JPH01301165A - Immunoassay - Google Patents

Abstract

PURPOSE:To rapidly and accurately measure a vital component by dispensing and mixing a monoclonal antibody B1 which labels a specific antigenic determinant or an immune diagnosis reagent contg. said antibody B1 to and with a specimen. CONSTITUTION:The first monoclonal antibody B1 or the immune diagnosing reagent contg. the same is first added to the specimen. The antibody or reagent is added at such a concn. at which the antibody and the antigen come into contact with each other in a relatively thick state. An immune complex C1 is rapidly formed in this way. The monoclonal antibody which is separate from the first monoclonal antibody and/or the polyclonal antibody B2 or the immune diagnosing reagent contg. said antibody is brought into reaction with this immune compelx C1, by which the immune complex C2 measurable by a spectrophotometer is formed. The immune complex C2 is rapidly formed as the immune complex C1 exists in the system. The agglutination reaction progresses in a short period of time and the absorbance of the reaction system increases. The accurate determination of the material to be measured in a short period of time is enabled by measuring the absorbance.

Description

[Detailed description of the invention] (Industrial application field) The present invention is based on immunoagglutination (immunoassay).

In particular, it relates to immunoassay methods with high measurement sensitivity.

(Prior art) As one method for measuring trace components in body fluids, a substance (antibody) capable of antigen-antibody reaction with the target analyte is applied to the target analyte, and the degree of agglutination that occurs is measured. Immunoassay is used. As such methods, immunoturbidimetry, latex a collection method, etc. are known.
Method 9 for Measuring Increase in Turbidity of Reaction System Due to Aggregation Measurement is performed by a method such as measuring changes in the number of particles or particle size distribution using a blood cell counter.

Various methods have been proposed to increase the measurement sensitivity in measuring the increase in turbidity in a reaction system due to aggregation1.
For example, Japanese Patent Publication No. 62-43138 discloses that the absorbance of a reaction system between a substance to be measured and a latex reagent is measured after the reaction has started.
A method of measuring at least twice is disclosed. In JP-A-59-187264.

A method is disclosed in which the intensity of transmitted light and scattered light incident on a reaction system is measured using an integrating sphere turbidity meter.

Various measurement systems and kits that include these and other improved methods and are used in the field of clinical testing are commercially available. For such a system, LPIΔ(
Diayatron Co., Ltd.).

LA System (Alphatic Co., Ltd.), EL-1
There are various dedicated measurement systems such as 000 (Kyowa Medex Co., Ltd.). As a measurement kit, a general-purpose biochemical automatic analyzer (for example, Hitachi 705 model 9, Hitachi 736 model 2, etc.) was used as an 8g measuring device, and various measurement kits using latex agglutination reaction (Kainos Co., Ltd., Kainos Co., Ltd.) were used as measurement kits. Yatron, etc.). As a method for measuring the change in the number of particles or the distribution of particle size in the above reaction system, for example, JP-A-60-111963 discloses a method of counting the number of particles on the particle size side in the reaction solution and measuring the degree of aggregation. A method is disclosed.

Among these methods, the agglutination reaction measurement method using the above system or kit is widely used. For example, in order to perform an antigen-antibody reaction (agglutination reaction) between an antigen substance contained in a sample and a reagent containing an antibody (immunodiagnostic reagent) using the above system, the following steps are generally adopted. Will be:
1-(a) Dispensing the sample into a reaction container; 1-(b)
A step of dispensing a diluent into a reaction container to dilute the specimen; and 1-(c) a step of adding an immunodiagnostic reagent to the reaction container to start one reaction. Alternatively, 2-(a) dispensing the immunodiagnostic reagent into a reaction container; and 2-(b) leaving the specimen as is.

Alternatively, the step of adding the diluent to the reaction container to start three reactions. And the measurement of the rate of this reaction is of the ninth order (
Performed by method 1), (2) or (3): (1
) After step 1-(b) above, measure once to obtain a sample blank value, and then measure once after step 1-(c) and use the obtained value to determine the sample blank value or the sample blank value. Subtract the value corrected for the change in volume at each step (this method is effective when the sample blank value is high due to chyle or hemolysis); measure once after step 2-(a) above. A blank value is obtained, and then, from the value obtained by measuring once after step 2-(b), the blank value or a value corrected for the change in capacity in each step for the blank value is subtracted. (3) 1-(c) or 2- above)
(b) Determine the reaction rate from the values measured two or more times after the step (in this method, the blank values of the analyte and reagent are irrelevant to the measured values).

In these measurement methods, the reaction between the antigen in the sample and the antibody in the immunoreagent is performed in step 1-(c) or 2-(b) above.
), and the antibodies used in the reagents in step 1-(c) or 2-1) are mainly polyclonal antibodies suitable for this reaction. Therefore, in order to obtain sufficient measurement sensitivity, the above steps 1-(c) or 2-(
It is necessary to set the reaction time carried out in b) to be long. However, in daily testing, rapid testing is often desired, and there is a limit to how long one reaction time can be extended.
It is desired to develop a highly sensitive immunoassay method that can be performed accurately with a shorter reaction time.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and its purpose is to rapidly and accurately measure biological components etc. by utilizing agglutination caused by antigen-antibody reactions. The goal is to provide a way to do so.

(Means for Solving the Problems) The immunoassay method of the present invention comprises (A) a specimen containing an antigen;
Antibody Bl is a type of antibody against the antigen.

A substance containing the antibody B1, a carrier-bound antibody in which the antibody B1 is bound to a microparticle carrier, or a carrier-bound antibody in which a substance containing the antibody B1 is bound to a microparticle carrier are mixed and reacted to form an immune complex. Step of forming body C1; (B)
An antibody 82. which is an antibody against the antigen and is different from the antibody B1 is added to the reaction solution containing the immune complex C5. A substance containing the antibody B2, a carrier-bound antibody in which the antibody B2 is bound to a microparticle carrier, or a carrier-bound antibody in which the substance containing antibody B2 is bound to a microparticle carrier are mixed and reacted to form an immune complex. forming C2; and (C
) Measuring the agglutination state of the obtained reaction solution and calculating the antigen concentration in the sample from the measured value, thereby achieving the above object.

The specimen to be measured by the method of the present invention is a sample containing a component that can be an antigen, particularly a biological sample (eg, serum or urine). The components to be measured in the above sample are:
Any substance that can be an antigen is included. In other words, all substances measured using conventional methods are included. Examples include alphafetoprotein (8FP), fifirinogen, C-reactive protein (CRP), human chorionic gonadotropin (IICG), carcinoembryonic antigen (CEA), and the like.

Antibody B is one or more types of monoclonal antibodies that can recognize and bind to specific antigenic determinants. As the antibody B2, either a monoclonal antibody or a polyclonal antibody can be used. These monoclonal and polyclonal antibodies are
- Obtained by known methods using extensive immunization and purification steps. For example, monoclonal antibodies can be obtained by using hybridomas; by using EB virus that causes lymphoma to increase specific B lymphocytes; or by genetically manipulating cultured cells derived from mouse bone marrow or microorganisms such as yeast. various types of chromatography (e.g. gel chromatography, ion exchange chromatography).

(e.g., affinity chromatography). Polyclonal antibodies can be obtained, for example, by immunizing a suitable animal with a substance that serves as an antigen, and purified by performing the same purification procedures as monoclonal antibodies.

These monoclonal and polyclonal antibodies can be used as such, or, if necessary, in the form of substances containing the antibodies.

in the form of a carrier-bound antibody, in which the antibody is bound to a microparticle carrier;
Alternatively, a substance containing the antibody is used in the form of a carrier-bound antibody bound to a microparticle carrier (hereinafter these are referred to as immunodiagnostic reagents). As the microparticle carrier, inert synthetic polymer particles (eg, polystyrene particles) or inorganic particles (eg, kaolin, bentonite) are used. In particular, latex in which polystyrene or styrene copolymer particles are uniformly suspended is preferably used. Latex reagents in which known antibodies are supported on latex particles are commercially available and can also be used.

To measure an antigen contained in a sample by the method of the present invention, 1. For example, first, a sample such as blood or urine is dispensed into a reaction container, and monoclonal antibody B that recognizes a specific antigenic determinant or the monoclonal antibody Dispense and mix the immunodiagnostic reagent containing antibody B1. Through this process, an immune complex C1 is formed (step (A)). In this step, the order of dispensing the specimen and reagent may be reversed or simultaneous (if an automatic biochemical analyzer is used). If the antigen concentration in the specimen is too high, the specimen may be diluted with an appropriate diluent before use. The diluent used is a liquid that does not contain the antigen and/or immunodiagnostic reagent and is chemically inert with the antigen and/or immunodiagnostic reagent (usually water, physiological saline, buffer, etc.). It will be done. It is preferable that the amount of immunodiagnostic reagent dispensed is as small as possible without interfering with the measurement.

This allows the antigen contained in the sample to come into contact with the monoclonal antibody B+ in a relatively concentrated state,
The immune complex formation reaction proceeds rapidly.

The concentration of the reagent (e.g., the titer of the antibody it contains,
It is preferable to set the amount of solid content of the latex to be optimal depending on the measurement conditions such as the reaction system and measuring equipment used. And what is the aggregation state of the immune complex formed by this reaction?

It is preferable that the amount is such that it cannot be measured by a spectrophotometer (the change in absorbance is almost 0).

Next, antibody B2 which is an antibody against the above-mentioned antigen and which recognizes a different antigenic determinant from the above-mentioned antibody or an immunodiagnostic reagent containing the antibody B2 is dispensed and mixed into this reaction solution. As a result, the antibody B2.

binds to the antigen and/or the immune complex C.

As a result, an immune complex C2 having a larger particle size than the immune complex C0 is formed (step (B)). The immunoconjugate C2 has a particle size sufficient to measure the absorbance in one spectrophotometer. However, if the two particle sizes are not large enough, or if you want to obtain an immune complex with a larger particle size to increase the measurement sensitivity.

If necessary, the operation of step (B) is further repeated n times (however, n is preferably 0 to 5) to obtain the immune complex C2.
1 can also be formed.

By measuring the aggregation state of the reaction solution containing the immune complex C2.7 thus formed.

The antigen concentration in the sample is calculated. Measurement of the aggregation state of the reaction solution is carried out by either of the following methods (1), (2) or (2): (1) Measure once after step (A) and use as a blank value.

Then, subtract the blank value or the value corrected for the change in the volume of the reaction solution in each step for the blank value from the value obtained by measuring once after step (B); Measure once later; or (2) Determine the reaction rate from the values measured two or more times after step (B).

The above measurements can be made with known equipment. For example, a conventional spectrophotometer for measuring absorbance, a device for measuring light scattering intensity, a device for measuring particle number and/or particle diameter, etc. are used.

As a dedicated device for measuring the above immunoagglutination reaction, 2
Automatic biochemical analyzer incorporating a spectrophotometer, dedicated equipment for measuring agglutination reactions using immunoturbidimetry, dedicated equipment for measuring agglutination reactions using flow injection, and for measuring latex agglutination reactions There are special equipment for this purpose. These devices can be any of the various commercially available dedicated measurement systems mentioned in the prior art section. Other 1. A method in which the agglutination reaction is carried out in the measuring device cell using a conventional method is also suitably used. When performing measurements using these various methods, in order to obtain the best measurement results (measurement sensitivity, accuracy, etc.), the dilution factor of the sample is 1.
It is desirable to find out in advance the optimal conditions such as reaction time and method of measuring the state of aggregation.

(Function) As mentioned above, in the method of the present invention, 1
The antibody components (mainly polyclonal antibodies) of the immunodiagnostic reagent that are added at the same time are divided into two or more types of antibodies that recognize different antigenic determinants, and these are sequentially added to the system containing the specimen. In other words.

In the method of the present invention, first monoclonal antibody B1 or an immunodiagnostic reagent containing it is added. These are preferably added at a concentration such that the antibody and antigen come into contact with each other in a relatively concentrated state, thereby rapidly forming an immune complex CI. The formation of this immune complex C.

It has minute changes in particle size that cannot be measured with a spectrophotometer. Next, this immune complex C1 is treated with another monoclonal antibody and/or polyclonal antibody B.
By reacting 2 or an immunodiagnostic reagent containing it, an immune complex C2 is formed which has a larger particle size than the immune complex C1 and can be measured with a spectrophotometer. This immune complex C2 is rapidly formed because the immune complex C1 is present in the system, and its production rate is extremely faster than when the antibody is added all at once as in the conventional method. Therefore, the amount of immune complexes produced per unit time increases to the extent that absorbance can be measured. In other words.

The aggregation reaction progresses in a short time and the absorbance of the two reaction system increases. By measuring this, it becomes possible to quantify the substance to be measured with high accuracy in a short time. In the method of the present invention, a method for measuring a reaction system containing immune complex C2 is usually most preferably used because of its simple operation. However, there is also a method of measuring a reaction system containing immune complex C2,1 by further adding n antibodies (or an immunoreagent containing the same) as necessary. Can be adopted. In this way 1 without improving the sensitivity of the immunodiagnostic reagent itself, such as latex reagents.

Highly accurate measurements are taken in a short time.

(Example) The invention is illustrated by the following examples.

AFP measurement was performed using the following AFP standard solution, immunodiagnostic reagent, and measuring device, and an AFP calibration curve was created.

AFP standard product (obtained from the National Institute of Preventive Health) was diluted with 5% bovine serum albumin (BSA) and used as an AFP standard solution.

For confirmation, the AFP concentration in this standard solution was measured using a commercially available radioimmunoassay kit.

Latex method 1υ-: Using mouse cells, two types of anti-Hippin FP monoclonal antibodies (MAB-1) with different antigen recognition sites were generated by a conventional cell fusion method.
and MAR-2) producing hybridomas were produced. These hybridomas were grown in the peritoneal cavity of mice, and their ascites fluid was obtained. These monoclonal antibodies in ascites,
Ig
It was purified to a G fraction. Separately, two pieces of polystyrene latex with a particle size of 0.15 μm were prepared, and this was diluted with PBS so that the solid content was 2%. The above monoclonal antibody M
AB-1 and MAR-2 at 200 μg/rr each
The mixture was diluted with PBS to give a total of 100 ml of polystyrene, mixed with an equal amount of the polystyrene latex solution, and gently stirred at 37°C for 1 hour. This mixture was then heated to 27.000×
The mixture was centrifuged at 100 g to remove unbound monoclonal antibodies. PB containing 1% BSA in polystyrene latex bound to anti-human AFP monoclonal antibody.
S was added to produce latex reagent-1 (containing MAR-1) at 0.5% and 1% latex solids and latex reagent-2 (containing MAR-1) at 0.5% and 1% latex solids.
MAR-2) was prepared.

Part 1: Hitachi Automated Analyzer Model 705 [Measurement of AFP] A two-reagent system measurement mode using a first reagent and a second reagent was adopted. Latex solid content 0.5 as the first reagent
A mixed solution containing % latex reagent-1 and diluent (PBS containing 1% R5A) in a ratio of 3:40 was used, and the second reagent was latex reagent-2 with a latex solid content of 0.5%. was used. The reaction and measurement were carried out as follows: The reaction was set so that all steps were carried out at 37°C, and the degree of aggregation of the two reaction systems was measured by absorbance measurement at 570 nm.

AFP and MAR-1 were allowed to react by dispensing 430 μl of the first reagent into 20 μβ of the AFP standard solution. The absorbance was measured 4 minutes and 40 seconds and 5 minutes after dispensing the first reagent, and the average value (A,) was used as a blank value.

Add the second reagent at any time between 5 and 5 minutes and 20 seconds.
0μ1 was dispensed and MAB-2 was further reacted. The absorbance of the reaction system was measured 5 minutes and 5 minutes and 20 seconds after the addition of the second reagent, and the average value (As) was determined. From AS+8, absorbance (corrected value) ΔA=A, -A.

(The volume of the reaction system before and after the addition of the second reagent is
450μ each! And 480μ! And the volume change before and after addition is 30μ! It is. What is the amount of this change?

Since it was approximately 10% or less of the volume of the reaction system before addition, no volume correction was made for the AsO value when calculating Δ. ). For each concentration of AFP standard solution.

ΔA was measured three times each, and the average value (7i) was determined.

From each concentration, subtract the absorbance value (absence) when the AFP concentration is O, and calculate the value (A AA A A o )
Figure 1 shows the results of plotting AFP; i4 degrees.
Shown in i).

Kitakai Yojo (Measurement of API' by conventional method) As the first reagent, only 430 μl of the diluted solution for the first reagent used in Example 1 was used, and as the second reagent.

Latex reagent-1 with a latex solid content of 1% and latex reagent-2 with a latex solid content of 1% prepared in Example 1 were mixed into 15μ! Using 30 μl of the mixture,
The procedure was carried out in the same manner as in Example 1, except that FP was allowed to react with MAR-1 and MAR-2 in one step. The results are shown in FIG. 1(ii).

From Figure 1, AFP is 5ng/mff using the conventional method.
It can be seen that at a concentration as low as 1, almost no change in absorbance is observed. In contrast, in the method of the present invention, 5
Even in the case of ng/ml, a detectable absorbance change is observed and the value is large, clearly indicating that the measurement sensitivity is improved.

Tenguse Masashi (Measurement of fibrinogen) Next fibrinogen standard solution, immunodiagnostic reagent.

Fibrinogen was measured using a measuring device and a calibration curve was created.

Fibrinogen W' was diluted to give lyophilized fibr/se, and this was used as a fibrinogen standard solution.

Anti-human fibrinogen monoclonal antibody was obtained from mouse ascites in which a monoclonal anti-human fibrinogen antibody-producing hybridoma prepared by a conventional method was grown, and purified using the same purification method as in Example 1. Purified. The anti-human fibrinogen polyclonal antibody was purified by immunizing a goat with human fibrinogen, fractionating the obtained antiserum with ammonium sulfate, and collecting the T-globulin fraction. These monoclonal and polyclonal antibodies were each
0 u g/ml and 500 B g/nd
The latex solid content was 0.5% by diluting with PBS to give a total of
and 0.83% latex reagent-1 (containing anti-human fibrinogen monoclonal antibody) and 0.5% and 0.78% latex solids latex reagent-1
2 (containing anti-human fibrinogen polyclonal antibody) was prepared.

■Motomata Znia Model 05 Hitachi automatic analyzer [Measurement of fibrinogen] A two-reagent system measurement mode using a first reagent and a second reagent was adopted. Latex solid content 0.5 as the first reagent
A mixed solution containing % latex reagent-1 and diluent (PBS containing 1% BSA) in a ratio of 3:40 was used, and the second reagent was latex reagent-2 with a latex solid content of 0.5%. was used. The reaction and measurement were carried out as follows: The reaction was set so that all steps were carried out at 37°C, and the degree of aggregation of the 9 reaction system was measured by absorbance measurement at 570 nm.

After dispensing 1 reagent of the first reagent into 10 μ2 of fibrinogen standard solution, dispense the second reagent at 50 μC at any time between 5 and 5 minutes and 20 seconds to the immune complex formed above. Furthermore, anti-human fibrinogen polyclonal antibody was reacted. The absorbance of the reaction system was measured 5 minutes and 5 minutes and 20 seconds after the addition of the second reagent, and the average value (A3) was determined. For each concentration of standard solution, measure 3 in triplicate.

The average value (A5) was determined. Figure 2 (i) shows the results of subtracting the absorbance value (■π) when the fibrinogen concentration is 0 from each density mark and plotting that value (^, -^, .) against the fibrinogen concentration. show.

As is clear from Figure 2, fibrinogen is 0.
．． Even at a very low concentration of 5 μg/rrdl,
A detectable absorbance change was obtained.

As the first reagent (measurement of fibrinogen by conventional method), 430μ of the diluted solution for the first reagent used in Example 2 was used.
! Stop only the second reagent.

18μ2 of latex reagent-1 prepared in Example 2 with a latex solid content of 0.83% and a latex solid content of 0.83%.
Example 2 except that 50 μl of a mixture of 32 μl of 78% latex reagent-2 was used to react fibrinogen with anti-human fibrinogen monoclonal antibody and anti-human fibrinogen polyclonal antibody in one step. Operate in the same way, As
The results of plotting Aso against fibrinogen concentration are shown in FIG. 2(ii) together with the results of Example 2.

From Figure 2, using the conventional method, fibrinogen is reduced to 1
．． It can be seen that at a low concentration of 0 μg/ml, almost no change in absorbance is observed. In contrast, in the method of the present invention, a detectable change in absorbance was observed even at half the concentration of 0.5 μg/ml, and the value was large, clearly indicating that the measurement sensitivity was improved. It is.

(Effects of the Invention) According to the method of the present invention, various substances can be quantified using immune reactions more quickly and with high sensitivity. Utilizing this method, 2 e.g. alpha-fetoprotein (8FP), fibrinogen.

C-reactive protein (CRP), human chorionic gonadotropin (HCG), carcinoembryonic antigen (CEA), etc. can be quantified effectively. Therefore, this method can be widely used in fields such as clinical testing for diagnosis and treatment of diseases.

Figure 1 shows 9 AI'l when AFP 4 degrees in a sample containing AFP was measured using the method of the present invention and the conventional method.
"A graph showing the relationship between concentration and absorbance (0,0,); and FIG. (0,0,
) is a graph showing the relationship between

that's all

Claims (1)

[Claims] 1. (A) A specimen containing an antigen, antibody B_1 which is a type of antibody against the antigen, a substance containing the antibody B_1, and a carrier bond in which the antibody B_1 is bound to a microparticle carrier. A step of mixing and reacting an antibody or a substance containing the antibody B_1 with a carrier-bound antibody bound to a microparticle carrier to form an immune complex C_1; (B) a step of forming the immune complex C_1;
An antibody B_2 which is an antibody against the antigen and is different from the antibody B_1, a substance containing the antibody B_2, a carrier-bound antibody in which the antibody B_2 is bound to a microparticle carrier, or the antibody B_2 is added to the reaction solution containing the antibody B_2. A substance containing a carrier-bound antibody bound to a microparticle carrier is mixed and reacted,
An immunoassay method comprising the steps of forming an immune complex C_2; and (C) measuring the agglutination state of the obtained reaction solution and calculating the antigen concentration in the specimen from the measured value. 2. Further, using an antibody different from the antibody used in the previous step, repeat the operation in step (B) n times (where n is 0 to 5
) The measuring method according to claim 1, which includes the step of repeating. 3. The method according to claim 1 or 2, wherein the antibody B_1 is at least one monoclonal antibody. 4. The method according to claim 1 or 2, wherein the antibody B_2 is at least one monoclonal antibody or polyclonal antibody.