JPH02122266A - Immunological measurement - Google Patents

Abstract

PURPOSE:To shorten the agglutination time required for an agglutination reaction without using a centrifuge and to obtain more distinct agglutination patterns by putting a reaction liquid contg. a material to be measured and the magnetic particles fixed with a material reacting or competing specifically therewith into a reaction vessel, then applying magnetic fields thereto. CONSTITUTION:A microplate 3 as a reaction vessel is disposed above a supporting base 1 and this microplate 3 has plural bottomed cylindrical wells 4 which have the U-shaped bottoms and are disposed in correspondence to respective magnets 2. The material reacting or competing specifically with the material to be measured is fixed to the wall of the microplate 3 at 0.2X10<6> to 1.2X10<6> cell/cm<2> density. The reaction liquid contg. the material to be mea sured and the magnetic particles fixed with the material reacting or competing specifically therewith is put into the microplate 3 and such magnetic fields as to focus the magnetic particles along the wall surfaces of a part of the microplate 3 are applied to the reaction liquid, by which the distribution patterns of the magnetic particles are formed and the bonding states among the materials are measured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for detecting and determining the presence of an antigen or antibody by immunological agglutination reaction.

[Conventional technology and issues]

In a method of forming and analyzing a distribution pattern of aggregated or non-aggregated particles based on an immunological agglutination reaction, antigens or antibodies in a sample are mixed and bound to antigens or antibodies immobilized on specific indicator particles. At the same time, these reaction components are moved to the wall surface of the reaction vessel, and unbound indicator particles are collected on a part of the wall surface and separated. Such a method is
Generally, mixed agglutination method (mixed agglutinat method)
ion), Wicnsr, A, S, and l
lerman, M., J. Immunol.
3G.

255 (1939), Co.
ombs.

R, R, A, and 13edford, D.
Voxsang, 5 111 (1955) and Coombs, R.R.A., et al., Lancet.
+461, (1,956) reported that it was developed and established to the point where blood type determination was performed. For example, USP 4,608,246 and USP 4,275,053 apply to various blood types.
No. 4221) and USP No. 4328183.

Further, there is USP No. 2,770,572 which attempts to improve the sensitivity by determining blood type on a solid surface. Furthermore, Rosenrield1? , IE,
et al., Paris, Proc., 15Th
Cong, Intl, 5ocBlood Tras
f'usion, 27 (197G) uses the principle of mixed agglutination to react red blood cell antigens and antibodies on a solid surface.

By the way, most of the conventional mixing aggregation methods are based on natural sedimentation of indicator particles in a reaction vessel having a convergence section at the bottom. Therefore, it took a long time for the indicator particles to settle uniformly on the bottom surface and for the unreacted particles to settle in the convergence part to form a pattern. In order to simply increase the sedimentation rate, it is possible to increase the particle size or use abrasive material with a high specific gravity, but there is a limit in that the particle size and specific gravity must be selected to the extent that the pattern does not become rough. , a significant time reduction cannot be expected.

Therefore, USP No. 4297104 (Specification
31299), antibodies (e.g. Ig
G) is fixed, an anti-1gG antibody against the antigen in the sample and antibodies on the surface of the red blood cells and the wall of the container is added in advance, and red blood cells that have not reacted with the antigen are brought to the convergence point of the container by the first and second centrifugation treatments. Patterns are formed by collecting them. However, although the agglutination reaction by centrifugation is effective in promoting sedimentation of particles, the necessity of a centrifuge itself poses operational constraints.

In other words, when performing sampling, reaction, determination, disposal, etc., the flow of handling the reaction container is not smooth, and in particular, the axis of symmetry of the reaction container must be precisely aligned with the direction in which centrifugal force is applied. .

In addition, when analyzing different items, it is necessary to change the sedimentation conditions for each particle, or it is difficult to set multiple centrifugation accuracy and timing during the same centrifugation process, so it is necessary to repeat the process each time, or Requires configuration such as connecting to multiple centrifuges. Furthermore, the use of a centrifuge inevitably increases the size of the apparatus.

The present invention has been made in order to solve the problems of the prior art described in 2.It is an object of the present invention to shorten the time required to determine an agglutination reaction without using a centrifuge, and to clarify the agglutination pattern. The purpose of this study is to provide a method for immunological measurement.

[Means and effects for solving the problems] The present invention provides a substance that specifically reacts with or competes with the substance to be measured on the wall of a reaction vessel at a rate of 0.2 x 100 cells/cTA to 1.
After fixing at a density of 2 × 106 cells/cell and placing a reaction solution containing a substance to be measured and magnetic particles that specifically react with or compete with the substance in this container, either the magnetic particles or the reaction vessel forming a distribution pattern of the magnetic particles by applying a converging magnetic field along the substance fixed on the inner surface of the container, and measuring the bonding state between the substance fixed on the inner surface of the container and the substance to be measured; This is an immunological measurement method characterized by:

Hereinafter, the present invention will be explained as shown in FIGS. 1(A) and (B) and FIGS.
This will be explained in detail with reference to the drawings. Here, FIG. 1(A) is a plan view showing an apparatus consisting of a microplate, a magnet, etc. used in the immunoassay method of the present invention, and FIG.
B) is a front view of the same figure (A). 1 in the figure is a support stand that supports a plurality of disc-shaped ferrite magnets 2 on two sides.

A microplate 3 as a reaction container is placed on both sides of the support 1, and the microplate 1
・3 denotes a plurality of cylindrical wells 4 with a U-shape at the bottom arranged corresponding to each of the magnesols 1-2.
I'm getting better.

First, a substance to be measured, such as a substance that specifically reacts with or competes with an antigen or antibody such as a virus, bacteria, or protein, such as an antibody or an antigen, is immobilized on the surface of a magnetic particle containing a known magnetic material. do. As magnetic particles, commercially available DYNABEADSM-450 (DYNAI
) or magnetic latte socks (cstapor)
[manufactured by R11ONE-POULENC], gelatin particles containing magnetic material (see Japanese Patent Application Laid-open No. 195161/1983), etc. can be used. Moreover, various known methods can be applied to immobilize the substance on the magnetic particles.

Next, a substance that specifically reacts with or competes with the substance to be measured in the sample is placed in each well 4 of the microplate 3 shown in FIGS.
As shown in the figure, the substance 5 is fixed on the inner surface of the well 4. In fixing such a substance 5 to the inner surface of the well 4,
0.2XIO' cell/crj~12x106 cell/c
Fix it so that the density is J. The reason for limiting the fixed density of the material is that its density is 0.2XIO'-'cell/
When the density is less than ci, the distribution pattern of magnetic particles becomes either a positive pattern or a negative pattern, whereas when the density exceeds 1.2 x 10' cells/crd, the distribution pattern of magnetic particles becomes a negative pattern. This is because it may result in a false positive. Note that conventional known methods can be used to immobilize a substance that specifically reacts with or competes with the substance to be measured on the well wall surface.

Subsequently, a sample is added to each well 4, the substance to be measured in the sample reacts with the substance immobilized on the inner surface of the well, and then the above-described magnetic particles are added. After that, the microplate 3 and the support 1 supported by the magnets 2 are moved relatively (for example, each magnet 2 of the support 1 is moved closer to the bottom of each well 4 of the microplate 3). A magnetic field is applied to converge the magnetic particles along at least a portion of the wall surface of the well 4.

By applying the magnetic field, the magnetic particles 6 attracted to the wall of the well 4 combine with the substance 5 fixed on the inner surface of the well 4 via the non-measurable substance 7 in the sample, as shown in FIG. A uniformly wide distribution pattern is formed on the inner surface of the well 4.

On the other hand, when there is no substance to be measured in the sample, the magnetic particles 6 are attracted to the wall surface of the well 4 by the magnetic field, move further along the inner surface, and move along the inner surface as shown in FIG. Converged and collected in the department. Note that a known aggregation suppression reaction may be used, in which a substance that competes with the substance to be measured in the sample is immobilized on the magnetic particles, and a certain amount of a substance that binds and reacts with the substance to be measured is added. .

Therefore, a substance that specifically reacts with or competes with the substance to be measured is immobilized on the inner surface of the reaction vessel at a specific density,
After placing a reaction solution containing a substance to be measured and magnetic particles that specifically react with or compete with the substance in this container, the magnetic particles converge along the substance fixed to the wall of the reaction vessel. By applying a magnetic field such as Can be formed quickly. Furthermore, by fixing a substance that reacts with or competes with the substance to be measured in the sample at a specific density on the inner surface of the reaction vessel, a clear distribution pattern of the magnetic particles can be formed. As a result, the time required to determine the agglutination reaction can be shortened, and the presence of the substance to be measured in the sample can be detected with high sensitivity from its clear distribution pattern.

〔Example〕

Embodiments of the present invention will be described in detail below.

Preparation of WGA (wheat germ lectin) plate for immobilization of red blood cells> NUNC U-shaped plate (Micro well
Module 2X 8 well round bo
ttom High binding Capacit
yNo, 469264) to WGA (Seikagaku Corporation)
Serial number 92107) to 0. OIM PI35,
Dispense 50 μl/well of 3 μg/min dissolved at pH 7.0, incubate for 30 minutes at room temperature, and then 0.00 μg/well.
Wells were filled with 01M PBS and washed twice. Next, O, DIM containing 0.05% Tween 20
After washing once with PBS and draining, it was air-dried and stored refrigerated.

<Negation of red blood cells on WGA plate> IgG, the substance to be measured, is treated as unsensitized blood cells by surge screen (
Ortho Inc., serial number 3SS888) and IgG, the substance to be measured, are sensitized blood cells.
- A roll (manufactured by Ortbo, serial number 804) was used. Each blood cell suspension was added to Ijss containing 0.01% sarcosinate LN (Kkko Chemical Co., Product No. 5083).
, diluted with pH 6,7, 1.0%, 09%, f
], 7%, 05%, 0.3%, 0.2%, 01%, 0.
08%, 0.06%, 004%, 0.02%, 0.01
adjusted to %. Add 50 μJ of each diluted blood cell to the WGA Play 1! Dispense 200ml/well into 0.01M PBS and incubate for 10 minutes at room temperature.
Washed three times with μA/well. After washing, 0, 01.
M]0 PBS was dispensed at 200 μi/well and stored refrigerated to prevent the blood cells from drying out. The thus prepared red blood cell coated plate was examined under a microscope at a magnification of 200 times, and the number of cells bound to the bottom of the well was determined by the number of cells bound to the well bottom surface, and the number of red blood cells per 1 d. The results are shown in Table 1 below.

Preparation of anti-human I-1gG sensitized magnetic beads> Particle size 1.7μ
m1 magnetic latex (estapor) [RO11N
E-POU L E N 0 company, serial number LMP
233] to Anti-HumanlgG [Capp
e1 company, Afrinjty Purrjf.

Goat-anti-11Mman IgG serial number 2
997+) and Anti-11Mman1gGF (
ab' ) 2 [Cappe1, F(ab' ) 2
Fragment or Af'rjnity Pur
rif, Goatanti-Human IgG production number 2959B). That is, O, 1M
The above biz was diluted to 0.4% and the antibody was diluted to 20 Mg/min in C1y-NaOH, pH 8.3, and each IIIL was mixed.
The reaction was carried out at 7°C for 1 hour. Next, 0.2% BSA
, 0.02M Gly containing O,14M NaCl
- Incubate each bead with 3 ml of NaOII, pH 8, 5, 2 ml.
After washing twice, the anti-human IgG magnetic pieces and the anti-human IgG F (ab') 2 magnetic beads were suspended in the same buffer and stored in a refrigerator.

Furthermore, the same operation was performed on the unsensitized magnetic piece without using the antibody.

Analysis using anti-human IgG pieces> Two types of anti-human IgG magnetic pieces [
Anti-human IgG magnetic piece, anti-human IgG F (a
b”) 25μ suspension of magnetic Beisco and unsensitized beads
1/well was dispensed. Continuing, Figure 1 (A), (
The support stand 1 shown in B) is attached to a magnet (diameter g).
mm, thickness 3 mm, residual magnetic flux density 2000 Gauss)2
The microplate was moved so as to approach the bottom of each well 4 of the microplate 3, a magnetic field was applied perpendicularly to the bottom of each well 4, and the reaction was carried out at room temperature for 2 minutes.

When we investigated the distribution patterns of anti-human IgG magnetic pieces, anti-human IgG F (ab') 2 magnetic screws, and unsensitized pieces formed on the bottom of each well, we obtained the results shown in Table 1. . The reaction pattern in Table 1 is as shown in Figure 5 (A), where the beads]2 are uniformly spread over the bottom of the well (positive), and the distribution of beads as shown in Figure 5 (B). A false positive (±) indicates a case where the beads are thinly distributed in the center and slightly densely distributed around the center, making it difficult to distinguish between negative and positive beads.As shown in the same figure (C), beads are concentrated in the center at the bottom of the well. This is the result of determining negative (-).

As is clear from Table 1, the density of blood cells fixed on the bottom of the well is 0.2 x 10 cells/cM ~ 1.2 x 10 cells.
By setting the cell/ci range, it is possible to clearly distinguish between positive and negative results. In contrast, the cell density of blood cells fixed on the bottom of the well was set to 0.2 x 106 cells/c.
If the blood cell density is less than 1j, what would normally be a positive (+) pattern becomes a negative (-) pattern; on the other hand, if the density of the same blood cells exceeds 1.2 x 106 cells/ci, it will become negative (-).
−) pattern becomes a false positive (±) pattern and becomes unclear.

〔Effect of the invention〕

As described in detail above, according to the immunoassay method of the present invention, it is possible to shorten the time required to determine an agglutination reaction without using a centrifuge, and to clarify the agglutination pattern. , it is possible to achieve two improvements in negative detection sensitivity, and it also has remarkable effects such as reducing the amount of substances (red blood cells, etc.) that specifically react with or compete with the substance to be measured, which is immobilized on the inner surface of the reaction vessel. play.

[Brief explanation of drawings]

Figures 1 (A) and (B) are microplay!・This figure shows an immunological measuring device equipped with a magnet, and the same figure (A) is a plan view, the same figure (B) is a front view of the same figure (A), and FIG. Schematic diagram showing a state in which a substance that specifically reacts with or competes with a substance is immobilized, 3rd
The figure is a schematic diagram showing the bonding state between particles when the present invention is positive. Figure 4 is a schematic diagram showing the bonding state between particles when the present invention is negative. Figure 5 (A5) is a schematic diagram showing the bonding state between particles when the present invention is negative. Figure (B) is a schematic diagram showing the bead reaction pattern at the time of false positive;
C) is a schematic diagram showing the reaction pattern of beads when negative. ■... Support stand, 2... Disc-shaped ferrite magnet, 3... Microplate, 4... Well, 5...
- Substance that specifically reacts with or competes with the substance to be measured, 6... Magnetic particles, 7... Substance to be measured. Applicant's representative Patent attorney Atsushi Tsuboi] 6 Figure 5 (C)

Claims (1)

[Claims] A substance that specifically reacts with or competes with the substance to be measured is placed on the wall of the reaction vessel at 0.2 x 10^6 cells/cm^.
Fixed at a density of 2 to 1.2 x 10^6 cells/cm^2,
After placing a reaction solution containing a substance to be measured and magnetic particles that specifically react with or compete with the substance in this container, the magnetic particles converge along the substance fixed on the inner surface of the reaction vessel. An immunoassay method, characterized in that a magnetic field is applied to form a distribution pattern of the magnetic particles, and the state of binding between the substance fixed on the inner surface of the container and the substance to be measured is measured.