen or antibody is determined and the concentration is determined. However, even if the amount of change cannot be read in a high-concentration sample or it can be determined that the antigen or antibody is excessive, the measurement must be performed twice to obtain the correct concentration.

[0007]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above problems, and an object of the present invention is to measure the amount of an antigen or antibody simply and accurately even in the antigen or antibody excess region. It is to provide a possible immunoassay method.

[0008]

[Means for Solving the Problems] When measuring the amount of a specific substance in a sample, it is generally carried out in a method of detecting an immune complex formed by an immune reaction with an antigen or an antibody corresponding to the object. In the method, the concentration curve of the antigen or antibody with respect to the degree of aggregation is obtained in advance and the concentration is obtained from the degree of aggregation of the sample.

When the object to be measured in this concentration curve is an antigen, the degree of aggregation and the antigen concentration show linearity in the low-concentration region of the antigen, but as the antigen concentration increases, the absorbance at a given time increases. It is known that the linearity is lost due to the change in the temperature and the reaction rate, and this phenomenon is generally called the hook phenomenon. The concentration curve region after the hook phenomenon occurs is generally called a prozone region or an antigen excess region.

In the immunoassay method of the present invention, two calibration curves are prepared for the region until the hook phenomenon occurs and the prozone region, and the amount of the object in the sample is obtained by using one of the calibration curves. And includes the following five steps.

In the first step, a known amount of the measurement target and an antigen or antibody corresponding to the measurement target are mixed. At this time, since the antigen-antibody reaction causes aggregation, the degree A of this aggregation is measured. The same operation as above is performed for various known amounts of the measurement object, and the degree of aggregation A
To measure.

In the second step, a calibration curve I showing the relationship between the degree A of aggregation and the amount of the object to be measured is prepared from the obtained values. This calibration curve I is used in the area until the hook phenomenon occurs.

In the third step, when the antigen or antibody corresponding to the measurement target substance is mixed with various known amounts of the measurement target substance used in the first step, it is used in the first step. An excess amount of antigen or antibody is mixed. Then, the degree B of aggregation is measured in the same manner as in the first step.

As a method for mixing an excess amount of the antigen or antibody, the substance to be measured may be mixed with an excess amount of the corresponding antigen or antibody at a time, or first, the excess amount of the antigen or antibody is mixed and then the second amount is mixed. The same amount of antigen or antibody as in the first step may be mixed, for example, two or more times, and the total amount may be an excess amount than that used in the first step.

In the fourth step, the ratio of A and B (hereinafter referred to as "A / B ratio") is obtained from the aggregation degrees A and B obtained in the first and third steps, and A / B A calibration curve II showing the relationship between the ratio and the amount of the measurement object is created.

From the above A / B ratio, the point where the hook phenomenon occurs in the measuring system can be estimated. In the area until the hook phenomenon occurs, the A / B ratio shows a low value,
When the hook phenomenon occurs, the A / B ratio increases linearly. A
The region where the / B ratio increases linearly is the prozone region, and the calibration curve II is used in this region.

In the fifth step, with respect to the sample containing the substance to be measured, the above first and third steps are repeated to measure the aggregation degrees A and B, and further the A / B ratio is obtained. From this value, the amount of the object to be measured in the sample can be obtained from the calibration curve I if the concentration is in the region until the hook phenomenon occurs in the sample, and from the calibration curve II if the concentration is in the prozone region. . In the immunoassay method of the present invention, the order of the above steps is not particularly limited.

When the sample is diluted as necessary, A
By obtaining the / B ratio, it is possible to select a dilution ratio such that the concentration is equal to or lower than the concentration corresponding to the A / B ratio estimated to cause the hook phenomenon.

The sample containing the measurement target is a sample containing an immunologically active substance to be measured such as an antigen and an antibody, particularly a biological sample. As the biological sample, for example,
Examples thereof include body fluids such as blood, pleural effusion, ascites and lymph, excrements such as urine, stool, sweat, and extracts of tissues.

As the substance to be measured, any immunologically active substance such as an antigen or an antibody capable of reacting with an antigen,
That is, any of the substances that have been conventionally measured is included, for example, proteins, polypeptides, polysaccharides, lipids,
Examples include steroids. As the above protein,
For example, alpha fetoprotein (AFP), fibrinogen degradation product (FDP), C-reactive protein (C
RP), blood proteins such as human chorionic gonadotropin (HCG), antigens against infectious diseases such as hepatitis B virus (HB), hepatitis C virus (HC), human immunodeficiency virus (HIV), and their antibodies, receptors , Enzymes, etc.

The antigen or antibody corresponding to the substance to be measured may be carried on an insoluble carrier. As the insoluble carrier, for example, organic polymer powder, inorganic substance powder,
Examples include microorganisms, blood cells and cell membrane pieces, and plastic microtiter plates. Examples of the organic polymer powder include natural polymer powders such as insoluble agarose, cellulose and insoluble dextran, methacrylic acid polymers, acrylic acid polymers, polystyrene, styrene-styrene sulfonate copolymers, acrylonitrile-butadiene copolymers. Polymers, acrylonitrile-butadiene-styrene copolymers, vinyl chloride-acrylic acid ester copolymers, vinyl acetate-acrylic acid ester copolymers and the like synthetic polymer powders, and the like, especially synthetic polymer powders uniformly Suspended latex is preferred.

As the above-mentioned inorganic substance powder, for example, gold,
Metal pieces such as titanium, iron, nickel, silica, alumina,
Carbon powder etc. are mentioned. The average particle size of the insoluble carrier varies depending on the measuring method and measuring equipment, but is usually 0.0
The thickness is 5 to 1.0 μm, preferably 0.05 to 0.5 μm. The concentration of the insoluble carrier used is usually 0.05 to 3.0 mg / m when used for optical observation.
1, preferably 0.2 to 1.0 mg / ml, and usually 0.5 to 20 mg / ml, preferably 1.0 to 5.0 mg / ml when used for visual observation. Examples of the method of supporting the antigen or antibody on the insoluble carrier include a method of sensitizing by chemical or physical binding.

In the immunoassay method of the present invention, the above-mentioned antigen-antibody reaction is carried out under ordinary conditions, and the pH in the reaction is usually 5-10, preferably 6-8. Examples of the buffer solution used include phosphate buffer solution, Tris buffer solution, glycine buffer solution, citrate buffer solution, and ammonia buffer solution. The reaction temperature is generally 0 to 50 ° C, preferably 20 to 40 ° C. The reaction time is usually 20 seconds to 30 minutes,
It is preferably 1 to 15 minutes.

As a measuring system for measuring the antigen-antibody reaction, for example, an immunoturbidimetric method, a latex agglutination method, a hemagglutination method and the like are used. Examples of the method for measuring the degree of aggregation generated by the above reaction include a method of optically observing the degree of aggregation and a method of visually observing the degree.

In the optical observation method, the intensity of scattered light, the absorbance, the intensity of transmitted light or the number of particles of the mixed solution after the above reaction is detected by a known optical instrument or the like. Measurement wavelength is 30
0 to 2400 nm can be used. According to a known method, the increase or decrease in scattered light intensity, absorbance, or transmitted light intensity is measured according to the particle size, concentration, and reaction time of the insoluble carrier used. When measuring in a shorter time, the agglutination reaction rate and the absolute value of absorbance can be used. In addition, these methods may be used alone or in combination. In the method of observing by visual observation, it is possible to make a determination by taking an image with a video camera and performing image processing, instead of simply making a determination with the naked eye when determining the presence or absence of aggregation.

If necessary, a reaction promoting substance may be added to the above measurement system. As the reaction promoting substance, for example,
Examples thereof include polyethylene glycol, polyglycosyl methacrylate, polyvinylpyrrolidone, carboxymethyl cellulose, dextran, pullulan and the like.

[0027]

EXAMPLES The present invention will be described with reference to examples. The following reagents were used in the examples. -Phosphate buffer (PBS): monosodium phosphate (dihydrate), disodium phosphate (dihydrate) and sodium chloride, the final concentration of phosphoric acid is 0.02M, the final concentration of sodium chloride Of 0.15 M and pH of 7.2 dissolved in purified water-1% BSA-PBS: 1 ml of bovine serum albumin (BSA, reagent grade) dissolved in 100 ml of PBS-5% BSA-PBS : A solution in which 5 g of bovine serum albumin (BSA, reagent grade) is dissolved in 100 ml of PBS

(Example 1) Measurement of human HBs antigen (hepatitis B surface antigen) by latex agglutination method [Preparation of antibody-bound latex solution] Anti-HBs monoclonal antibody (Seikagaku Corporation, code No. 230853) was used in PBS.
900 μl of solution prepared to 0.5 mg / ml
And 50 μl of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) having a particle size of 0.2 μm were mixed and left at 4 ° C. overnight. Then, this solution is centrifuged and the supernatant is removed to obtain a precipitate.
1 ml of% BSA-PBS was added, and blocking was performed at 37 ° C. for 2 hours. Then, wash 3 times with physiological saline, and add 1% B
2 ml of SA-PBS was added to obtain an antibody-bound latex solution.

[Preparation of calibration curve] An HBs antigen standard product (HBs antigen standard product kit manufactured by Mitsubishi Chemical Co., Ltd.) was prepared with 5% BSA-PBS so as to have a concentration of 500 U / ml, and then serially diluted, 400, 3
00, 200, 100, 50, 0 (5% BSA-PBS
Only) U / ml standards were prepared.

100 μl of each of the above standards was added to 1 ml of 1% B
Add to SA-PBS and mix. Next, 200 μl of the above antibody-bonded latex solution was added thereto, and the amount of change in absorbance at a measurement wavelength of 570 nm for 5 minutes was measured by a biochemical automatic analyzer (Hitachi U-3200 type, manufactured by Hitachi, Ltd.) using a cell with an optical path length of 1 cm. A was measured (Table 1), and a graph (FIG. 1) showing the relationship between the standard product concentration and the absorbance change amount A was created.

Anti-HBs monoclonal antibody 0.1 μm
1 ml of a solution prepared with 1% BSA-PBS to give g / ml and the above 500, 400, 300, 200, 10
100 μl of 0, 50, 0 U / ml standard was mixed.
Next, 200 μl of the above antibody-bonded latex solution was added thereto, and the amount of change in absorbance at a measurement wavelength of 570 nm for 5 minutes was measured by a biochemical automatic analyzer (Hitachi U-3200 type, manufactured by Hitachi, Ltd.) using a cell with an optical path length of 1 cm. B was measured (Table 1).

B / A × 100 was determined from the above A and B (Table 1). From this value and also in Figure 1 created above
It is considered that the hook phenomenon is occurring at 300 U / ml or more from the graph shown in, so 0-200 U / ml
A calibration curve I (Fig. 2) showing the relationship between the standard concentration and the absorbance change amount A was prepared from the measured value A of (y = 0.15x +3.4, r = 0.9
9) For 300 to 500 U / ml, a calibration curve II (FIG. 3) showing the relationship between the standard product concentration and B / A × 100 was prepared (y = 8.6x + 114.4, r = 0.99). In this measurement system, the calibration curve I was used for B / A × 100 of 10 or less, and the calibration curve II was used for B / A × 100 of more than 10.

[0033]

[Table 1]

[Measurement of Specimens] With respect to the specimens determined to be HBs antigen-positive, the absorbance was measured in the same manner as described above, and A, B and B / A × 100 were determined for each specimen. Since the B / A of the samples 1 and 3 was 10 or less, the antigen concentration in the sample was obtained from the calibration curve I, and the samples 2 and 4 had the B / A × 100 of more than 10, the calibration curve II. Table 2 shows the results.

Further, the specimens 2 and 4 were diluted 10 times and the absorbance was measured again in the same manner as above, and A ′, B ′ and B ′ / A ′ × 100 were obtained for each specimen.
The antibody concentration in the sample was determined from the calibration curve I. Table 2 shows the results. This value almost agrees with the value obtained above, and according to the method of the present invention, it is not necessary to dilute and measure again a high-concentration sample.

[0036]

[Table 2]

(Example 2) Measurement of human CRP (C-reactive protein) by immunoturbidimetric method [Preparation of antibody solution for measurement] Anti-CRP antibody (immunized to sheep,
Seikagaku Corporation, code No.370443) 0.1 mg / m
It diluted with 1% BSA-PBS so that it might become 1 and prepared the antibody solution for a measurement.

[Preparation of calibration curve] CRP standard product (CRP standard solution manufactured by Denka Seiken Co., Ltd.) 20
mg / dl is serially diluted with 5% BSA-PBS, and 1
5, 10, 5, 2, 1, 0 (5% BSA-PBS only)
A mg / dl standard was prepared.

100 μl of each standard above and 1% BSA-P
1 ml of BS was mixed. Next, 300 μl of the above-mentioned antibody solution for measurement was added to this, and a biochemical automatic analyzer (Hitachi U-3
Model 200, manufactured by Hitachi, Ltd.) was used to measure the change A in absorbance for 5 minutes at a measurement wavelength of 340 nm using a cell having an optical path length of 1 cm (Table 3).

An anti-CRP antibody was diluted with 1% BSA-PBS so as to have an amount of 0.5 μg / ml to prepare an antibody solution for addition. 1 ml of this addition antibody solution and the above 20, 15,
100 μl of 10, 5, 2, 1, 0 mg / dl standard was mixed. Next, 300 μl of the above-mentioned antibody solution for measurement was added thereto, and the amount of change in absorbance at a measurement wavelength of 340 nm for 5 minutes was measured by a biochemical automatic analyzer (Hitachi U-3200, manufactured by Hitachi, Ltd.) using a cell having an optical path length of 1 cm. B was measured (Table 3).

B / A × 100 was determined from the above A and B (Table 3). From this value, it is considered that the hook phenomenon occurs at 10 mg / dl or more, so 0 to 5 mg / d
A calibration curve I (Fig. 4) showing the relationship between the standard product concentration and the absorbance change amount A was prepared from the measured value A of 1 (y = 0.0028x -0.047,
r = 0.99) and 10 to 20 mg / dl, a calibration curve II (FIG. 5) showing the relationship between the standard concentration and B / A × 100 was prepared (y = 0.87x + 5.3, r = 0.99). In this measurement system, the calibration curve I was used for B / A × 100 of 5 or less.
The calibration curve II was used for those exceeding 5.

[0042]

[Table 3]

[Measurement of Specimens] Regarding the specimens judged to be CRP positive, the absorbance was measured in the same manner as above and the A, B and B / A × 100 of each specimen were determined. Samples 1, 2 and 4 had a B / A x 100 of 5 or less, so the CRP concentration in the sample was obtained from the calibration curve I, and since Samples 3 and 5 had a B / A of more than 5, the calibration curve II It was
The results are shown in Table 4.

Further, the samples 3 and 5 having B / A × 100 exceeding 5 were diluted 10 times and the absorbance was measured again in the same manner as above, and A ′, B ′ and B ′ / A ′ were obtained for each sample. × 100 is calculated, and the CR in the sample is determined from the calibration curve
The P concentration was determined. The results are shown in Table 4. This value almost agrees with the value obtained above, and according to the method of the present invention, it is not necessary to dilute and measure again a high-concentration sample.

[0045]

[Table 4]

[0046]

EFFECT OF THE INVENTION The immunoassay method of the present invention is as described above, and the amount of the target substance can be measured easily and accurately,
It is not necessary to dilute and measure again even a sample having a concentration in the antigen or antibody excess region. Even when the sample needs to be diluted, an appropriate dilution ratio can be easily obtained.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the concentration of a standard product and the degree of aggregation caused by an antigen-antibody reaction in Example 1. The vertical axis represents the absorbance change amount A (mABS × 10), and the horizontal axis represents the HBs concentration (U / ml).

FIG. 2 is a calibration curve I in the region until the hook phenomenon occurs in Example 1. Vertical axis shows HBs concentration (U / ml)
The horizontal axis represents the amount of change in absorbance A (mABS × 10).

FIG. 3 is a calibration curve II in the prozone region of Example 1. The vertical axis is HBs concentration (U / ml), the horizontal axis is B / A
Indicates × 100.

FIG. 4 is a calibration curve I in a region until the hook phenomenon of Example 2 occurs. Vertical axis shows CRP concentration (mg / dl)
The horizontal axis represents the amount of change in absorbance A (mABS × 10).

5 is a calibration curve II in the prozone region of Example 2. FIG. The vertical axis represents CRP concentration (mg / dl) and the horizontal axis represents B /
A × 100 is shown.

Claims (1)

[Claims] 1. A step of mixing [1] an object to be measured and an antigen or antibody corresponding to the object to be measured, and measuring the degree A of aggregation caused by an antigen-antibody reaction; [2] The degree A of aggregation and measurement A step of creating a calibration curve I showing the relationship with the amount of the object; [3] a measurement object and an excess of the antigen or antibody corresponding to the measurement object used in the step [1] are mixed, A step of measuring the degree of aggregation B caused by the antigen-antibody reaction; [4] A calibration showing the relationship between the ratio of A and B and the amount of the object to be measured by obtaining the ratio of A and B from the above-mentioned degrees of aggregation A and B. line
Step of creating II; [5] Regarding the sample containing the measurement object, the above [1]
And the step [3] is repeated to obtain A, B, and the ratio of A and B, and obtain the amount of the measurement object in the sample from the calibration curve I or II; Law.