JPH0359459A - Method for measuring antigen and antibody - Google Patents

Abstract

PURPOSE:To enable measurement with high sensitivity by specifying the average grain size of insoluble carrier particles which do not contain a magnetic material to 1.5 to 3 mum and the average grain size of an insoluble carrier which contains the magnetic material to 0.1 to 2mum, specifying the ratio of using both and making measurement at 600 to 1,000 nm wavelength. CONSTITUTION:The average grain size of the insoluble carrier particles which do not contain the magnetic material is 1.5 to 3 mum and the average grain size of the insoluble carrier particles which contains the magnetic material is 0.1 to 2 mum. These particles are used at 1:4 to 4:1 using ratios of both. The quantity of the remaining insoluble carrier particles which do not contain the magnetic material is measured at 600 to 1,000 nm wavelength. Polystyrene latex is particularly useful as the insoluble carrier particles which contain the magnetic material and iron and magnetic iron oxide are preferable as the mag netic material to be incorporated into the insoluble carrier particles. The particles contg. 30 to 50% magnetic material is used. Polyvinyl toluene latex is particularly useful as the insoluble carrier particles which do not contain the magnetic material.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an antigen/antibody measuring method using insoluble carrier particles containing an antibody or a magnetic material carrying an antigen. In particular, the present invention relates to a method for quantitatively measuring proteins and related substances contained in serum, plasma, urine, etc., particularly in the field of medical clinical testing.

[Conventional technology]

Currently, in the field of immunodiagnosis in clinical testing, methods for measuring antigens or antibodies by supporting an antibody or antigen on insoluble carrier particles such as latex and reacting with the antigen or antibody in a specimen include: The latex particles are reacted with the antigen or antibody in the specimen, the resulting aggregated latex particles are irradiated with light of a specific wavelength, the intensity of the scattered light or transmitted light is measured, and from the increase in intensity, the antigen or antibody in the specimen is Method for quantifying antibodies (for example, Japanese Patent Publication No. 5B-11175)
.

(b) Antibodies that do not agglutinate against the antigen in the sample (
A monoclonal antibody) is supported on insoluble carrier particles containing a magnetic substance, and reacted with the antigen in the sample. (hereinafter referred to as "sensitized insoluble carrier particles").By applying a magnetic field from outside the reaction vessel, the sensitized insoluble carrier particles containing unreacted magnetic material and the sensitized insoluble carrier particles containing magnetic material are separated. The aggregate containing the sensitized insoluble carrier particles is separated from the reaction mixture, the amount of the remaining sensitized insoluble carrier particles that do not contain the magnetic substance is optically measured, and the antigen in the sample is detected from the decrease in turbidity. There are methods for quantifying (W089101161), etc.

[Problem that the invention seeks to solve]

Antigens using magnetic materials, such as those described in (b) above,
In the antibody measurement method, the reaction mixture theoretically contains sensitized insoluble carrier particles that do not contain unreacted magnetic material (hereinafter referred to as A), and sensitized insoluble carrier particles that contain unreacted magnetic material (hereinafter referred to as A). (hereinafter referred to as B), aggregated particles of sensitized insoluble carrier particles that do not contain a magnetic substance (hereinafter referred to as C), aggregated particles of sensitized insoluble carrier particles that contain a magnetic substance (hereinafter referred to as below), Aggregated particles (hereinafter referred to as E
) exists. Of these, B, D, and E are removed by applying a magnetic field, and A and C remain and are optically measured.

Incidentally, latex particles having an average particle size of 0.8 μm have been conventionally used as sensitized insoluble carrier particles that do not contain a magnetic substance. However, the average particle size of the particles used was 1μ
m or less, 600 nm-1 with little influence of sample components
When measured at a wavelength of 0.000 nm, C among the remaining particles is detected as an increase in turbidity. The increase in the turbidity of C is a factor that significantly lowers the sensitivity in the method of measuring and quantifying the decrease in turbidity. In particular, when the amount of antibodies or antigens supported on insoluble carrier particles that do not contain a magnetic material is extremely larger than the amount of antibodies or antigens supported on insoluble carrier particles that contain a magnetic material, specifically, magnetic If the proportion of sensitized insoluble carrier particles that do not contain a magnetic substance is significantly higher than that of sensitized insoluble carrier particles that contain a magnetic substance, or if the average particle size of the sensitized insoluble carrier particles that contain a magnetic substance is This becomes noticeable when the average particle size of the insoluble carrier particles is significantly larger than the average particle size of the insoluble carrier particles.

[Means for solving problems]

In order to solve the conventional problems and provide a measurement method with even higher sensitivity, the present inventors have conducted intensive studies and have developed a method for optically detecting antigen-antibody reactions using sensitized insoluble carrier particles that do not contain magnetic substances. As a method for detecting an aggregation reaction, insoluble carrier particles with a large average particle size of 1.5 μa+ to 3 μ−, which has not been generally used in the past, and sensitized insoluble carrier particles containing a magnetic substance with an average particle size of 0.1 μm are used. Particles of ~2 μm were used in a ratio of l:4 to 4i1, and 600 nm-100
The inventors have learned that the above-mentioned problems can be solved by optically measuring at a wavelength of 0 nm, and have completed the present invention.

That is, the gist of the present invention is that insoluble carrier particles that do not contain magnetic material and insoluble carrier particles that contain magnetic material particles, respectively,
An insoluble carrier containing unreacted magnetic material is prepared by carrying an antibody or antigen, reacting the carried antibody or antigen with the antigen or antibody, or a mixture thereof in a solution, and then applying a magnetic field to the reaction mixture. The aggregate containing particles and insoluble carrier particles containing a magnetic material is separated from the reaction mixture, and the amount of remaining insoluble carrier particles not containing a magnetic material is determined by optically detecting turbidity or scattered light. In the method for quantitatively measuring an antigen or antibody, the average particle size of the insoluble carrier particles not containing a magnetic substance is 1.5.
μn is in the range of 1 to 3 μm, and the average particle size of the insoluble carrier particles containing the magnetic material is 0.1 μ1 to 2 μm.
and the usage ratio of both is 1:4 to 4:
The present invention relates to an antigen/antibody measuring method characterized by measuring the amount of insoluble carrier particles within the range of 1 and containing no residual magnetic material at a wavelength of 600 to 11,000 nm.

The present invention will be explained in detail below.

As the insoluble carrier particles containing the magnetic substance used in the present invention, organic polymer substances and inorganic substances can be used.

Examples of organic polymer substances include latexes of organic polymer substances obtained by emulsion polymerization such as gelatin particles and styrene polymer particles such as polystyrene and styrene-butadiene copolymers, and polystyrene latex is particularly useful.

The magnetic material to be contained in the insoluble carrier particles is preferably iron and magnetic iron oxide, and those prepared to contain 5% to 100%, preferably 30% to 50% of this are used.

The average particle size of the insoluble carrier particles containing the magnetic material of the present invention is 0.1 μl to 2 μl, preferably 0.5 μl to 2 μl.
A thickness within the range of 1.5 μm is used.

If the average particle size is smaller than 0.1 am, separation using a magnetic field takes a long time and is not practical. Furthermore, if the average particle size is larger than 2 μm, the amount of antibody or antigen supported per unit weight will be reduced, and the sensitized insoluble carrier particles that do not contain a magnetic substance will tend to aggregate with each other, which is not preferable.

Examples of insoluble carrier particles that do not contain magnetic substances include gelatin particles, polystyrene, polyvinyltoluene, and styrene particles.
A latex of an organic polymer material obtained by emulsion polymerization such as butadiene copolymer particles is used, and polyvinyltoluene latex is particularly useful.

The particle size of such insoluble carrier particles is in the range of 1.5 μm to 3 μm.

When the average particle size is smaller than 1.5 μ■, the surface area per unit weight becomes large, and the amount of antibody or antigen supported increases.
This tends to cause aggregation of sensitized insoluble carrier particles that do not contain a magnetic substance. In addition, when measuring the agglutination reaction of the sensitized insoluble carrier particles IEiJ that does not contain a magnetic substance at a wavelength of 600 nm to 11000 nm, which is less affected by sample components, it is detected as an increase in turbidity, which causes a decrease in sensitivity. So I don't like it.

Moreover, if the average particle size is larger than 3 μm, natural sedimentation will be rapid, which is not practical.

On the other hand, according to the present invention, when sensitized insoluble carrier particles containing no magnetic material have an average particle diameter of 1.5 μm to 3 μm and are measured at a wavelength of 600 to 11000 nm,
Since the aggregation of sensitized insoluble carrier particles that do not contain a magnetic substance is unexpectedly detected as a decrease in turbidity, it acts additively to the original turbidity reduction, and the sensitized insoluble carrier particles that do not contain a magnetic substance are unexpectedly detected as a decrease in turbidity. Even if aggregation occurs, the sensitivity does not decrease, and on the contrary, measurement can be performed with high sensitivity.

The above-mentioned method for supporting an antibody or an antigen on an insoluble carrier particle is common to both insoluble carrier particles containing a magnetic substance and insoluble carrier particles not containing a magnetic substance. This is carried out by physically adsorbing or chemically supporting the antibody or antigen against the antibody.

A polyclonal antibody or a monoclonal antibody is used as the supporting antibody, and its form is γ globulin, I
It may be any of gG, IgM, F (ab')z, Fab', etc.

When an antigen is used as the carrier, cell fragments, haptens, antigen proteins, immune complexes, natural or synthetic polymer antigens, etc. are used.

Generally, the amount of antigen or antibody supported is the same for both types of insoluble carrier particles, and is 0.01μ/ll11-21
IIg/1ml, preferably 0.05mg/ve1
-0°5111 g/lN.

The combination of antibodies or antigens supported on the above two types of insoluble carrier particles can be varied depending on the purpose, and in the case of antibodies, the following combinations may be mentioned, for example.

(1) A polyclonal antibody is supported on insoluble carrier particles containing a magnetic substance, and a polyclonal antibody is supported on insoluble carrier particles that do not contain a magnetic substance.

(2) A monoclonal antibody is supported on insoluble carrier particles containing a magnetic substance, and a polyclonal antibody is supported on insoluble carrier particles that do not contain a magnetic substance.

(3) A polyclonal antibody is supported on insoluble carrier particles containing a magnetic substance, and a monoclonal antibody is supported on an insoluble carrier particle that does not contain a magnetic substance.

(4) A monoclonal antibody is supported on insoluble carrier particles containing a magnetic substance, and monoclonal antibodies with different recognition sites are supported on insoluble carrier particles that do not contain a magnetic substance.

(5) Two or more types of monoclonal antibodies with different recognition sites are carried on insoluble carrier particles containing a magnetic substance, and the same monoclonal antibodies as the antibodies carried on insoluble carrier particles containing a magnetic substance are carried on insoluble carrier particles that do not contain a magnetic substance. Carrying antibodies.

The insoluble carrier particles carrying antibodies or antigens as described above were prepared using 0.1 M IJ acid buffer (pt17).
It is dispersed in an aqueous solvent such as 0.1M Tris-HCl buffer (pH 8,0) to a concentration of 0.01 to 10% by weight and used as a suspension.

The ratio of the sensitized insoluble carrier particles that do not contain a magnetic substance and the sensitized insoluble carrier particles that contain a magnetic substance is 1:4 to 1:4.
Selected from a range of 4:1.

If the proportion of the sensitized insoluble carrier particles that do not contain a magnetic substance increases beyond the above range, reactions between the sensitized insoluble carrier particles that do not contain a magnetic substance will increase, which is not preferable. Also,
If the proportion of the sensitized insoluble carrier particles containing a magnetic substance increases, reactions between the sensitized insoluble carrier particles containing a magnetic substance occur exclusively, which is undesirable because it causes a decrease in sensitivity.

Next, a specific measurement method will be explained.

(1) A method of mixing in advance sensitized insoluble carrier particles containing a magnetic substance and sensitized insoluble carrier particles not containing a magnetic substance.

First, sensitized insoluble carrier particles containing a magnetic substance and sensitized insoluble carrier particles not containing a magnetic substance are mixed in advance in the above-mentioned usage ratio. This mixed suspension is reacted with an antigen or antibody (hereinafter referred to as a specimen) contained in plasma, serum, or urea to be measured. The sample is 0.1MTrts-
Hydrochloric acid buffer (pH 8) or 0.1 M phosphate buffer (pH 8)
It may be diluted with a buffer such as 7). The reaction is carried out at room temperature until equilibrium is reached. Also, in order to speed up the reaction rate, 25~
The reaction may be carried out in a constant temperature bath at 50°C. It usually takes about 30 minutes for the reaction to reach equilibrium at room temperature.

(2) A method in which a sample is reacted with sensitized insoluble carrier particles that do not contain a magnetic substance, and then sensitized insoluble carrier particles that contain a magnetic substance are added.

First, sensitized insoluble carrier particles that do not contain a magnetic substance are reacted with a specimen or a specimen diluted with a buffer solution. The reaction varies depending on the antigen or antibody being measured, but is generally carried out at room temperature for about 10 minutes. The reaction time is selected so that the antibody-antigen reaction between the antibody or antigen supported on the sensitized insoluble carrier particles not containing a magnetic substance and the antigen or antibody in the specimen does not reach equilibrium. Subsequently, sensitized insoluble carrier particles containing a magnetic substance are added at the above-mentioned usage ratio, and an antibody/antigen reaction is further performed. The reaction is carried out at room temperature until equilibrium.

Further, in order to speed up the reaction rate, the reaction may be carried out in a constant temperature bath at 25 to 50''C.

(3) Add a certain amount of an antigen to the antibody to be measured (or an antibody to the antigen) to the reaction system, and allow the added antigen (or antibody) to react in advance with the antibody (or antigen) in the specimen to be measured. Afterward (or at the same time), a method of reacting sensitized insoluble carrier particles that do not contain a magnetic substance and sensitized insoluble carrier particles that contain a magnetic substance in the same manner as in (1) or (2) above.

For example, when attempting to measure antibodies contained in a specimen, a buffer solution (for example, 0.1M Tris-HCl p118) containing a certain amount of an antigen for the antibody of interest is prepared in advance. The antibody in the sample is reacted with this buffer solution, and then the sensitized insoluble carrier particles containing the magnetic material carrying the antibody and the sensitized insoluble carrier particles carrying the antibody but not containing the magnetic material are combined with the above ((
Mix and react in the same manner as in 1) or (2). At this time, the antibody in the sample, the buffer solution, and two types of insoluble carrier particles may be mixed together to perform a competitive reaction. The reaction time is carried out at room temperature until equilibrium is reached. Further, in order to speed up the reaction rate, the reaction may be carried out in a constant temperature bath at 25°C to 50°C.

For methods (1), (2), and (3), the reactions are generally carried out in a spectrometer cell, 966 microplate,
When the reaction reaches equilibrium, a magnetic material is contained in the container at a position where it does not obstruct photometry by applying a magnetic field from outside the container or by introducing or inserting magnetic powder or small pieces of magnetic material into the reaction mixture. Aggregates of sensitized insoluble carrier particles and sensitized insoluble carrier particles not containing magnetic material and sensitized insoluble carrier particles containing unreacted magnetic material are collected. As the magnet, a permanent magnet is used, and 1 is a magnet. The sensitized insoluble carrier, which does not contain a floating magnetic substance, remains in the reaction solution. This residual amount depends on the amount of antigen in the sample in the case of antigen measurement, and on the amount of antibody in the sample in the case of antibody measurement. This residual amount is determined by externally measuring the absorbance or scattered light intensity of light at a certain wavelength in the range of 600 nm to 11000 nm, preferably 800 nm to 1000 nm. In the method of the present invention, a calibration curve is created by measuring the absorbance or the intensity of scattered light for samples with different concentrations of known concentrations, and this calibration curve is used to measure the absorbance of samples with unknown concentrations. Alternatively, the concentration of an unknown antigen or antibody can be determined from the scattered light intensity.

(Effects of the Invention) The method of the present invention has the following advantages compared to conventional techniques using particles containing magnetic material.

That is, in the present invention, by using an insoluble carrier having an average particle diameter of 1.5 μm to 3 μm, which has not been used conventionally, as the sensitized insoluble carrier particles that do not contain a magnetic material, the sensitized insoluble carrier particles do not contain a magnetic material. This method eliminates the problem of reduced sensitivity due to reactions between insoluble carrier particles, making it possible to perform measurements with higher sensitivity than before. Further, the present invention is very easy to operate and can be carried out in a short time. These advantages make the system ideal for automation and promising for processing multiple samples.

[Example],) Detection of AFP (Reagent preparation method) - Preparation method of anti-AFP sensitized latex containing magnetic material.

0.1 M Tris-hydrochloric acid buffer (pH 8,0) in which 1% latex separated from 10% (W/V) 0.7 μm magnetic latex (Estapol SML266 manufactured by Rhone Poulenc) was dispersed 1 c c ni, h) AFP
Add 0.25 μg of anti-AFP specific antibody obtained by immunizing animals with (alpha-fetobutyin) and stir for about 1 hour.
After sensitization, centrifugation (16°000 rpm for 10 minutes) was performed, the supernatant was removed, and the separated latex was mixed with 0.3% bovine serum albumin 10°1M Tris-HCl buffer (p
H8,0> and further stirred for 1 hour. After centrifuging again (16,00 rpm for 10 minutes), remove the supernatant and remove the supernatant. Redisperse in IM phosphate buffer (pH 7).

- Preparation method of anti-AFP sensitized latex that does not contain magnetic material.

A 0.1 M Tris-HCl buffer (pH 18,0
) l c c, anti-AFP specific antibody 0.1 mg/ml
was added, stirred for about 1 hour, sensitized, and then centrifuged (16,
00 rpm for 10 minutes), remove the supernatant, and add the separated latex to 0.1% bovine serum albumin 10.1M T.
Redisperse in ris-hydrochloric acid buffer (pi18.0),
Stir for an hour. Centrifuge again (16,00 rpm for 1
After 0 minutes), remove the supernatant and redisperse in 0.1M phosphate buffer (pH 7).

(Procedure) After mixing 1% anti-AFP sensitized latex containing a magnetic substance and 1% AFP sensitized latex not containing a magnetic substance at a ratio of 1:1, the mixture was mixed with 0.1 M IJ acid buffer. Dilute 1:2. This is used as a latex preparation reagent.

AFP standard product (250ng/n+ffi, manufactured by Diatron) was mixed with human serum that does not contain AFP at 30ng/n.
After diluting the sample to 100 ml, a 2-fold dilution series is prepared and used as a sample.

Sample 1 in a cell for LPIA100 (manufactured by Mitsubishi Kaei Co., Ltd.)
0ul, buffer solution (0.1M Tris-HCl (pH 8.0)
)) Add 50 ui of 250μ11 Latex Preparation Reagent, mix well, and react at room temperature for 1 hour. next,
A permanent magnet is applied from the bottom of the cell to collect latex aggregates containing magnetic material and unreacted latex containing magnetic material at the bottom. Aggregates containing magnetic material and unreacted latex containing magnetic material can be collected in about 10 minutes. Then, the concentration of the insoluble carrier particles that do not contain magnetic material remaining on the microplate is measured using a fully automatic immunodiagnosis device LPIA.
100 (manufactured by Mitsubishi Kakai Co., Ltd.), wavelength 950
It was determined by measuring its absorbance at nm. Figure 1 shows the calibration curve data of absorbance and AFP concentration. For the comparison data with the present invention shown in the same figure, commercially available products of EIA (Ono Immunoball: manufactured by Ono Pharmaceutical Co., Ltd.) and LPIA-100 (Elpia Ace AFP, manufactured by Mitsubishi Kasei Corporation) were used.

[Example 2] Detection of HBs (Reagent preparation method) - Preparation method of anti-HBs sensitized latex containing magnetic material.

0.1 M Tris in which 1% latex separated from 10% (W/V) 0.7 μ-magnetic latex (Estaball SML266 manufactured by Rhone-Poulenc) was dispersed.
= Hydrochloric acid buffer (pH 8,0) 1 cc, human HB
Add 0.25μ of anti-HBs polyclonal antibody obtained by immunizing an animal with the SADW antigen, stir for about 1 hour,
After sensitization, centrifugation (16,00 rpm for 10 minutes) was performed, the supernatant was removed, and the separated latex was diluted with 0.3% bovine serum albumin in 70.1M Tris-HCl buffer (p
H8,0) and further stirred for 1 hour. After centrifugation again (16°00 rpm for 10 minutes), the supernatant was removed and redispersed in 0.1 M phosphate buffer (pH 7).

・Preparation method of anti-HBs sensitized latex containing no magnetic material.

A 0.1 M Trls-hydrochloric acid buffer (pH
8,0) lcc, anti-HBs (d type) monoclonal antibody 0.1/ml. was added, stirred for about 1 hour, sensitized, centrifuged (16,000 rpm for 10 minutes), the supernatant was removed, and the separated latex was diluted with 0.1%
Bovine serum albumin 10. LM Tris-HCl buffer (
(pH 8.0) and stirred for 1 hour. After centrifuging again (16,00 rpm for 10 minutes),
Remove the supernatant and redisperse in 0.1M phosphate buffer (pH 7).

(Procedure) After mixing 1% anti-AFP sensitized latex containing a magnetic substance with 1% AFP sensitized latex not containing a magnetic substance at a ratio of 1:1, the mixture was diluted 8 times with 0.1 M phosphate buffer. dilute to This is used as a latex preparation reagent.

HBs standard product 570U/mjl! (manufactured by Mitsubishi Kakai Co., Ltd.) is diluted 2 times with normal rabbit serum to prepare a series and use this as a sample.

96-hole microplay) (Falcon Flexi
ble As5ay Plate flat bottom) 50u1 sample
．． , buffer solution (0,1M Tris-HCl (pH 8,0)
) Add 50 μl of latex preparation reagent and 50 μl of latex preparation reagent, mix well, and react at room temperature for 30 minutes. Next, apply a permanent magnet to the side of the sample dispensing part of the microplate from the outside.
Collect latex aggregates containing magnetic material and unreacted latex containing magnetic material on the measuring surface. Aggregates containing magnetic material and unreacted latex containing magnetic material can be collected in about 10 minutes. Then, the concentration of the insoluble carrier particles that do not contain any magnetic material remaining on the microplate was measured using a microplate reader (NJ Intermed Co., Ltd.).
2000), the absorbance was measured at a wavelength of 620 nm. Figure 2 shows the calibration curve data of absorbance and HBs concentration.

[Example 3] Detection of TSH (Reagent preparation method) - Preparation method of anti-TSH sensitized latex containing magnetic material.

0.1 M Trls in which 1% latex separated from 10% (W/V) 0.7 μ-magnetic latex (Rhone-Poulenc Estaball SML266) was dispersed.
- Hydrochloric acid buffer (pH 8,0) lcc, anti-human T
Add 0.25μ of SH monoclonal antibody, stir for about 1 hour to sensitize, and then centrifuge (16°, 000 rpm for 1 hour).
0 min), remove the supernatant, and drain the separated latex at 0.0 min).
3% bovine serum albumin 10° was redispersed in 1M Tris-HCl buffer (pH 8,0) and stirred for an additional hour.

After centrifuging again (16°00 rpm for 10 minutes), remove the supernatant and add 0.1 M phosphate buffer (pH 7).
redisperse.

- Preparation method of anti-human TSH sensitized latex that does not contain magnetic material.

0, IM Tris-HCl buffer (pH 8,0) in which 1% latex separated from 10% (W/V) 2.02 μm polystyrene latex (manufactured by Dow) was dispersed.
I c c, anti-human TSH monoclonal antibody (
hTsH, hTsHβ-chain) 0, 1 mg/
tal was added, stirred for about 1 hour, sensitized, centrifuged (16.00 rpm for 10 minutes), the supernatant was removed, and the separated latex was mixed with 0.11% bovine serum albumin 10.0.
Redisperse in 1M Tris-HCl buffer (pH 8,0) and stir for 1 hour. Centrifuge again (16,000 rpm)
m for 10 minutes), then remove the supernatant and redisperse in 0.1M phosphate buffer (pH 7).

(Procedure) Add 1% of anti-human I-TSH sensitized latex containing 1% of magnetic material.
AFP-sensitized latex containing no magnetic material is mixed at a lit ratio, and then diluted 8 times with 0.1M phosphate buffer. This is used as a latex preparation reagent.

Human TSH standard product (25μlU/a/!, manufactured by Sigma)
pH 80, 1m Tri s-HCl buffer (0°1%
Prepare a 2-fold dilution series with BSA (containing BSA) and use this as the sample.

966 microplate (Falcon Flexi
ble As5ay Plate flat bottom) sample 50uQ
, buffer solution (0.1M Tris-HCl (pH 8.0))
Add 50 μl of the latex preparation reagent, mix well, and allow to react at room temperature for 90 minutes. Next, a permanent magnet is applied from the outside to the side surface of the sample dispensing part of the microplate, and the latex aggregate containing the magnetic substance and the unreacted latex containing the magnetic substance are collected on the surface. Aggregates containing magnetic material and unreacted latex containing magnetic material can be collected in about 10 minutes. Then, the concentration of the insoluble carrier particles that do not contain magnetic substances remaining on the microplate was measured using a microplate reader (NJ20 manufactured by Nippon Intermed Co., Ltd.).
00) at a wavelength of 620 nm by measuring its absorbance. Figure 3 shows the calibration curve data of absorbance and concentration in human TS.

[Example 4] Detection of ATLAGP'24 antibody (reagent preparation method) - Preparation method of magnetic substance-containing anti-ATLAGP24 sensitized latex.

10% (W/V) of 0.1 M Tris dispersed in 1% latex separated from 0.7 μm magnetic latex (Estapol SML266 manufactured by Rhone-Poulenc).
- Hydrochloric acid buffer (pH 8,0) lcc to adult T1
Anti-ATLAGP24 obtained by immunizing animals with 11-cell leukemia virus antigen GP24 recombinant product (manufactured by Triton)
After adding 0.25 μ of antibody and stirring for about 1 hour to sensitize,
Centrifuge (16,000 rpm for 10 minutes), remove the supernatant, redisperse the separated latex in 0.3% bovine serum albumin 10.1M Tris-HCl buffer (pH 8,0), and further Stir for an hour. Centrifuge again (16
, 00 Orpm for 10 minutes), remove the supernatant,
061M phosphate buffer (pH 7).

- Preparation method of anti-ATLAGP24 sensitized latex that does not contain magnetic material.

A 0.1 M Tris-HCl buffer (pH 8,
0) 1 c c, anti-ATLAGP24 antibody 0, 11
After adding 11g/ml and stirring for about 1 hour to sensitize, centrifuge #1 (16,000 rpm for 10 minutes), remove the supernatant, and transfer the separated latex to Tris containing 0.1% bovine serum albumin 10.1M. -Redisperse in hydrochloric acid buffer (pH 8,0) and stir for 1 hour. Centrifuge again (16,0
After 10 minutes at 0 rpm, remove the supernatant and
Redisperse in lM phosphate buffer (pH 7).

(Procedure) 1% of the anti-ATLAGP24 sensitized latex containing a magnetic substance is mixed with 1% of the AFP sensitized latex that does not contain a magnetic substance.
After mixing at a ratio of t, dilute 8 times with O, 1 M phosphate buffer. This is used as a latex preparation reagent.

Gelatin agglutination method and EIA method positive specimens at pH 80, 1M
A 2-fold dilution series is prepared with Tris=hydrochloric acid buffer (containing 0.1% BSA) and used as a sample.

Sample 10μe in a cell for LPIA100 (manufactured by Mitsubishi Chemical Corporation)
, a buffer containing 50 μ/ml of GP24 recombinant protein (0
, 1MTrts-250μi of hydrochloric acid (pH 8,0)) and 50μl of latex preparation reagent were added, and after mixing well,
Allow to react at room temperature. Next, a permanent magnet is applied to the bottom of the cell from the outside, and the latex aggregate containing the magnetic substance and the unreacted latex containing the magnetic substance are collected at the bottom. Aggregates containing magnetic material and unreacted latex containing magnetic material can be collected in about 10 minutes. Then, the concentration of the insoluble carrier particles containing no magnetic material remaining on the microplate was measured by measuring the absorbance at a wavelength of 950 nm using LPIAloo (manufactured by Mitsubishi Kasei Corporation). Figure 4 shows the absorbance and sample dilution ratio.
Indicates sponsecurve.

[Brief explanation of drawings]

FIG. 1 shows the calibration curve of AFPLK drug by latex EIA method and LPIA method (latex agglutination reaction method). The reaction intensity ratio (%) in the figure is defined as follows. Reaction intensity ratio (%) = FIG. 2 is a diagram showing the calibration curve of the HBs antigen detection reagent in Honzuke. FIG. 3 is a diagram showing a calibration curve of the human TS)i reagent detection reagent in Honzuke. Figure 4 shows the dose response of ATLAGP24 (adult T-cell leukemia antigen protein) antibody in Honzuke.
It is a figure showing an e curve. Applicant: Mitsubishi Kakai Co., Ltd. Representative: Patent attorney: Haka Hase (1 person)

Claims (1)

[Claims] (1) Antibodies or antigens are supported on insoluble carrier particles that do not contain magnetic material and insoluble carrier particles that contain magnetic material particles, respectively, and the antigen, antibody, or a mixture thereof is added to the carried antibodies or antigens in a solution. After the reaction, by applying a magnetic field to the reaction mixture, insoluble carrier particles containing unreacted magnetic material and aggregates containing insoluble carrier particles containing magnetic material are separated from the reaction mixture, and the remaining magnetic material is separated from the reaction mixture. In a method for quantitatively measuring an antigen or antibody in a solution by optically detecting turbidity or scattered light, the average particle size of the insoluble carrier particles not containing the magnetic substance is determined. The diameter is in the range of 1.5 μm to 3 μm, and the average particle diameter of the insoluble carrier particles containing the magnetic substance is in the range of 0.1 μm to 2 μm, and the ratio of both is 1:4 to 2 μm. An antigen/antibody measuring method characterized by measuring the amount of insoluble carrier particles in the range of 4:1 and containing no residual magnetic material at a wavelength of 600 to 1000 nm.