JPH01158354A - Reagent and method for immunological measurement of many components - Google Patents

Abstract

PURPOSE:To achieve simultaneous measurement, by allowing a mixture of a plurality of sensitized fine particles, which are obtained by respectively sensitizing a plurality of fine particle carriers having different average particle sizes with different antibodies or antigens, to generate coagulation reaction between the antigens or antibodies of a liquid specimen. CONSTITUTION:Each sensitized fine particle is prepared by sensitizing a fine particle carrier with an antigen or antibody in a physical adsorbing manner or covalent bond manner. Next, when three kinds of substances A, B, C to be examined are measured, three kinds of antibody sensitized fine particles of anti-A, anti-B, anti-C, wherein fine particle carriers respectively different in an average particle size are sensitized with the specific antibodies corresponding to the substances to be examined, are separately prepared and mixed to prepare a measuring particle suspension. This suspension is reacted with a liquid specimen such as serum to coagulate the fine particles each other corresponding to the concns. of the substances A, B, C to be examined and the concns. of said substances are measured from particle size distributions to be collated with a preliminarily calculated calibration curve. Therefore, many components can be simultaneously measured.

Description

[Detailed Description of the Invention] [Field of Industrial Application] This invention relates to an immunological multi-component measurement reagent for measuring two or more substances present in a test fluid such as serum, urine, or other body fluids. It is related to the measurement method.

[-Conventional technology]

Detects and measures antigens and antibodies present in serum, urine, body fluids, etc.
Diagnosis of diseases is generally widely practiced.

One of the measurement methods is to react the antigen or antibody contained in the sample with sensitized fine particles that have been sensitized with a specific antibody or antigen, and observe the agglutination reaction or agglutination inhibition reaction of the fine particles with the naked eye. Measuring methods (so-called agglutination tests) are widely used because their reaction time is short and they are sensitive.

In this case, in normal clinical tests, one component is quantified in a single measurement operation, but with recent advances in clinical medicine, a small amount of specimen can be used and a large number of test items can be performed in a shorter time. Measurement is required.

For this purpose, a method is used in which two or more types of sensitized fine particles obtained by separately sensitizing antibodies corresponding to two or more different antigens to be measured are mixed and used. 40166) and a method using sensitized fine particles in which the same carrier is sensitized with antibodies corresponding to two or more types of antigens to be measured (Japanese Unexamined Patent Publication No. 59-24256).

[Problem that the invention seeks to solve]

Although the method disclosed in the above-mentioned publication can measure the sum of the components to be measured, there is a problem in that the individual components cannot be quantitatively measured in one measurement.

Therefore, in order to quantitatively measure multiple components individually, it is necessary to repeat the measurement for the number of test items. However, this requires a large amount of specimen (mainly serum and body fluids), which increases patient pain. Furthermore, processing a large number of samples requires a lot of time and effort, and there is a risk that items necessary for the patient may be omitted.

Therefore, an object of the present invention is to provide a reagent for immunological multicomponent measurement and a measurement method that can simultaneously measure multiple components in a single measurement operation.

[Means for Solving the Problems] The reagent for immunological multi-component assay of the present invention comprises a plurality of sensitized microparticles in which a plurality of microparticle carriers having different average particle diameters are sensitized with different antibodies or antigens. It consists of a mixture.

The immunological multicomponent measurement method of the present invention causes an agglutination reaction between a mixture of a plurality of sensitized fine particles in the reagent and a plurality of antigens or antibodies contained in a test liquid, and is based on the agglutination reaction. The change in particle size distribution is measured to measure the antigen or antibody contained in the test liquid.

Examples of the particulate carrier used for sensitizing antigens or antibodies in this invention include synthetic latex such as polystyrene latex, collodion particles, activated carbon, bentonite, kaolin, and human or animal red blood cells. Among these particles, synthetic latex, animal red blood cells, etc. have different average particle diameters, and each particle group can be used as a particle group with a narrow particle size distribution, so they can be suitably used in the present invention. Furthermore, synthetic latex generally has excellent performance as a carrier and storage stability.

The sensitized fine particles used in this invention can be prepared by conventionally known means. That is, each sensitized fine particle is created by sensitizing an antigen or antibody to a fine particle carrier such as polystyrene latex by physical adsorption or covalent bonding.

The antigens mentioned here include, for example, serum proteins such as human albumin, human immunoglobulin G, human fibrinogen,
Examples include alpha-fetoprotein and hormones. Furthermore, the term "antibody" refers to a specific antibody against the above-mentioned antigens. Note that the antigens and antibodies in this invention are not limited to the above-mentioned antigens and antibodies.

The difference in average particle diameter of the multiple microparticle carriers used is
In order to distinguish between aggregated particles based on the aggregation of each sensitized fine particle, it is preferable that the difference in average particle diameter is about 0.018 or more, especially < 0.1 u or more, and when the difference in average particle diameter is smaller than this range, Considering the discrimination ability of devices currently in practical use, it is not desirable because the particle size distributions of each particle are too close to each other, making it difficult to distinguish. However, depending on future improvements to the device, it will be difficult to distinguish between particles of about 0.001μ. It is thought that this will make it possible to distinguish without any difference. Also, although there is no upper limit to the difference in average particle diameter, if the difference becomes too large,
For example, when particles are optically detected using a laser-based light scattering measuring device that uses a laser as a light source, two wavelengths of laser light are required for measurement, which makes the measuring device complicated and increases costs. Therefore, it is preferable that the average particle diameter is about 20 microns or less, particularly 10 microns or less.

In addition, the mixing ratio of each sensitized fine particle carrier is preferably such that the concentration of each particle in the reaction tank after mixing is approximately the same and does not differ by at least one order of magnitude.
When the i degree is more than one digit, particle counting errors are likely to occur. The concentration of each particle in the reaction tank should be the optimum concentration for the measuring device used. For example, when using a light scattering particle counter, the concentration of each particle group in the reaction tank should be 0.01 to
O.1 w/V% is appropriate.

The measuring method of the present invention is individually based on a particle agglutination reaction. In other words, the antigen or antibody contained in the test liquid is reacted with a sensitized particulate carrier sensitized with a specific antibody or antigen, and the particle size distribution is determined based on the agglutination reaction or agglutination inhibition reaction of the particulate carrier. This method measures the change in the amount of water, and then quantifies the substance of interest.

The particle size distribution measuring device used in this invention may be any device that measures the particle size distribution of a particle group by measuring the particle size or particle volume of each particle agglomerate in the particle group. For example, a capacitance type particle size distribution measuring device, an electrical resistance type particle size distribution measuring device, a light scattering particle counter (see JP-A-60-111963), etc. can be used alone or in combination.

As a method for analyzing changes in particle size distribution of this invention,
Any method can be used as long as the change in particle size distribution can be analyzed as directly or indirectly reflecting the agglutination reaction rate of particles based on antigen-antibody reaction, for example, The concentration of a specific particle size particle group during a reaction time, (2) the ratio of a specific particle size particle group during a certain reaction time, etc. are determined as measured values corresponding to the aggregation reaction rate. At this time, by creating a calibration curve in advance using samples with known concentrations of the target component, and then obtaining the measured values of the test liquid with unknown target components and comparing them with the calibration curve, the target component to be measured can be quantified. .

Note that the specific particle size particle group here refers to a group of unagglomerated unit particles having a single particle size, a group of two agglomerated particles consisting of two unit particles, or a group of three agglomerated particles consisting of three unit particles. It means a group etc.

[Effect]

The simultaneous multi-component measurement method in this invention will be schematically explained.

When measuring three types of test substances A, B, and C, anti-A antibody sensitization is performed by sensitizing fine particle carriers with different average particle diameters with specific antibodies (or antigens) corresponding to these test substances. A and B are prepared by separately preparing three types of microparticles, anti-B antibody-sensitized microparticles, and anti-C antibody-sensitized microparticles, and mixing these in an appropriate ratio.

Prepare particle suspension for C measurement. This suspension is reacted with a test liquid such as serum or urine, and the particle size distribution in the reaction mixture is measured after a predetermined period of time. Then, depending on the concentration of test substances A, B, and C in the test liquid, aggregation occurs between fine particles sensitized with the corresponding antibodies.

From the particle size distribution obtained in this way, the temporal change or concentration or ratio of the particle group corresponding to each measurement item is measured,
By comparing with a calibration curve determined in advance, each of the test substances A, B, and C in the sample can be quantified simultaneously.

Furthermore, when using an agglutination inhibition reaction, A antigen-sensitized fine particles are used instead of anti-A antibody-sensitized fine particles.

In this case, if anti-A antibody is added to the reaction system, A
An antiaggregation reaction occurs that is related to the concentration of antigen. In other words, when A antigen-sensitized fine particles and anti-A antibody are added to a specimen and reacted, the agglutination reaction between A antigen-sensitized fine particles and anti-A antibody is blocked depending on the amount of A antigen in the specimen. , the aggregation between microparticles is reduced compared to when no analyte is included. Since this amount of decrease correlates with the amount of A antigen in the sample, if a standard curve is prepared in advance, the A antigen can be quantified. Therefore, the aggregation inhibition reaction is used for the purpose of quantifying habuten, which cannot be determined by normal aggregation reactions.

In addition, if the optimum reaction time for each sensitized fine particle is different, after allowing the mixed particle suspension and the test liquid to react to some extent, a portion of the reaction liquid is taken out at each optimum reaction time to complete the reaction. You can quantify each component by measuring the particle size distribution individually, or you can add sensitized particles to the reaction system sequentially starting with the longest reaction time, so that the optimal reaction time for each sensitized particle is reached at the end of the reaction. By adjusting, the particle size distribution can be measured at the end of the reaction and the concentration of the test substance can be determined simultaneously.

〔Effect of the invention〕

According to this invention, multiple components can be quantified simultaneously in a single measurement, thereby reducing the time required for measurement and reducing the amount of sample required.

〔Example〕

Examples will be explained below.

Examples: (a) Preparation of anti-alpha-fetoprotein (AFP) antibody-sensitized microparticles Polystyrene with a particle diameter of 1.0μ and a concentration of 5-/ν% suspended in 0 and 1 M glycine buffer (pl+8.2) To 1 volume of latex suspension, anti-AFP rabbit serum γ-globulin fraction was added to 0.1 M glycine buffer (p
Add 4 volumes of a solution dissolved in H8, 2) to 1 w/v%, incubate at 37°C for 2 hours, remove the supernatant by centrifugation, and add Q, 5 w/v% bovine serum albumin,
Resuspend in 10 volumes of 0.1M Grinsci buffer (pH 8.2) containing 0.05% sodium azide to obtain 0.5% sodium azide.
A w/v% anti-AFP antibody sensitized microparticle suspension was obtained.

(1)) Preparation of anti-carcinoembryonic antigen (CEA) antibody-sensitized microparticle carrier The same method as in (a) above was carried out. Note that polystyrene latex with a particle size of 0.7 μm was used as the carrier.

(Preparation of sensitized fine particle suspension for CI AFP and CEA measurement The anti-AFP antibody-sensitized fine particle suspension and the anti-CEA antibody-sensitized fine particle suspension prepared in (a) and (b) above were mixed at 1=1). A sensitized particle suspension for AFP and CEA measurement was prepared by mixing at a ratio of 1:1.

(d) Preparation of BiYl# solution■ AFP standard solution: CEA 50μg/a+1.1
AFP was added to a saline phosphate buffer (PBS buffer) containing % bovine serum albumin (BSA) at a concentration of 0.
．． 50.100.200. The solution was dissolved to a concentration of 500 μg/ml.

■ CEA standard solution: AFP 50μg/m+,
CEA was added to a saline phosphate buffer (PBS buffer) containing 1% bovine serum albumin (BSA) at concentrations of 0.50, 100, and 200, respectively. It was dissolved to a concentration of 500 μg/ml.

(e) Measurement of particle size distribution ■ 25μ of the anti-AFP antibody-sensitized fine particle suspension of (a) above
! to 0.5 w/% bovine serum albumin and 0.5 w/% bovine serum albumin.
25 μl of 0.1 M glycine buffer (pH 8,2) containing 0.5% sodium azide and the AFP standard '4i8i'& (AFP concentration 100 ng/m
100 μl of P) containing 1% bovine serum albumin
Add 100μe of BS buffer and heat at 37°Ct'30
The reaction was allowed to proceed for a minute with gentle stirring, and immediately after the reaction was completed, the particle size distribution was measured using a light scattering particle counter.

The results are shown in FIG. 1, where A indicates a group of non-agglomerated particles and B indicates a group of two aggregated particles. Note that the relative particle diameter shown on the horizontal axis of the graph is a relative value based on the signal intensity (the same applies to FIGS. 2 and 3).

■ 25μ of the anti-CEA antibody sensitized fine particle suspension of (b) above
l, 0.5 w/v% bovine serum albumin and 0.0
0.1M glycine buffer containing 5% sodium azide (
25μ2 of pH 8,2) and 100μ of the AFP standard solution (AFP concentration I Q OB/ml) in (d)■ above! and PBS buffer 10 containing 1% bovine serum albumin.
0 .mu.l was added and allowed to react with gentle stirring at 37.degree. C. for 30 minutes. Immediately after the reaction was completed, the particle size distribution was measured using a light scattering particle counter.

The results are shown in FIG. 2, where C indicates a group of non-agglomerated particles and D indicates a group of two aggregated particles.

■ Add the AFP standard solution (AFPfi) of (d) ■ above to 50 μl of the sensitized particle suspension for AFP and CEA measurement of (C) above.
50 ul of 200 ng/ml) and CE of (d)■
50 of A standard solution (CEA'a degree 200 ng/ml)
μffi and PBS buffer lOOμe containing 1% bovine serum albumin were added, and the mixture was allowed to react at 37°C for 30 minutes with gentle stirring. Immediately after the reaction was completed, the particle size distribution was measured using a light scattering particle counter.

The results are shown in FIG. The particle size distribution shown in the figure is a superposition of the particle size distribution in Figure 1 and the particle size distribution in Figure 2, and each peak is in order from the left to the non-agglomerated particle group C1 with a particle size of 0.7μ. Particle size l, non-agglomerated particle group A with particle size 0μ, two agglomerated particle group with particle size 0.7μ, and particle size 1.0μ
The two agglomerated 'particles group B are shown respectively.

(“) Creation of a calibration curve 50 μl of each fine particle suspension for AFP-CEA measurement was added at 1%
Place 100μ2 of AFP standard solution at the specified concentration in a reaction test tube containing 100μ2 of PBS buffer containing BSA.
was added and reacted at 37°C for 30 minutes with gentle stirring. Immediately after the completion of the reaction, the particle size distribution of the reaction mixture was measured using a light scattering particle counter. Here, the average particle diameter is 1.
The number of particles measured in the 0μ particle group and the number of particles measured in the two agglomerated particle group, in which two particles of this average particle diameter particle group are aggregated, are each expressed as X 1. If X 2, the average particle diameter is 1.0
The aggregation rate Y (%) of the particle group μ, that is, the anti-AFP antibody-sensitized fine particles, is determined from the following formula.

X, + (2XXl) From the obtained aggregation rate and the AFPfi degree of the standard solution, AFP
A calibration curve was obtained. This is shown in FIG.

In the same manner, the aggregation rate of the particle group with an average particle diameter of 0.7μ, that is, the anti-CEA antibody-sensitized particles, and the CE of the standard solution.
A calibration curve of CEA was determined from the AiJ degree.

This is shown in FIG.

The calibration curve shown in Figure 4 is based on C at a concentration of 50 μg/ml.
Although this was in the presence of EA, the same calibration curve was obtained even if the CEA concentration was changed. Furthermore, the calibration curve shown in Figure 5 is in the presence of AFP at a concentration of 50 μg/ml.
In this case as well, the same calibration curve was obtained even if the AFPfi degree changed. This shows that AFP and CEA in the reaction solution aggregate only with particles sensitized with the respective antibodies, do not affect mutual agglutination reactions, and do not cause cross-agglutination reactions.

[Brief explanation of the drawing]

Figure 1 is a graph showing the particle size distribution measured by reacting an AFP standard solution with an AFP antibody-sensitized particle suspension, and Figure 2 is a graph showing the particle size distribution measured by reacting an AFP antibody-sensitized particle suspension with an AFP standard solution. A graph showing the particle size distribution measured by reacting CEA standard solution with liquid A, Figure 3 is AFP.
, A graph showing the particle size distribution measured by reacting a sensitized particle suspension for CEA measurement with 8 standard AFP standard solutions and an EA standard solution. Figure 4 shows the relationship between AFP concentration and aggregation rate.
FP Calibration Curve FIG. 5 is a CEA calibration curve showing the relationship between CEA concentration and agglutination rate. Silk 'rruff + diameter → Figure 1 Phase lantern cover + diameter → Figure 2 String lamp Fi-Jf! -=- Figure 3 AFPJ 1 degree (ng/ml) Figure 4 CEAJ degree (ng/ml) (procedural amendment exclusive use) January 29, 1985

Claims (2)

[Claims] (1) A reagent for immunological multi-component assay consisting of a mixture of a plurality of sensitized microparticles in which a plurality of microparticle carriers having different average particle diameters are sensitized with different antibodies or antigens. (2) An agglutination reaction occurs between a mixture of multiple sensitized microparticles in which the microparticle carriers of multiple particle groups with different average particle diameters are sensitized with different antibodies or antigens, and the antigen or antibody contained in the test liquid. an immunological multi-component measurement method comprising the steps of: causing the sensitized fine particles to undergo an agglutination reaction; and measuring an antigen or antibody contained in the test liquid from a change in particle size distribution based on an agglutination reaction of each of the sensitized fine particles.