JP2003156494A - Immunological diagnostics latex particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide highly sensitive latex particles suitable for immunological diagnostics material. SOLUTION: A graft chain having a functional group is bonded onto the latex particle surface by graft polymerization, or the functional group of the bonded graft chain is converted into another functional group and then an antibody or an antigen is sensitized to the functional group in the graft chain bonded onto the latex particle surface, to thereby improve sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to latex particles suitable for immunological diagnostic material.

[0002]

2. Description of the Related Art At present, one of the important means for diagnosis is an immunological test method using latex particles. This is an inspection method in which latex particles having a substance (antibody or antigen) that reacts with a substance to be inspected (antigen or antibody) bound thereto are mixed with a sample to determine the presence or absence of specific aggregation.

Conventionally, methods such as an agglutination reaction for fixing tuberculin polysaccharides and proteins to erythrocytes have been developed.
Since erythrocytes have a large particle size and high specific gravity, they have the advantage that the agglutination reaction time is short and the sensitivity is relatively high, but they have the drawback that various antigens are present on the surface and individual differences are large. In addition, latex particles for immunological diagnosis using polystyrene, styrene-acrylic acid copolymer, etc. as carrier particles have been developed.

Since these latex particles have functional groups such as a sulfate group, a carboxyl group, an epoxy group and an amino group introduced on the surface of the latex particles, an antigen or an antibody can be immobilized by a chemical bond. However, the sensitivity of immunologically-diagnosed latex particles in which a functional group is directly introduced onto the surface of latex particles and antigens or antibodies are sensitized by chemical bonds depending on the concentration of the measurement object or sample and the measurement conditions is not satisfied It was impossible in some cases.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention have made earnest studies to overcome the drawbacks of the prior art, and as a result, have grafted a graft chain having a functional group on the surface of latex particles by graft polymerization or further bonded thereto. By converting the functional group of the chain to another functional group and then immobilizing the antibody or the antigen on the functional group in the graft chain bonded to the surface of the latex particle, it was found that the sensitivity is improved, and the present invention is completed. Came to.

[0006]

Thus, according to the present invention, after the graft chain having a functional group is bonded to the surface of the latex particle by graft polymerization, or after the functional group of the further bonded graft chain is converted into another functional group. Provided is an immunological diagnostic drug particle which improves sensitivity by immobilizing an antibody or an antigen on a functional group in a graft chain bonded to the surface of the latex particle.

Conventionally, the functional group is directly bonded to the particle surface,
Compared with the method of immobilizing the antigen or antibody to the functional group, after the graft chain having a functional group is bonded to the latex particle surface by graft polymerization or the functional group of the further bonded graft chain is converted into another functional group, Immobilizing an antibody or an antigen on the functional group in the graft chain bonded to the surface of the latex particle can increase the number of functional groups that can be introduced, so that the antigen or antibody that can be immobilized can be increased. Thereby, the active sites of the antigen-antibody reaction can be increased.

Compared with the conventional antigen-antibody reaction of an antigen or antibody immobilized on the surface of a particle by a functional group, the antigen-antibody reaction of an antigen or antibody via a functional group introduced into a graft chain is The graft group moves flexibly, and the reaction point of the antigen-antibody reaction develops reactivity as if it were a solution reaction. Further, steric hindrance that is often seen in the conventional reaction on the solid surface rarely causes a decrease in reactivity due to the generation of unreacted points.

Due to the increase of the active sites and the increase of the reactivity thereof, the sensitivity is greatly improved as compared with the conventional immunological latex particles. Also, if the sensitivity is sufficient, the measurement time can be shortened.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The carrier particles in the present invention are carrier particles of an organic polymer substance which are substantially insoluble in a liquid medium used for measurement, such as polystyrene, polystyrene-divinylbenzene copolymer and styrene-butadiene. It may be synthetic polymer particles obtained by emulsion polymerization or the like such as copolymers or particles of natural origin or inorganic type. The average particle size is 0.001 to 100 μm, preferably 0.02 to 1
It is possible to use a micrometer type.

The method of binding the graft chains to the carrier particles is carried out by a graft polymerization reaction. Graft polymerization is a reaction in which a branch polymer having a chemical structure different from that of the trunk is bonded to the trunk polymer to form a graft copolymer.

For graft polymerization, (1) a method of forming a polymerization initiation point in a trunk polymer and polymerizing a monomer forming a branch polymer (graft (co) polymerization method), (2) a branch polymer prepared in advance There is a coupling method in which a backbone polymer is bonded by a reaction between polymers.

The graft polymerization reaction of the present invention can be applied to both of the above graft polymerization methods, but the graft chain is formed only on the solid surface in the heterogeneous system (solid-liquid reaction) of the latex particle surface and the liquid. This is done by the method of surface grafting.

In the graft polymerization reaction for bonding the graft chain to the latex particles of the present invention, (1) a method utilizing a chain transfer reaction to a polymer, (2) a peroxy group introduced by oxidizing a trunk polymer, or A method in which a decomposable group that decomposes a diazo group introduced by diazotizing a side chain amino group to generate a free radical is used as a polymerization initiation point, (3) by irradiation with electron beams or γ rays Method of utilizing polymerization initiation reaction and photochemical reaction, (4)
A method utilizing a polymerization initiation reaction by a redox mechanism of a hydroxyl group or a thiol group of a chain-measuring and a metal ion such as cerium (IV) salt, (5) an epoxy group having an active group of a side chain such as a hydroxyl group, an amino group and a carboxyl group Graft polymerization reaction methods such as those that utilize the polymerization initiation reaction of lactams and polar vinyl monomers can be used.

A specific method of graft polymerization will be exemplified below. Radicals are created in advance by irradiating the carrier particles with a radical initiator, electron beam or gamma ray, and then, for example, acrylate ester, ethylene glycol monomer, epoxy group-containing glycicyl methacrylate, 2-
A method of graft-polymerizing a monomer such as hydroxyethyl methacrylate.

Further, polystyrene has N, N, N ', N'-
Treatment with an alkyllithium compound in the presence of a tetraethylenediamine (TMEDA) complexing agent lithiates the C—H bond of these functional groups. When such a polymer is used for the backbone, anionic polymerization of the vinyl polymer is initiated from the polymer main chain, so that efficient graft polymerization can be performed. Further, a polymer or oligomer having a predetermined molecular weight such as ethylene glycol may be previously graft-bonded to the surface of the carrier by a conventional method.

Tsuchida (Tsuchida,
E. et al. , Makromol. Chem. , R
rapid Commun. 2, 621-626 (198
1) discloses grafting polystyrene by reacting an activated polystyrene derivative with polyethylene glycol.

Becker, H .; , H .; Lucas,
J. Maul, V.M. N. R. Pillai, H .; Anz
inger, M .; Mutter, Makromol. C
hem. , Rapid Commun. , 3, 217-
223 (1982)) discloses polyethylene glycol (PEG) grafted onto polystyrene for use as a polymeric support for peptide synthesis.

Bayer and Rapp (US Pat. No. 4,908,405, E. Bayer and W. Ra).
"Graft Copolym, issued to pp
ersof Crosslinked Polymer
s and Polyoxethylene, Proc
esses For Ther Product
n, and Ther Usage "(1990))
Discloses cross-linked polymers and graft copolymers of poly (ethylene glycol) and provides disclosure of their manufacture and use.

H. Becker, H .; Lucas, J .;
Maul, V.M. N. R. Pillai, H .; Anzin
ger, M.G. Mutter, Makromol. Che
m. , Rapid Commun. , 3, 217-22
3 (1982)) describes the preparation of chloromethylated crosslinked polystyrene-divinylbenzene by reaction with ethylene glycol or low molecular weight poly (ethylene glycol) oligomers.

Candau, Francoise et al.
R. Hebd. Sequences Acad. Sci. , S
Er. C (1976), 283 (11), 453 is a chloromethylated polystyrene grafted with polyoxymethylene having a K-alkofolate group at the end.

As a method of using radiation, a Co60 γ-ray source and electron beam irradiation are used as the radiation source. Pre-irradiate carrier particles with electron beams or γ rays,
And chloromethylstyrene (CMS), acrylic acid (A
A), graft chains of polychloromethylstyrene, acrylic acid polymer, polyglycidyl methacrylate, etc. can be introduced into the carrier particles by contact with a reaction liquid such as glycidyl methacrylate.

When the introduced functional group can fix the antigen or antibody, the antigen or antibody is fixed as it is. However, if the functional group is inactive or insufficient with respect to the antigen or antibody, then a functional group capable of newly immobilizing the antigen or antibody after graft polymerization should be introduced or replaced by a chemical reaction. You can

For example, in the case of chloromethylstyrene (CMS), a tertiary amine can be introduced by reacting with dimethylamine. Also, instead of styrene and chloromethylstyrene, glycidyl methacrylate (GM
There is also a method of using A). In this method, the glycidyl group of glycidyl methacrylate (GMA) can be used to react with sodium sulfite or diethanolamine to introduce a sulfonate or a tertiary amine.

After graft-polymerizing polyethylene glycol of polystyrene particles, the hydroxyl groups of polyethylene glycol can be converted to amino groups by sulfonation with chlorosulfonic acid and treatment with ammonia.

When a functional group capable of newly immobilizing an antigen or antibody is introduced or replaced by a chemical reaction or the like after this graft polymerization, the reaction should be performed as much as possible depending on the conditions for immobilizing the antigen or antibody. You can do it, or you can limit it to one reaction.

The antigen or antibody in the present invention is not particularly limited as long as it is a component generally contained in a sample, but various proteins, polysaccharides, lipids and the like can be mentioned.

More specifically, for example, denatured gamma globumin, rheumatoid factor, antinuclear factor, albumin, IgG,
IgA, IgM, IgE, IgD, streptolysin O, C-reactive protein, alphafetoprotein (AFT), carcinoembryonic antigen (CEA), placental lactogen (HPL), chorionic gonadotropin (HCG), estrogen antibody, insulin antibody. , Hepatitis B surface antigen (H
BS), anti-HBS antibody, Treponema pallidum antigen, rubella antigen, complement components C1, C3, C4, HBs antibody, HIV antibody, HTLV-1 antibody, plasminogen, antithrombin-III, D-dimer, TAT, PPI. Coagulation / fibrinolysis markers such as β2-microglobulin, ferritin, TPA, IAP, urinary polyamine, α-fetoprotein, CA19-9, CA-50, CA15-3, prostate specific antigen (PA), CA-125, CA-130,
CA602, CA72-4, APO-AI, APO-A
II, APO-B, APO-CII, APO-CII
I, APO-E) and other polypoproteins, α1-macroglobulin, α2-macroglobulin, and the like.

The term “antigen” refers to all substances capable of inducing antibody production in humans or animals, and contains one or more substances selected for a particular purpose such as diagnosis, or contains them. Means a mixture of It is the antibody that normally binds specifically to the antigen.
As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.

It is also possible to use a DNA oligomer that specifically binds to methotrexate (an anticancer agent) as an immunoactive substance, and label the purified oligomer with a fluorescent substance or a luminescent substance to measure an antigen or the like. . The latex particles can also be applied to analytical reagents such as agricultural chemicals and environmental pollutants. As an example of this, it can be used for measurement of dioxin pointed out as an environmental pollutant, a herbicide applied to fields and golf courses, an insecticide applied to forests, and various pesticides attached to vegetables.

Similarly, it is possible to measure the antigens and antibodies of animals such as dogs, cats, cows, horses and pigs.

An immunological measurement method for measuring an immunologically active substance in a biological sample by utilizing an antigen-antibody reaction is widely used in the field of clinical examination. For example, single immunodiffusion method (SR
ID), immunoturbidimetric assay (TIA), immunonephelometric assay, haemagglutination assay (HA), latex agglutination assay (LA), enzyme immunoassay (EIA, ELISA), fluorescent antibody assay (FI)
A), radioimmunoassay (RIA), chemiluminescence (RL)
U) etc. are fisted, and each is easy to operate. There are merits and demerits in sensitivity, accuracy, etc., and they are used properly according to the purpose of use.

The latex particles of the present invention are used in a latex agglutination method and an immunoturbidimetric method. Latex agglutination reaction
This is an indirect agglutination reaction in which an antigen is adsorbed on polystyrene particles and an antibody is detected. Typical ones are CPR, CEA,
There are AFT and RA tests.

The immunoturbidimetric method is a method of optically titrating an insoluble immune complex, which is the main component of an antigen-antibody reaction, and it is easy to operate without requiring a B / F separation operation and has a medium sensitivity. ,
It is widely used because it can be used in general spectrometers and general-purpose automatic analyzers. For example, immunoglobulin G, A, M, transferrin, complement, apolipoprotein and β-lipoprotein, Lp (a), C-reactive protein (CR) in serum samples.
P)), anti-streptolysin 0 (ASO), rheumatoid factor (RF), plasminogen, albumin and the like.

The principle is that the antigen / antibody in the sample reacts with the antibody / antigen in the reagent, and as a result of the antigen-antibody reaction, a reaction product (suspended particles) consisting of an insoluble antigen-antibody complex is quantitatively produced. This suspended particle is an analysis method that optically quantifies by utilizing the fact that it blocks (scatters) the transmission of light.
Therefore, the sensitivity, accuracy, and time required for analysis depend largely on the degree of formation of suspended particles. Generally, when an antigen solution and an antibody specific to the antigen are mixed, an antigen-antibody reaction occurs in the order of 10 to 3 seconds, but after that, several times are required until optically measurable suspended particles are formed. It may take from minutes to hours, or even days.

In the latex agglutination reaction which is generally carried out, an optical measurement method is to sensitize an antibody against an antigen to be quantified or an antigen against an antibody to be quantified into a latex having a certain average particle size, This sensitized latex is reacted in a cell using plastic, glass or the like in a water solvent containing urine, fecal dispersion, serum or plasma of the patient. When the target substance is present in the cell, the latex agglomerates, and 0.3 to 2.4 is introduced from the outside of the cell.
The amount of antibody or antigen in the cell can be quantified by irradiating with light of an appropriate wavelength selected from the wavelength of μm and measuring the change in absorbance. In practice, using a calibration curve of concentration and average reaction rate or concentration and absorbance change obtained by measuring different levels of known analytes in the method of optically measuring latex agglutination reaction, unknown concentration The concentration can be quantified by measuring the reaction rate or the change in absorbance of the sample.

To measure an actual sample, the same sensitized carrier particles as those used in the preparation of the calibration curve were used, and an unknown sample and the sensitized carrier were prepared under substantially the same conditions as in the preparation. By reacting with and measuring the absorbance, and comparing and matching this absorbance with the calibration curve, the amount (or concentration) of the antigen and / or antibody in the unknown sample can be quantified.

Alternatively, when preparing the calibration curve, for example, the reaction time required to reach a certain absorbance or the change in absorbance with time (reaction rate) and the amount of the antigen or antibody in the standard sample used ( If a calibration curve showing the relationship with (concentration) is created, the reaction time required to reach the above-mentioned constant absorbance by reacting the sensitized carrier particles with an unknown sample under substantially the same conditions as these. Alternatively, the amount (or concentration) of the antigen and / or antibody in the unknown sample can be quantified by reading the reaction rate.

The method using the latex agglutination reaction of the present invention has the advantages of safety and short measurement time, and the use of measuring devices that have been widely used in the past.

However, the present invention is not limited by the latex agglutination reaction method. As an effect of the present invention, improvement in sensitivity and / or reduction in measurement time extends to other measurement methods using carrier particles such as a radioimmunoassay method, a fluorescence method and a luminescence method.

Recently, in immunodiagnosis, trace substances contained in urine, fecal dispersion, serum, plasma, etc., such as α-fetoprotein (AFP), which is a cancer marker, and Cartenoembrio uitquantiteien (CFA), etc. ng / m
It is desired to quantify to a concentration such as 1. The concentration range that needs to be measured varies depending on the substance, and a wide range of concentrations from several ng / ml to several μmg / ml must be quantified.

In order to carry out a highly sensitive measurement of the latex agglutination reagent, a latex having a relatively large average particle size is used. Increase the amount of antibody or antigen sensitization to latex. Use an antibody or antigen that sensitizes with high specificity and affinity. It has been practiced to increase the concentration of sensitized particles within a range where the absorbance can be measured.

The method using a latex having a relatively large average particle size has a drawback that the sensitivity is improved at a low concentration, but the linearity of the calibration curve is lost at a high concentration and the measurement range is narrowed. . In addition, using a large amount of antibody or antigen or increasing the concentration of sensitizing particles may not always give the corresponding effect to the antigen, antibody or high-concentration particles to be used, or may increase the cost of reagents. become. As described above, in the conventional method, the trace amount of the target sample was still unsatisfactory or impossible in terms of sensitivity, measurement range, measurement time, and the like.

[0044]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

Example 1 Using the carrier particles prepared from a styrene-divinylbenzene copolymer having an average particle diameter of 0.05 micron as a base material, the Lee, Yoon-Sik method (I
nnovation Present. Solid
Phase Sybth. Comb. Libr. , Co
llect. Pap. , Int Symp. , 5th
(1999), Meetin Date 1997, 3
33, Mayflowee Scientific
Ltd. , Kingswind, UK), the surface of polystyrene particles is treated with ozone, hydroperoxide groups are introduced on the surface of polystyrene, and then acrylic acid is thermally polymerized by a conventional method to graft-polymerize a polyacrylic acid chain on the surface of polystyrene. did.

The styrene-divinylbenzene copolymer latex particles graft-polymerized with acrylic acid were sensitized with an anti-human β2-microglobulin antibody.

When the suspension of the sample and the latex particles is reacted, β2-microglobulin in the sample and the anti-β2-microglobulin antibody adsorbed on the latex particles start an antigen-antibody reaction, and the latex particles start to aggregate. This agglutination reaction is measured by absorbance (or turbidity) at a wavelength of 570 nm.
When grasped as a change, the amount of change is proportional to the β2-microglobulin concentration in the sample, so the β2-microglobulin concentration in the sample can be obtained from a calibration curve prepared using a standard solution having a known concentration. .

As a comparative example, it is used as an anti-human β2-microglobulin antibody-sensitized latex particle used as a reagent for measuring β2-microglobulin in urine / serum used for early detection and progress monitoring of renal damage. Latex reagent for β2-microglobulin measurement (Superior BETAII, Intermedic Corporation)
Company) was used.

Using a Hitachi absorbance measurement device (Model 7150), the main wavelength of measurement is 570 nm and the sub-wavelength is 800 n.
20 μl of β2-microglobulin, which was prepared by diluting standard samples of known concentration with various dilution factors at various times, was used as a sample, and 150 μl of tris- (human loquinmetel) -aminomethane was used as a buffer solution.
The amount of change in absorbance was measured using 150 μl of the graft latex particle suspension sensitized with the anti-human β2-microglobulin antibody.

Table 1 shows the relationship between the amount of change in absorbance and the concentration.

[Table 1]

Example 2 Using the carrier particles prepared from the styrene-divinylbenzene copolymer used in Example 1 as a base material, H. Becker method Makromol. ch
em. , Rapid Commun. 3,217 (19
According to 82), polyethylene glycol (PEG) chains were grafted. In the grafting, first, a chloromethyl group was introduced into the benzene ring of the styrene-divinylbenzene copolymer by a known method. Separately, PEG having a molecular weight of 400 was treated with sodium naphthalate in THF to obtain sodium polyethylene glycolate.
Styrene-divinylbenzene copolymer particles introduced with chloromethyl groups were added to this solution, and the mixture was reacted under reflux for grafting.

The hydroxyl groups of polyethylene glycol of the obtained styrene-divinylbenzene copolymer particles grafted with polyethylene glycol chains were converted into amino groups by a known method.

For the amination, the hydroxyl groups of the polyethylene glycol chains grafted on the styrene-divinylbenzene copolymer particles were alkylsulfonated with thionyl chloride and subsequently treated with ammonia to be converted into amino groups.

The styrene-divinylbenzene copolymer particles grafted with polyethylene glycol chains converted to the amino terminal were sensitized with an anti-human hemoglobin antibody.

When the sample is reacted with anti-human hemoglobin rabbit polyclonal antibody-sensitized latex particles, tohemoglobin in the sample and the anti-human hemoglobin rabbit polyclonal antibody adsorbed on the latex particles start an antigen-antibody reaction, and the latex particles aggregate. start. When this agglutination reaction is taken as the amount of change in absorbance at a wavelength of 450 nm,
Since the amount of change is proportional to the concentration of human hemoglobin in the sample, from the calibration curve created using the standard wave of known concentration,
The quantitative value of human hemoglobin in the sample can be obtained.

As a comparative example 2, a latex reagent for measuring human hemoglobin (Hemo. II, which uses latex particles sensitized with anti-human hemoglobin antibody used as a diagnostic agent for measuring human hemoglobin in feces used as a screening test for gastrointestinal bleeding) Intermedic Corporation
Company) was used.

Using the same automatic analyzer as in Example 1, using a main wavelength of measurement of 450 nm and a sub-wavelength of 800 nm,
Standard samples with known concentrations were prepared as various diluted standard samples diluted with a predetermined magnification, and human hemoglobin was adjusted to 12 μm.
1. Grafted latex particle suspension 1 of the present invention, which was sensitized with an anti-human hemoglobin antibody using 100 μl of tris- (human loquinmeter) -aminomethane as a buffer
The amount of change in absorbance was measured using 00 μl. Table 2 shows the relationship between the change in absorbance and the concentration.

[Table 2]

The minimum detection sensitivity of the comparative example was 10 ng / ml, while the minimum detection sensitivity of Example 2 was 5 ng / ml.

[0059]

EFFECT OF THE INVENTION Grafted latex particles in which a functional group is introduced by graft polymerization of the present invention to sensitize an antigen or an antibody have conventional functional groups directly present on the latex surface, and the functional group and the antibody or the antigen are present. Compared with latex particles sensitized with, the sensitivity as a diagnostic agent is superior, and the antigen-antibody reaction can be measured with high sensitivity over a wide concentration range.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) C08G 81/02 C08G 81/02

Claims (5)

[Claims] 1. Latex particles for immunological diagnosis, characterized in that a graft chain having a functional group is bound to the surface of the latex particle by graft polymerization to sensitize the functional group with an antibody or an antigen. 2. A graft chain having a functional group is bonded to the latex particle surface by graft polymerization, the functional group of the bonded graft chain is converted into another functional group, and the graft chain in the graft chain bonded to the latex particle surface is then converted into another functional group. Latex particles for immunological diagnosis characterized by sensitizing a functional group with an antibody or an antigen 3. The latex particle for immunological diagnosis according to claim 1, wherein the graft chain is a polymer of acrylic acid. 4. The latex particle for immunological diagnosis according to claim 2, wherein the graft chain is polyethylene glycol. 5. The latex particle for immunological diagnosis according to claim 1, wherein the graft chain is obtained by polymerizing glycidyl methacrylate.