JPH07117541B2 - Immunological measurement method using magnetic particles - Google Patents

Description

The present invention relates to a method for measuring a substance to be measured involved in an immunological reaction by using magnetic particles.

[Conventional technology]

A mixed agglutination method is a method for forming and analyzing a distribution pattern of aggregated or non-aggregated particles based on an immunological reaction. This is to determine positive or negative from the difference in the aggregation pattern caused by the reaction of the antigen or antibody in the sample with the antibody or antigen immobilized on the carrier and the aggregation or non-aggregated particles settling on the bottom of the reaction vessel. . This mixed agglutination method is described by Wiener, AS and Herman, M., J. Immunol., 36 , 255 (1939).
Since being reported in Coombs, RRA and Bedford, D.,
Voxsang., 5 , 111 (1955) and Coombs, RRA et al., Lance
It was developed and established until the blood type was determined by the report of t, i, 461 (1956). For example, U.S. Pat. No. 4,608,246, U.S. Pat. No. 4,275,053 (Japanese Patent Publication No. 62-44221) and U.S. Pat. No. 4,328,183 correspond to various blood types. By holding the antibody that reacts with the antigen in the sample in advance on the solid surface in a dry state, it is possible to determine the blood group on the solid surface to improve the sensitivity, and US Patent No. 2770572 is disclosed. is there. Rosenfield R.
E. et al., Paris, Proc. 15Th Cong. Intl. Soc. Blood Transfusi
On, 27 (1976) uses the principle of the mixed agglutination method to react erythrocyte antibody with antibody on the solid surface.

[Problems to be Solved by the Invention]

In the conventional technique, the aggregation pattern formed by spontaneously sedimenting aggregated or non-aggregated particles is measured, and thus there is a problem that it takes time to form the aggregated pattern.

The present invention aims to form an aggregation pattern in a shorter time than the conventional techniques.

[Means and Actions for Solving Problems]

 FIG. 1 is a conceptual diagram of the present invention.

A magnetic particle 1 having on its surface a substance to be measured and a substance that binds to the substance to be measured or competes with the substance to be measured is housed in a reaction container 2. Next, when a magnetic pole having a needle-like tip is positioned below the container 2, the magnetic particles 1 in the reaction container 2 receive the force attracted by the needle-shaped magnetic pole 3 and move in the reaction solution to the bottom of the container. The magnetic particles 1 react while moving to the bottom surface of the container. Since the distribution pattern of reacted magnetic particles and unreacted magnetic particles is formed on the bottom surface of the container, the distribution pattern is visually or measured with an optical measuring device to determine whether there is a substance to be measured, Determine the amount.

Magnetic particles include DYNABEADS M-450 (manufactured by DYNAL),
Magnetic latex particles such as Estapol LMP233 (manufactured by Rhone Poulenc) or gelatin particles containing a ferromagnetic material (see Japanese Patent Laid-Open No. 59-195161), and a ferromagnetic material such as iron powder in cells such as red blood cells. Incorporated cells can also be used as magnetic particles. Furthermore, the magnetic particles are sensitized magnetic particles,
A substance that binds to the substance to be measured or a substance that competes with the substance to be measured is immobilized on the surface of the magnetic particles by binding.

The substance to be measured is a specific binding substance represented by a ligand and a receptor, and has a substance having antigenicity, an antibody that binds to an antigen, an immunoglobulin, an antibody against immunoglobulin, a cell membrane protein, a cell, a cell Examples thereof include antibodies against surface antigens.

The reaction vessel used in the present invention is preferably a test tube or a microplate.

The distribution pattern of magnetic particles differs depending on the reaction form. When an aggregate is formed by the reaction between the substance to be measured in the sample and the sensitized magnetic particles, an image of the particles is observed uniformly on the bottom surface of the reaction vessel in the aggregation pattern.

In the non-aggregation pattern, an image in which unbonded magnetic particles are concentrated and gathered in the vicinity of the needle-shaped magnetic pole positioned below the reaction vessel due to the magnetic force is observed.

A positive reaction occurs when a substance that binds to the substance to be measured in the sample is immobilized on the inner surface of the reaction vessel, and the sample and magnetic particles with the substance that binds to the substance to be measured in the sample immobilized on the surface are added to this reaction vessel. Is a reaction vessel inner surface on which a substance that binds to the measurement substance is immobilized, and the magnetic particles sandwich the measurement substance in the sample. Therefore, a uniform image of the magnetic particles is formed on the reaction vessel inner surface.

In the negative reaction, since the measurement substance does not exist, the magnetic particles cannot bind to the substance that binds to the measurement substance immobilized on the entire inner surface of the reaction container. Therefore, due to the magnetic force, the magnetic particles form an image that is concentrated and gathered in the vicinity of the needle-shaped magnetic pole positioned in the lower direction of the reaction vessel.

A substance that binds to the measurement substance in the sample is immobilized on the inner surface of the reaction container, and the sample and the substance that competes with the measurement substance in the sample, for example, magnetic particles on the surface of which the same substance as the measurement substance is immobilized, are immobilized in this reaction container. When added, if the amount of the measurement substance in the sample is large, the measurement substance in the sample will bind to the substance that binds to the measurement substance immobilized on the inner surface of the reaction vessel, so the unbound magnetic particles are magnetically affected. , A concentrated image is formed in the vicinity of the needle-shaped magnetic pole located below the reaction vessel.

In the absence of the substance to be measured, magnetic particles are bound to the inner surface of the reaction vessel on which the substance that binds to the substance to be measured is immobilized, so that a uniform image of the magnetic particles is formed on the inner surface of the reaction vessel.

In addition, a substance that competes with the measurement substance in the sample, for example, the same substance as the measurement substance, is immobilized on the inner surface of the reaction container, and the sample and the substance that binds to the measurement substance in the sample are immobilized on the surface. When particles are added, if the amount of the substance to be measured is large in the sample, the substance to be measured in the sample will bind to the substance that binds to the substance to be measured immobilized on the surface of the magnetic particles. Since it cannot bind to the measurement substance immobilized on the magnetic particles, the magnetic particles form a concentrated image by the magnetic force in the vicinity of the needle-shaped magnetic pole positioned below the reaction vessel.

When the substance to be measured does not exist, magnetic particles are bound to the inner surface of the reaction container on which a substance that competes with the substance to be measured is immobilized, so that a uniform image of the magnetic particles is formed on the inner surface of the reaction container.

In the present invention, since the needle-shaped magnetic pole is used, a distribution pattern of unbonded magnetic particles can be clearly formed.

〔Example〕

 The present invention will be described based on examples.

Example 1. Detection of irregular antibody by antiglobulin test (Cooms method) <Preparation of O-type erythrocyte solid phase plate> U-shaped bottom microplate (NUNC, serial number 464394)
Wheat germ lectin (WGA) (manufactured by Seikagaku Corporation) adjusted to 10 μg / ml with 0.01 M phosphate buffer (PBS), pH 7.0 was added to each well in an amount of 100 μm and treated at room temperature for 30 minutes. Next, a WGA-treated plate was obtained by washing 5 times with 0.01 M PBS, pH 7.0, 300 μm each, and drying at room temperature. Next, surge screen (Ortho, serial number 3SS837), which is a human O type red blood cell having each blood group antigen in Table 1, was adjusted to 0.3% with physiological saline, and this was adjusted to 25 μ / well per WGA.
The surge screen was adsorbed on the microplate by adding it to the treated microplate and allowing it to stand at room temperature for 10 minutes. Contains 0.1% bovine serum albumin (BSA)
It was washed 3 times with 200 μl of 0.01MPBS, pH 7.0.

<Preparation of anti-human IgG sensitized latex beads> Magnetic brown latex estapor LMP233 (RH
ONE-POULENC) and anti-human IgG (Capple, production number 0601-0081) were adjusted to 0.4% and 1 μg / ml with 0.1 M glycine / sodium hydroxide buffer (hereinafter glycine buffer) pH 8.3, respectively. Then, 2 ml of each was mixed and reacted at 37 ° C. for 1 hour to sensitize the latex with anti-human IgG. Next, such a sensitized latex was washed three times with 2 ml of 0.1 M glycine buffer containing 1% BSA and pH 8.3, and then non-specific by reacting 2 ml of the same buffer at 37 ° C for 30 minutes. Blocking was performed to suppress aggregation. Furthermore, 0.01 ml of 0.01% sarcosinate LN (surfactant manufactured by Nikko Chemicals Co., Ltd.), 0.01 ml of PBS containing 0.1% BSA, pH 7.0 was washed 3 times with 2 ml each and stored in 4 ml of the same PBS.

<Detection of Irregular Antibodies> On the O-type red blood cell solid-phase plate, 60 μl each of Liss solution and anti-, anti-K, anti-Fy ² , anti-antisera (manufactured by Ortho) as samples or 0.01 M as negative control
PBS was added to each well in an amount of 30 μm and reacted at 37 ° C. for 30 minutes. Each well was washed 4 times with 200 μM of 0.01MPBS, pH 7.0, and then the anti-human IgG latex beads were added to 25 wells.
μ / well was added. This microplate was placed on the reaction accelerator shown in FIG. 2 and incubated for 20 seconds.

FIG. 2 is a perspective view (including a partial cross-sectional view) showing the structure of the reaction promoting device.

The support base 4 having a raised outside so that the microplate waiting for 8 × 12 wells can be placed without being displaced, is provided with columnar grooves 5 in a matrix at positions corresponding to the respective wells of the microplate. A cone-shaped acicular ferrite magnet (magnetic flux density 2200 gauss) 6 is housed inside. When the microplate is placed on the support base 4,
The needle-shaped magnetic pole 6 is arranged in the center of the U-shaped bottom surface.

According to the distribution pattern of the anti-human IgG latex formed on the bottom surface of the well, Table 2 shows that the cells uniformly spread on the bottom surface of the well are positive (+), and those gathered at the center of the well are negative (-).
Is the result of the determination. As shown in Table 2, the determined antiserum results were consistent with the phenotype of the immobilized antigen shown in Table 1.

Example 2. Detection of autoantibodies by direct antiglobulin test <Preparation of WGA-treated plate for red blood cell solid phase> A WGA-treated plate is prepared by the procedure described in Example 1.

<Preparation of anti-rabbit IgG-sensitized latex beads> Preparation is performed by the same procedure as in Example 1, except that anti-human
Here, instead of IgG, anti-rabbit IgG (Kirkegaard & Pe
rry Laboratories, Inc., serial number 50-0115-16) is used for sensitization.

<Detection of autoantibodies> Erythrocytes are washed from the test blood containing an anticoagulant with physiological saline and adjusted to 0.3%, and 25 μ each is added to the WGA-treated plate at room temperature for 10 minutes. Let stand. Next, 0.01MPBS containing 0.2% gelatin, pH 7.0 to 200
After washing once with μ, 25 μm each of rabbit-derived Coombs serum (manufactured by Ortho) is added and reacted at room temperature for 10 minutes. After washing three times with 0.01MPBS, pH7.0, 200μ, the above anti-rabbit IgG latex beads were added at 25μ / well, and a magnetic field was applied in the same manner as in Example 1 to form a reaction pattern for determination. You can

Example <Preparation of Antibodies HB _S antibody-immobilized plate> 3.HB _S antigen of the detection (1) U-bottom micro-plates (NUNC, Inc., product number 464394) to the rabbit anti-HB _S antibody affinity purified 0.01M
Adjust to 10 μg / ml with PBS, pH 7.0, add 100 μ / well, react at 37 ° C. for 1 hour, and adsorb to microplate. Wash with 0.01MPBS, pH7.0 three times using 250μ of each well, and add 200μ of 0.01MPBS, pH7.0 containing 0.2% BSA.
/ Well is added and reacted at 37 ° C for 1 hour to perform blocking. After washing 3 times with 0.01μPBS containing 0.05% Tween20, pH 7.0 using 250μ of each well, it is dried at room temperature and 4 ℃.
Save to.

<Anti-HB _S adjustment of antibody sensitized latex beads> same magnetic latex as used in Example 1 and anti-HB
_S- antibody was added 0.4M with 0.1M glycine buffer, pH 8.3.
% And 2.5 μg / ml, mix 2 ml each and mix at 37 ℃
React with time sensitization. 2 ml of 0.1 M glycine buffer, pH 8.3 containing 1% BSA is washed 3 times, and 2 ml of the same glycine buffer is reacted at 37 ° C. for 30 minutes for blocking. 0.01M PBS with 0.05% Tween 20 and 0.1% BSA, pH
After washing 3 times with 2 ml of 7.5, 4 ml of the same PBS is added and used for the reaction.

<HB _s detection of antigen> HB _S antigen positive sera and respectively negative serum to the HB _S antibody solid phase plate undiluted reacting 25.mu. / well added 15 minutes at room temperature as a control. The added serum is discarded or washed as it is with 0.01MPBS, pH 7.5 once with 200μ. It said here the anti-HB _S antibody-sensitized latex beads 25μ
Is added to form a distribution pattern in the same manner as in Example 1.

In the above examples, the substance to be measured in the sample was bound to the magnetic particles and gathered at the center of the bottom surface of the well of the microplate by the needle-shaped magnetic poles to form a clear distribution pattern of the magnetic particles. You can make various judgments.

Further, as shown in FIGS. 3 (A) and 3 (C), a substance 9 that binds to a substance to be measured such as an antibody is previously fixed to the bottom surface of the well 8 of the microplate 7, so that after the reaction with the sample. By washing the wells 8 to remove unreacted substances, and reacting the magnetic particles 10 there, the substance 11 to be measured is present (FIG. 3 (A)) and not present (third). Each bonded state of FIG. (C)) is formed. In this way, the binding sensitivity to the magnetic particles can be improved and a stable and clear pattern can be obtained.

Example 4. Detection of HB _S antigen (2) In the same manner as in Example 3, preparation of anti-HB _S antibody solid-phase plate and preparation of anti-HB _S antibody-sensitized latex beads are performed.

And HB _S antigen positive sera and remained negative sera each stock as a control anti-HB _S antibody solid phase plate to 25.mu. / well added <Detection of HB _S antigen>, the addition of anti-HB _S antibody sensitized latex beads one by 25.mu. further Incubate for 15 minutes at room temperature. 0.01MPBS containing 0.05% Tween20 and 0.1% BSA, distribution by collecting anti-HB _S antibody sensitized latex beads in the wells topped with 150 mu / well added to the micro-off plate to the support 4 to pH7.5 microplate bottom Form a pattern.

In this embodiment, as shown in FIGS. 3 (B) and 3 (C), the rapid reaction of the magnetic particles 10 and the needle-shaped magnetic pole is utilized to
By immobilizing the substance 9 that binds the substance to be measured on the bottom surface of the well 8 of the microplate 7, and by diluting it after reacting with a sample containing various precipitating or insoluble substances such as stock solution serum, The binding sensitivity to the magnetic particles 10 is improved to form each binding state in the case where the substance 11 to be measured is present (FIG. 3 (B)) and not present (FIG. 3 (C)), and is stable and stable. A clear pattern is obtained. Therefore, highly sensitive analysis can be expected even with a high-concentration sample. Furthermore, false positives (false positives) due to non-specific agglutination that often cause problems in serum
Is reduced by increasing the dilution ratio.

Example 5. Detection of HB _S antigen using whole blood as a sample An anti-HB _S antibody solid phase plate is prepared in the same manner as in Example 3.

<Anti-HB adjustment of _S antibody-sensitized latex beads> having a particle size of 1μ magnetic latex beads estapor (RHONE-POUL
ENC, serial number LMP233) 0.1M glycine buffer, pH
8.3 at 0.4%, was adjusted to 2 ml, whereas, 1 [mu] g / m of rabbit anti-HB _S antibody affinity purified with the same glycine buffer
Adjust to l and mix 2 ml of each and incubate for 1 hour at 37 ° C. Next, 0.1 M glycine buffer containing 1% BSA, pH 8.3
Wash 3 times with 2 ml each, add another 2 ml and
The latex surface is blocked by reacting for a minute so that non-specific aggregation is suppressed. Then 0.01% sarcosinate
Washed 3 times with 2 ml of 0.01M PBS containing 0.1% BSA and LN (surfactant manufactured by Nikko Chemicals Co., Ltd.), pH 7.5, and then PB
Prepare the reaction by adding S4 ml.

<Whole blood sample> HB _S antigen (made by Midori Cross Co.) whose subtype is ad is added to negative anticoagulated blood as an anode sample, and anticoagulated blood without addition is used as a negative sample.

To anti-HB _S antibody solid phase microplate <Detection of HB _S antigen>, 0.1% BSA and 0.01% sarcosinates LN (manufactured by Nikko Chemicals Co., Ltd.) and 5% dextran (molecular weight 200,000～300,000, Wako Pure Chemical Industries, Ltd.) 0.01MPBS, pH7.5 containing 100 μ / well is added. Then, the whole blood samples of positive and negative each 5 [mu], are mixed each further added each of a solution of anti-HB _S antibody sensitized latex beads 25.mu., is reacted at room temperature for 5 minutes. Then, applying a magnetic field to collect the anti HB _S antibody sensitized latex beads well bottom of a microplate,
Form a distribution pattern due to aggregation. Such distribution pattern detect the presence of HB _S antigen by observing such a well bottom from below of a microplate, it can be confirmed.

In the present embodiment, unlike the case of centrifugation, it does not act on other sedimentary substances in whole blood and promotes only the sedimentation of magnetic particles to form a pattern that can be set in a short time. . Therefore, a solution having a higher specific gravity than the magnetic particles can be used as the reaction solution. As described above, it is possible to measure even a sample containing various sedimentable or insoluble substances such as whole blood without washing or separating.

The present invention is not limited to the above-described embodiment, and the electromagnet 12 or the like as shown in FIG. 4 can be used in addition to the permanent magnet. FIG. 4 is a view showing a needle-shaped magnetic pole composed of an electromagnet.

The electromagnet 12 is formed by winding a coil around a ferromagnetic body having a conical tip. This electromagnet 12 is arranged under each V-bottom well 8 of the microplate 7, and an attractive force can be applied to the magnetic particles in the well 8 by applying a voltage + V to the coil for a certain time during the reaction. Further, while the magnetic field is applied, the reaction container may be in a stationary state or a transfer state.

Further, in the above-mentioned embodiment, the U-bottom and V-bottom microplates were used as the reaction vessel, but the shape of the reaction vessel is not limited to this, and the needle-shaped magnetic poles positioned below the reaction vessel are used. If the magnetic particles settle out due to the magnetic force of, the positive and negative distribution patterns are formed.

〔The invention's effect〕

According to the present invention, since the needle-shaped magnetic pole is positioned below the bottom of the reaction vessel, the magnetic particles can be forcibly settled on the bottom of the reaction vessel, and a clear distribution pattern can be obtained in a short time.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram of the present invention, FIG. 2 is a perspective view of a support base in which needle-shaped magnetic poles are arranged in a matrix, and FIG. 3 (A) is a sample in Examples 1 to 3. FIG. 3 (B) is a schematic diagram showing the binding state of particles at the time of positive reaction when the bottom surface of the well was washed after the reaction with, and FIG. 3 (B) is the binding state of particles at the time of positive reaction when diluted after the reaction with the sample FIG. 3 (C) is a schematic diagram showing the binding state of particles at the time of negative in Examples 1 to 4, and FIG. 4 is a diagram showing needle-like magnetic poles composed of electromagnets. 1,10 …… Magnetic particles 2 …… Reaction vessel 3,6 …… Needle magnetic pole 4 …… Support base 5 …… Groove 7 …… Micro plate 8 …… Well 9 …… Substance that binds to the substance to be measured 11 …… Matter to be measured 12 …… Electromagnet

Claims (3)

[Claims] 1. A substance to be measured and magnetic particles having on the surface thereof a substance that binds to the substance to be measured or competes with the substance to be measured are housed in a reaction vessel, and a needle-shaped magnetic pole is provided below the reaction vessel. An immunological measurement method using magnetic particles, which comprises accelerating the sedimentation of positioned magnetic particles and measuring the distribution pattern of the magnetic particles formed at the bottom of the reaction vessel. 2. The immunological measurement method using magnetic particles according to claim 1, wherein a substance that binds to the substance to be measured is fixed on the inner surface of the reaction vessel. 3. A substance that competes with the substance to be measured is fixed on the inner surface of the reaction container, and the substance to be measured and the magnetic particles having the substance binding to the substance to be measured held on the surface are housed in the reaction container. An immunological measurement method using magnetic particles, which comprises arranging a needle-shaped magnetic pole below the reaction container to promote the precipitation of the magnetic particles and measuring the distribution pattern of the magnetic particles formed at the bottom of the reaction container.