阅读说明：本技术 富含亮氨酸的α2糖蛋白的免疫测定法和测定试剂 (Immunoassay method and assay reagent for leucine-rich alpha 2glycoprotein ) 是由 高山茂雄 仲哲治 世良田聪 于 2019-10-08 设计创作，主要内容包括：本发明的目的是提供一种能够在短时间内简单地测定生物样品中的LRG的测定方法和测定试剂。本发明人进行了深入研究以实现该目的并发现,可以通过使生物样品与携带第一抗LRG单克隆抗体的不溶性载体颗粒和携带第二抗LRG单克隆抗体的不溶性载体颗粒在液相中接触的免疫凝集测定方法在短时间内简单地测定生物样品中的LRG,从而完成了本发明。(The purpose of the present invention is to provide a measurement method and a measurement reagent that enable the LRG in a biological sample to be easily measured in a short time. The present inventors have conducted intensive studies to achieve the object and found that LRG in a biological sample can be simply measured in a short time by an immunoagglutination assay method in which a biological sample is brought into contact with an insoluble carrier particle carrying a first anti-LRG monoclonal antibody and an insoluble carrier particle carrying a second anti-LRG monoclonal antibody in a liquid phase, thereby completing the present invention.)

1. An immunological assay for leucine rich alpha 2glycoprotein (LRG) in a sample of biological origin, comprising:

contacting the sample with at least insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody in a liquid phase.

2. The assay according to claim 1, wherein said insoluble carrier particles are latex particles having an average particle diameter of 100nm to 340nm, respectively.

3. The assay according to claim 1 or 2, wherein the insoluble carrier particles are latex particles having a critical agglutination concentration of 65mM to 270mM, respectively.

4. The assay method according to any one of claims 1 to 3, wherein the average particle size of the insoluble support particles carrying the first anti-LRG monoclonal antibody and the insoluble support particles carrying the second anti-LRG monoclonal antibody is the same.

5. The assay method according to any one of claims 1 to 4, wherein the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody are contained in the same amount in the liquid phase.

6. The assay according to any one of claims 1 to 5, wherein the sample of biological origin is serum.

7. The assay according to any one of claims 1 to 6, wherein the immunoagglutination assay is a homogeneous method-based method.

8. The assay method according to any one of claims 1 to 7, wherein the step of contacting a sample of biological origin with insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody in a liquid phase comprises the steps of:

(1) contacting a sample and a first reagent comprising a buffer in a liquid phase; and

(2) after step (1), a second reagent comprising insoluble support particles carrying a first anti-LRG monoclonal antibody and insoluble support particles carrying a second anti-LRG monoclonal antibody is added to the liquid phase.

9. An assay according to claim 8, wherein the salt concentration of the liquid phase of (1) is from 700 to 900 mM.

10. The assay method according to any one of claims 1 to 9, further comprising:

the degree of agglutination of the complexes of LRG with insoluble carrier particles carrying anti-LRG monoclonal antibodies was determined optically.

11. The measuring method according to claim 10, wherein the step of optically measuring is a step of measuring the intensity of scattered light, absorbance or intensity of transmitted light by an optical device.

12. A reagent for determining LRG in a blood sample by an immunological assay method, comprising at least:

insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody.

13. The assay reagent according to claim 12, wherein the insoluble carrier particles are latex particles each having an average particle diameter of 100nm to 340 nm.

14. The assay reagent according to claim 12 or 13, wherein the insoluble carrier particles are latex particles having a critical agglutination concentration of 65mM to 270mM, respectively.

15. The assay reagent according to any one of claims 12 to 14, wherein the average particle size of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody is the same.

16. The assay reagent according to any one of claims 12 to 15, wherein the content of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody is the same.

17. The assay reagent according to any one of claims 12 to 16, wherein the sample of biological origin is serum.

18. The assay reagent according to any one of claims 12 to 17, wherein the immunoagglutination assay is a homogeneous method-based method.

19. A kit for determining LRG in a blood sample by an immunoagglutination assay, comprising:

(1) a first reagent comprising a buffer, and

(2) a second reagent comprising insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody.

20. The kit according to claim 19, wherein the insoluble carrier particles are latex particles having average particle diameters of 100nm to 340nm, respectively.

21. The kit according to claim 19 or 20, wherein the insoluble carrier particles are latex particles having a critical agglutination concentration of 65mM to 270mM, respectively.

22. The kit according to any one of claims 19 to 21, wherein the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody have the same average particle size.

23. The kit according to any one of claims 19 to 22, wherein the second reagent of (2) comprises the same amount of insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody.

24. The kit according to any one of claims 19 to 23, wherein the first reagent of (1) comprises a salt amount such that the salt concentration of the reaction liquid phase becomes 700 to 900 mM.

25. The kit according to any one of claims 19 to 24, further comprising:

(3) LRG as standard antigen and/or control.

Technical Field

The present invention relates to an immunoassay and an immunoassay reagent for leucine-rich alpha 2 glycoprotein. In particular, the present invention relates to immunoassays and immunoassay reagents using insoluble carrier particles carrying a substance having specific affinity for leucine rich alpha 2 glycoprotein.

Background

It has been reported that a leucine-rich α 2glycoprotein (hereinafter, may be abbreviated as LRG) is one of serum proteins, is a glycoprotein of about 50kDa, and is secreted from neutrophils (non-patent document 1).

Further, it is disclosed that LRG can be used as a biomarker for testing autoimmune diseases such as Behcet's disease (patent document 1).

Patent document 1 shows that detection and quantitative determination of LRG have been performed by proteomic analysis and immunological methods, and the immunological methods include ELISA, western blotting, and the like using an anti-LRG monoclonal antibody.

As an assay reagent for ELISA, for example, human LRG assay kit IBL (IBL Co, Ltd.) is commercially available. The ELISA reagent for measuring human LRG can quantitatively determine the LRG concentration of a sample by forming an immune complex between an antibody binding to LRG and LRG through an antigen-antibody reaction and measuring the amount of a label in the immune complex.

Typically, the concentration of assay targets suitable for determination by ELISA is in the range of pg/mL to ng/mL. Therefore, when the substance to be measured is present in a biological sample at a relatively high concentration on the order of μ g/mL, pretreatment is required to dilute the sample to a concentration at which the ability to measure a reagent can be exerted.

Moreover, it is necessary to dilute the sample through a plurality of separate stages to prevent errors due to high-rate dilution, and complexity of the steps becomes a problem. Specifically, with the above-mentioned commercially available reagent, it is necessary to dilute the sample so that the LRG concentration of the sample falls within a constant range of 1.56 to 100 ng/mL. For example, in the case of ulcerative colitis, where the maximum concentration value in human serum is 100 μ g/mL, the dilution means that a 1000-fold dilution is required.

In addition, for a conventional ELISA reagent for LRG assay, it is necessary to react a sample to be tested with a solid-phase antibody for a long time. For example, the above-mentioned commercial kit requires a first-order reaction to be carried out overnight to bring the solid-phase antibody and the sample into contact with each other, and there is no reagent that can measure the LRG in the sample in a short time.

List of citations

Patent document

Patent document 1: JP-A-2010-286279

Non-patent document

Non-patent document 1: j Leukoc biol.2002, 72(3), 478-85, 2002

Disclosure of Invention

Technical problem

The purpose of the present invention is to provide a measurement method and a measurement reagent that can easily measure LRG in a biological sample in a short time.

Means for solving the problems

The present inventors have conducted intensive studies in order to achieve the object, and have found that in an immunological assay method in which a sample is brought into contact with an insoluble carrier particle carrying a first anti-LRG monoclonal antibody and an insoluble carrier particle carrying a second anti-LRG monoclonal antibody, specifically, in an immunoagglutination assay, when a reagent has a sufficiently considered composition, LRG in a biological sample can be simply assayed in a short time without performing pretreatment such as dilution, and have completed the present invention.

That is, the present invention has the following configuration.

<1> an immunological assay method for leucine-rich alpha 2glycoprotein (LRG) in a sample of biological origin, comprising:

contacting the sample with at least insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody in a liquid phase.

<2> the assay method according to <1>, wherein the insoluble carrier particles are latex particles having average particle diameters of 100nm to 340nm, respectively.

<3> the assay method according to <1> or <2>, wherein the insoluble carrier particles are latex particles having a critical agglutination concentration of 65mM to 270mM, respectively.

<4> the assay method according to any one of <1> to <3>, wherein the average particle size of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody is the same.

<5> the assay method according to any one of <1> to <4>, wherein the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody are contained in the same amount in the liquid phase.

<6> the assay method according to any one of <1> to <5>, wherein the sample of biological origin is serum.

<7> the assay method according to any one of <1> to <6>, wherein the immunoagglutination assay is a homogeneous method-based method.

<8> the assay method according to any one of <1> to <7>, wherein the step of contacting a sample of biological origin with insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody in a liquid phase comprises the steps of:

(1) contacting a sample and a first reagent comprising a buffer in a liquid phase; and

(2) after step (1), a second reagent comprising insoluble support particles carrying a first anti-LRG monoclonal antibody and insoluble support particles carrying a second anti-LRG monoclonal antibody is added to the liquid phase.

<9> the assay method according to <8>, wherein the salt concentration of the liquid phase of (1) is 700 to 900 mM.

<10> the assay method according to any one of <1> to <9>, further comprising:

the degree of agglutination of the complexes of LRG with insoluble carrier particles carrying anti-LRG monoclonal antibodies was determined optically.

<11> the measuring method according to <10>, wherein the step of optically measuring is a step of measuring intensity of scattered light, absorbance or intensity of transmitted light by an optical device.

<12> a reagent for measuring LRG in a blood sample by an immunological measuring method, comprising at least:

insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody.

<13> the assay reagent according to <12>, wherein the insoluble carrier particles are latex particles having average particle diameters of 100nm to 340nm, respectively.

<14> the assay reagent according to <12> or <13>, wherein the insoluble carrier particles are latex particles having a critical agglutination concentration of 65mM to 270mM, respectively.

<15> the assay reagent according to any one of <12> to <14>, wherein the average particle diameter of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody is the same.

<16> the assay reagent according to any one of <12> to <15>, wherein the content of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody is the same.

<17> the assay reagent according to any one of <12> to <16>, wherein the sample of biological origin is serum.

<18> the assay reagent according to any one of <12> to <17>, wherein the immunoagglutination assay is a homogeneous method-based method.

<19> a kit for determining LRG in a blood sample by an immunoagglutination assay, comprising:

(1) a first reagent comprising a buffer, and

(2) a second reagent comprising insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody.

<20> the kit according to <19>, wherein the insoluble carrier particles are latex particles having average particle diameters of 100nm to 340nm, respectively.

<21> the kit according to <19> or <20>, wherein the insoluble carrier particles are latex particles having a critical agglutination concentration of 65mM to 270mM, respectively.

<22> the kit according to any one of <19> to <21>, wherein the average particle diameter of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody is the same.

<23> the kit according to any one of <19> to <22>, wherein the second agent of (2) comprises the same amount of insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody.

<24> the kit according to any one of <19> to <23>, wherein the first reagent of (1) contains a salt in an amount such that the salt concentration of the reaction liquid phase becomes 700 to 900 mM.

<25> the kit according to any one of <19> to <24>, further comprising:

(3) LRG as standard antigen and/or control.

Advantageous effects of the invention

According to the present invention, there are provided a method and a reagent for measuring LRG in a biological sample, which can provide a result more simply in a short time.

Drawings

FIG. 1 is a graph showing the results of measuring a sample containing a known concentration of LRG using an autoanalyzer using a combination of the first reagent and the second reagent of formulation example 1.

FIG. 2 is a graph showing the results of measuring a sample containing a known concentration of LRG using an autoanalyzer using a combination of the first reagent and the second reagent of formulation example 2.

FIG. 3 is a graph showing the results of measuring a sample containing a known concentration of LRG using an autoanalyzer using a combination of the first reagent and the second reagent of formulation example 3.

FIG. 4 is a graph showing the results of measuring a sample containing a known concentration of LRG using an autoanalyzer using a combination of the first reagent and the second reagent of formulation example 4.

FIG. 5 is a graph showing the results of measuring a sample containing a known concentration of LRG using an autoanalyzer using a combination of the first reagent and the second reagent of formulation example 5.

FIG. 6 is a graph showing the results of measuring a sample containing a known concentration of LRG using an autoanalyzer using a combination of the first reagent and the second reagent of formulation example 6.

FIG. 7 is a graph showing the results of measurement of the LRG concentration of serum by the measurement method of the present invention using a sample of a healthy individual and a sample of a patient.

Detailed Description

(immunoagglutination assay method)

The immunoagglutination assay is an immunoassay which uses insoluble carrier particles on which a substance having a specific affinity for an object to be measured, such as an antigen or an antibody, is immobilized and measures the antigen or the antibody, and is widely used in the field of clinical examination. Latex and the like are mainly used for insoluble carrier particles, and in this case, the assay is specifically referred to as a latex immunoagglutination assay (LTIA).

The method of measuring LRG by LTIA can be roughly classified into a method in which latex particles to which a monoclonal antibody against LRG is immobilized react with LRG as an antigen to form a sandwich-like immune complex and LRG is measured by the degree of agglutination of the latex particles due to immune complex formation, and a method in which antigen-immobilized latex particles compete with LRG in a sample to inhibit the formation of immune complexes of the latex particles and the antibody and LRG is measured by the degree of agglutination inhibition of the latex particles due to inhibition of immune complex formation.

(sample)

The sample of the present invention is not particularly limited as long as the sample is a biological sample, but is usually a blood sample, and examples include whole blood, serum, and plasma. The plasma includes heparin plasma, EDTA plasma, etc.

(test substance)

The substance to be tested in the present invention is leucine-rich α 2glycoprotein (LRG).

(measurement method)

In a measurement method using immunoagglutination, represented by LTIA, a substance to be measured can be measured by optically or electrochemically observing the degree of agglutination that occurs. The optical observation method includes a method of measuring the intensity of scattered light, absorbance or intensity of transmitted light by an optical device (an end point method, a rate method, etc.). The concentration (quantitative value) of LRG contained in a sample is calculated by comparing a measured value such as absorbance obtained by measuring the sample with a measured value such as absorbance obtained by measuring a standard substance (a sample having a known LRG concentration). In this regard, the absorbance or the like of transmitted light, scattered light or the like can be measured by single-wavelength measurement or two-wavelength measurement (difference or ratio due to two wavelengths). The wavelength used for the measurement is generally determined in the range of 500nm to 800 nm.

LRG in a sample of the invention may be determined by manual processing or using a device such as an assay device. The assay device may be a general purpose autoanalyzer or a monocular assay device (dedicated device). Preferably, the determination is carried out by a method using a plurality of operation steps, for example, a two-step method (two-reagent method).

(anti-LRG monoclonal antibody)

The monoclonal antibodies of the invention can be obtained by methods known to those skilled in the art. That is, monoclonal antibodies can be easily produced by dissolving human LRG as an antigen in a solvent such as phosphate-buffered physiological saline and administering the solution to animals for immunization. If necessary, an appropriate adjuvant may be added to the solution, and then the animal may be immunized with the emulsion. As the adjuvant, commonly used adjuvants such as water-in-oil emulsion, water-in-oil-in-water emulsion, liposome and aluminum hydroxide gel, and protein and peptide substances derived from biological components, and the like can be used. For example, Freund's incomplete adjuvant, Freund's complete adjuvant, or the like can be suitably used. The administration route, dose and administration time of the adjuvant are not particularly limited, but are desirably appropriately selected so that a desired immune response can be enhanced in an animal to be immunized with the antigen.

The kind of animal used for immunization is also not particularly limited, but is preferably a mammal. For example, a mouse, rat, cow, rabbit, goat, sheep, or the like may be used, and a mouse may be more preferably used. The animal may be immunized according to a general method, and the immunization may be performed, for example, by injecting the animal subcutaneously, intradermally, intravenously or intraperitoneally with a solution of the antigen, preferably in a mixture with an adjuvant. Since the immune response generally varies depending on the species and strain of the animal to be immunized, it is desirable to appropriately set the immunization schedule depending on the animal used. Preferably, administration of the antigen is repeated several times after the first immunization.

When obtaining a monoclonal antibody, the following procedure is followed, but the method of producing the monoclonal antibody itself is not limited to this procedure, and may be carried out according to, for example, the method described in Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988)).

After the final immunization, spleen cells or lymph node cells as antibody-producing cells are removed from the immunized animal, and hybridomas are produced by cell fusion with myeloma cells having high growth potential. Cells with high antibody-producing potential (mass/amount) are preferably used for cell fusion, and myeloma cells are preferably compatible with the animal from which the antibody-producing cells to be fused are obtained. The cells can be fused according to a method known in the art, but for example, a polyethylene glycol method, a method using Sendai virus, a method using electric current, or the like can be used. The obtained hybridomas can be proliferated according to a known method, and desired hybridomas can be selected while examining the properties of the produced antibodies. Hybridomas can be cloned by known methods, such as limiting dilution analysis or soft agar analysis.

Selection of hybridomas producing the first and second monoclonal antibodies is described. Hybridomas can also be efficiently selected in the selection stage, taking into consideration the conditions under which actual measurement is performed using the produced antibodies. For example, hybridomas are obtained by selecting hybridomas that produce antibodies reactive with human LRG by ELISA, RIA, Biacore analysis, and the like. Specifically, monoclonal antibodies in the culture supernatant were first reacted with immobilized human LRG, and then hybridomas producing monoclonal antibodies highly reactive with human LRG were selected by antigen-immobilized ELISA in which labeled anti-IgG antibodies were reacted.

By culturing the hybridomas selected as described above on a large scale, monoclonal antibodies having desired properties can be produced. The large-scale culture method is not particularly limited, but examples include, for example, a method of culturing a hybridoma in an appropriate medium and thereby producing a monoclonal antibody in the medium, a method of intraperitoneally injecting the hybridoma into a mammal, allowing the hybridoma to proliferate and producing the antibody in the ascites, and the like. The monoclonal antibody can be purified by, for example, appropriate combination of anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, and the like.

As the antibody of the present invention, in addition to the whole antibody molecule, a functional fragment of an antibody having antigen-antibody reactivity, an antibody obtained by the above-mentioned immunization step of an animal, an antibody obtained by using a gene recombination technique, and a chimeric antibody can be used. Examples of functional fragments of antibodies include F (ab')₂Fab', etc., and such functional fragments can be produced by treating an antibody obtained as described above (e.g., pepsin, papain, etc.) with a protease.

(insoluble Carrier particle)

The insoluble carrier particle used in the present invention may be an insoluble carrier particle that can carry an anti-LRG monoclonal antibody and can measure LRG in a sample. Examples include latex particles, magnetic particles, metal particles, and the like, but latex particles are preferred among these examples.

The average particle diameter and the critical aggregation concentration of the insoluble carrier particles are appropriately determined in consideration of the LRG concentration in the sample, the detection sensitivity of the measuring apparatus, and the like.

The insoluble carrier particles preferably have an average particle diameter of 100nm to 340nm, more preferably 150nm to 260 nm.

Regarding the critical agglutination concentration of the insoluble carrier particles, those having a critical agglutination concentration of preferably 65mM to 270mM, more preferably 150mM to 270mM are appropriately selected.

(latex particles)

The latex particles as a preferable example of the insoluble carrier particles used in the present invention are not particularly limited as long as they are latex particles generally used as an immunoassay reagent. The latex particles may be obtained by polymerization or copolymerization of various monomers. Examples of the monomer herein include polymerizable unsaturated aromatic compounds such as polymerizable monomers having a phenyl group (including styrene, α -methylstyrene, o-methylstyrene, p-chlorostyrene, 4-vinylbenzoate, divinylbenzene, vinyltoluene and the like), polymerizable monomers having a phenyl group and a sulfonate (including styrene sulfonate, divinylbenzene sulfonate, o-methylstyrene sulfonate, p-methylstyrene sulfonate and the like) and polymerizable monomers having a naphthyl group (including 1-vinylnaphthalene, 2-vinylnaphthalene, α -naphthyl (meth) acrylate, β -naphthyl (meth) acrylate and the like)), polymerizable unsaturated carboxylic acids such as (meth) acrylic acid, itaconic acid, maleic acid, fumaric acid and the like, polymerizable unsaturated carboxylic acid esters such as methyl (meth) acrylate, methyl (meth), Ethyl (meth) acrylate, N-butyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, glycidyl (meth) acrylate, ethylene glycol-di- (meth) acrylate, and tribromophenyl (meth) acrylate), unsaturated carboxylic acid amides, polymerizable unsaturated nitriles, vinyl halides, and conjugated dienes (e.g., (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide, N-hydroxymethyl- (meth) acrylamide, methylenebis (meth) acrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, and vinyl bromide, etc.). The monomer is appropriately selected by the desired surface characteristics, specific gravity, and the like. They may be used alone or in admixture of two or more.

The average particle size of the latex particles can be analyzed using a laser diffraction/scattering method (LS method), the coulter principle, dynamic light scattering-photon correlation spectroscopy, an electron microscope, or the like.

As for the average particle diameter of the latex particles, those having an average particle diameter of preferably 100nm to 340nm, more preferably 150nm to 260nm are appropriately selected. The reason is as follows: when the average particle diameter is less than 100nm, the sensitivity in the low-to-medium concentration range is lowered and it is difficult to accurately measure; when the average particle diameter exceeds 340nm, although the sensitivity is increased at a low concentration, a so-called hook effect (hook effect) is caused at a high concentration, which causes a phenomenon that the obtained value is lower than the actual concentration.

The critical coagulation concentration of the latex particles means the maximum salt concentration at which the latex does not coagulate, and means a salt concentration that is one step lower than the salt concentration at which the latex particles completely self-coagulate when salt is gradually added to the latex particles that are not sensitized with the antibody. For example, an aqueous sodium phosphate solution (pH7.4) having a concentration of 10mM different from that of 10 to 400mM is prepared, and latex particles in an amount resulting in a final concentration of 1% (W/V) are added to the aqueous sodium phosphate solution of different concentrations and stirred. After one minute, the solution was visually observed to determine whether the latex had self-coagulated, and the concentration of an aqueous sodium phosphate solution one step lower than the concentration at which the latex had completely self-coagulated was determined as the critical coagulation concentration (maximum non-coagulation concentration).

With respect to the critical coagulation concentration of the latex particles (concentration of the sodium phosphate aqueous solution), those latex particles whose critical coagulation concentration is preferably 65mM to 270mM, more preferably 150mM to 270mM, are appropriately selected. When the critical aggregation concentration is less than 65mM, it is difficult to maintain the dispersion of the latex particles, and when the critical aggregation concentration is 270mM or more, an immune response is not easily caused, and the latex particles are not aggregated.

The critical coagulation concentration of the latex particles can be controlled by appropriately changing the weight ratio of the raw materials. For example, a styrene latex is obtained by copolymerizing an amount of a polymerizable monomer having a phenyl group such as styrene with an amount of a polymerizable monomer having a phenyl group and a sulfonate such as sodium styrenesulfonate in an aqueous medium, and in this case, the critical coagulation concentration can be controlled by changing the mixing ratio of styrene and sodium styrenesulfonate.

The material and particle size of the latex particles used may be appropriately selected to obtain desired properties such as increased sensitivity, and those having different materials and different particle sizes may also be used in combination.

The concentration of the latex particles in the agglutination reaction measurement in the present invention is not particularly limited, and may be appropriately set according to the desired sensitivity and properties.

(insoluble Carrier particles carrying anti-LRG monoclonal antibodies)

The anti-LRG antibody can be immobilized and carried on an insoluble carrier particle such as a latex particle by a known method such as a physical adsorption (hydrophobic binding) method, a chemical binding method, or a combination thereof. The following description is given using latex as a typical example of insoluble carrier particles.

The physical adsorption method can be performed according to a known method by mixing an anti-LRG antibody and latex particles in a solution such as a buffer and contacting the antibody and latex particles with each other, or by contacting an LRG antibody dissolved in a buffer or the like with a carrier.

May be prepared according to the protocol "Extra Special Issue of Clinical Pathology, No.53, Immunoassay for Clinical Examino-Techniques and Applications-" edited by the society of Clinical Pathology, 1983; the chemical binding method is carried out by a known method described in "New Courses in Biochemical Experimental 1, Protein IV" and the like, published by Tokyo Kagaku Dojin in 1991, edited by the Japan Biochemical society, by mixing and contacting the substance specifically binding to LRG and the carrier with a divalent crosslinking agent (e.g., glutaraldehyde, carbodiimide, imide ester or maleimide), and reacting the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group and the like of the substance specifically binding to LRG and the carrier with the divalent crosslinking agent.

At least two antibodies against a specific substance carried on the latex particle are required to form a sandwich shape. That is, two or more monoclonal antibodies having different recognition sites were used as the anti-LRG monoclonal antibody.

The first monoclonal antibody and the second monoclonal antibody need to be carried on different insoluble carrier particles.

In the present invention, as described above, the concentrations of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody can be appropriately set according to the desired sensitivity and properties, but the insoluble carrier particles are preferably contained in the same amount in the reaction liquid phase.

Further, in the present invention, as described above, the average particle diameter of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody is preferably 100nm to 340nm, respectively. The average particle diameters are preferably similar, more preferably the same. The average particle diameters are similar means that one of the average particle diameters falls within a range of 80% to 120% of the other average particle diameter, and the average particle diameters are the same means that one of the average particle diameters falls within a range of 90% to 110% of the other average particle diameter.

When a treatment is required to suppress spontaneous aggregation, nonspecific reactions, or the like of the latex particles, the carrier may be blocked (masked) by a known method, for example, by contacting or covering the surface of the latex particles with or with a protein (e.g., Bovine Serum Albumin (BSA), casein, gelatin, egg white protein or a salt thereof), a surfactant, skim milk powder, or the like.

(contact)

Bringing a sample containing LRG and a latex particle carrying an anti-LRG monoclonal antibody into contact with each other in a liquid phase generally refers to the following (1) to (4).

(1) An embodiment in which latex particles carrying the first anti-LRG monoclonal antibody and the second anti-LRG monoclonal antibody are mixed into a mixed solution after mixing the sample and the buffer.

(2) An embodiment wherein the sample, buffer, latex particles carrying a first anti-LRG monoclonal antibody and latex particles carrying a second anti-LRG monoclonal antibody are mixed simultaneously.

(3) An embodiment in which after mixing the sample and the buffer, the latex particles carrying the first anti-LRG monoclonal antibody are mixed, and then the latex particles carrying the second anti-LRG monoclonal antibody are mixed.

(4) Embodiments in which the sample is added and mixed at the end of (1) to (3).

(measurement reagent)

The measurement reagent of the present invention is a reagent for measuring LRG in a blood sample by an immunoagglutination assay, and comprises at least an insoluble carrier particle carrying a first anti-LRG monoclonal antibody and an insoluble carrier particle carrying a second anti-LRG monoclonal antibody. Typically, the reagent comprises two or more constituent reagents. At least one of the constituent reagents comprises latex particles carrying anti-LRG monoclonal antibodies, and the other constituent reagent comprises a buffer. Further, the assay reagent of the present invention is a liquid reagent.

In particular, the assay reagent of the present invention is preferably of a dual reagent type comprising a first reagent and a second reagent. For example, a first reagent of the two-reagent type comprises a buffer for diluting a biologically derived sample comprising LRG, and a second reagent is a reagent solution comprising latex particles carrying a first anti-LRG monoclonal antibody and latex particles carrying a second anti-LRG monoclonal antibody.

When the assay reagent of the present invention is a three-reagent type including a first reagent, a second reagent and a third reagent, for example, the first reagent contains a buffer or the like, the second reagent is a reagent solution containing latex particles carrying a first anti-LRG monoclonal antibody, and the third reagent is a reagent solution containing latex particles carrying a second anti-LRG monoclonal antibody.

The buffer contained in the assay reagent of the present invention may be a commonly used buffer, and examples include Tris-hydrochloric acid, boric acid, phosphoric acid, acetic acid, citric acid, succinic acid, phthalic acid, glutaric acid, maleic acid, glycine, salts thereof and the like, and Good's buffers such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES and the like.

The concentration of the buffer component may be in a range of concentration that does not cause spontaneous aggregation of insoluble carrier particles in the reagent and causes a desired immune response, and the concentration in the reaction solution may be 100mM or more, preferably 200mM or more, more preferably 300mM or more, and still more preferably 400mM or more.

It is desirable that the assay reagent of the present invention further comprises a salt. The kind of the salt is desirably an inorganic salt, and examples of the inorganic salt include sodium chloride, calcium chloride and the like.

The salt concentration may be within a concentration range that does not cause spontaneous aggregation of insoluble carrier particles in the reagent and causes a desired immune response, and the lower limit of the concentration may be 100mM or more, preferably 300mM or more, more preferably 500mM or more, further more preferably 700mM or more in the reaction solution. The upper limit of the concentration in the reaction solution is preferably 2000mM or less, more preferably 1500mM or less, still more preferably 1000mM or less, and most preferably 900mM or less.

The concentration in the reaction solution is preferably in the range of 100 to 1500mM, more preferably 300 to 1000mM, further more preferably 500 to 1000mM, most preferably 700 to 900 mM.

The measurable range (measurement range) of the measurement reagent of the present invention may be about 10 to 80. mu.g/mL, desirably 5.0 to 100. mu.g/mL, to measure LRG as a diagnostic marker.

(kit)

The kit of the present invention is characterized by comprising at least the following elements (1) and (2).

(1) A first reagent comprising a buffer.

(2) A second reagent comprising at least insoluble carrier particles carrying a first anti-LRG monoclonal antibody and insoluble carrier particles carrying a second anti-LRG monoclonal antibody.

In the kit of the present invention, (3) an LRG as a standard antigen or control may be included in addition to the above-mentioned measurement reagent.

A sample pretreatment reagent for pretreating a sample may also be included. The sample pretreatment reagent may be contained in the first reagent containing a buffer solution in (1), or may be contained as an element other than (1) and (2).

The kit may further include user instructions, sample collection tools (collection pipettes, syringes, swabs, filters, etc.), sample diluents, and sample extraction solutions.

(homogeneous phase method)

In the present invention, the homogeneous method refers to a measurement method for specifically detecting a reaction occurring in a mixed solution (reaction solution) of a sample and a reagent solution without performing B/F (binding/dissociation) separation, and refers to a measurement method named as a comparison with a heterogeneous measurement method in which a main reaction is detected after completely washing/removing excess components not involved in the measurement reaction by a B/F separation operation. Therefore, the "immunoagglutination assay is a method based on a homogeneous method" in the present invention means that the step (3) is a typical step of (1) to (3) "a step of measuring an agglutination reaction of LRG with insoluble carrier particles without performing a washing/separating step in the step (2) or after the step (2)".

(1) A step of bringing the sample and the buffer into contact with each other in a liquid phase.

(2) A step of adding insoluble carrier particles carrying an anti-LRG monoclonal antibody to the liquid phase after step (1).

(3) A step of measuring the agglutination reaction of the LRG with the insoluble carrier particles after the step (2).

(other reagent Components)

The agent of the present invention may contain a polymer such as polyethylene glycol, polyvinylpyrrolidone and phospholipid polymer as a component for complementing the aggregation formation of insoluble carrier particles. The component for controlling the formation of aggregation may contain one or a combination of generally used components such as proteins, amino acids, saccharides, metal salts, surfactants, reducing substances, and chaotropic substances (chaotropic substances).

Examples

The present invention is explained in detail below with reference to examples, but the present invention should not be construed as being limited to these examples.

[ test example 1]

The particle size of the insoluble carrier particles used to determine LRG concentration was varied and it was examined whether a concentration-dependent change in absorbance was observed and whether a calibration curve could be drawn.

1. Reagent

(1) A first reagent

The reagent contains the following components. The pH was controlled to 6.5 to 7.5.

·HEPES

·1M NaCl

·1.0％BSA

·0.05％Proclin950

(2) Second reagent

(2-1) formulation example 1

The following two anti-LRG monoclonal antibody-sensitized latex particle solutions were mixed in the same amount and diluted with 10mM HEPES buffer (ph7.2) so that the absorbance thereof at a wavelength of 570nm became 4.5Abs, thereby obtaining a second reagent.

(i) Emulsion particle solution sensitized by first anti-LRG monoclonal antibody (Kan10)

To a 1% polystyrene latex solution (20mM glycine buffer) having an average particle diameter of 343nm and a critical agglutination concentration of 60mM was added the same amount of an anti-LRG monoclonal antibody (Kan10) solution which had been diluted to 0.71mg/mL with 50mM glycine buffer, and the mixture was stirred at 4 ℃ for 2 hours. Then, the same amount of a synthetic polymer (Blockmaster CE210 manufactured by JSR) was added, and the mixture was stirred at 4 ℃ for 1 hour. Subsequently, 1/10 amount of 10% BSA solution in purified water was added and the mixture was stirred at 4 ℃ for 1 hour. This produced a latex solution sensitized with an anti-LRG monoclonal antibody (Kan 10).

(ii) Emulsion particle solution sensitized by second anti-LRG monoclonal antibody

A latex particle solution sensitized with an anti-LRG monoclonal antibody (Kan11) was prepared in the same manner as in (i) above using a polystyrene latex having an average particle diameter of 343nm and a critical coagulation concentration of 60 mM.

(iii) Physical Properties of the latex particles

(a) Method for measuring particle diameter of latex particles

The latex particle diameter was analyzed with a laser diffraction/scattering particle diameter distribution analyzer LS13320 (manufactured by Beckman Coulter).

(b) Method for measuring critical agglutination concentration of latex particles

Aqueous sodium phosphate solutions (pH7.4) were prepared at concentrations varying by 10mM from 10 to 400mM, and an amount of latex particles was added to the aqueous sodium phosphate solutions at various concentrations with stirring to give a final concentration of 1% (W/V). Then, the solution was visually observed after one minute to determine whether or not the latex was self-aggregated, and the concentration of an aqueous sodium phosphate solution one order lower than the concentration at which the latex was completely self-aggregated was determined as a critical aggregation concentration.

(2-2) formulation examples 2 to 6

The same procedure was carried out except that the latex particles of formulation example 1 were changed as follows.

Formulation example 2: the average particle size was 305nm, and the critical agglutination concentration was 70 mM.

Formulation example 3: the average particle size was 251nm, and the critical coagulation concentration was 260 mM.

Formulation example 4: the average particle diameter was 207nm, and the critical agglutination concentration was 180 mM.

Formulation example 5: the average particle size was 194nm, and the critical coagulation concentration was 230 mM.

Formulation example 6: the average particle diameter was 121nm, and the critical agglutination concentration was 210 mM.

2. Sample to be tested

Purified LRG (prepared by Bio vector Laboratory Medicine) was added to physiological saline in amounts of 0, 5.0, 10.0, 23.0, 50.0, and 100.0. mu.g/mL.

3. Measurement method

The first reagent and the second reagent of the formulation examples were mixed separately, and a sample containing LRG of a known concentration was measured using a Hitachi 7180 automatic analyzer. Specifically, 150. mu.L of the first reagent was added to 2.5. mu.L of the sample, and the mixture was incubated at 37 ℃ for 5 minutes. Then, 50. mu.L of the second reagent was added, and the mixture was stirred. Then, after 5 minutes, the change in absorbance due to the formation of aggregates was measured at a main wavelength of 570nm and a complementary wavelength of 800nm, and the change in absorbance was measured. A calibration curve was drawn based on the measured absorbance, and R2 (measurement coefficient) was determined.

4. Measurement conditions

The parameter conditions of the Hitachi 7180 automatic analyzer are as follows.

(1) Liquid amount: sample-first reagent-second reagent; 2.5-150-50. mu.L

(2) The analysis method comprises the following steps: 2 Point end point method (light measuring points 19-34)

(3) Measuring wavelength: dominant wavelength 570 nm/secondary wavelength 800nm

(4) Calibration: sample strip

5. Results and discussion of the assay

The results are shown in fig. 1 to 6.

According to the results, in prescription example 1, although the hook effect was observed at a high LRG concentration (100 μ g/ml), the absorbance increased in a concentration-dependent manner at low to medium concentrations. In formulation examples 2 to 6, the absorbance increased in a concentration-dependent manner at low to high concentrations, and the R2 value was 0.99 or more. In particular, the R2 values of formulation examples 3, 4, and 5 were 0.996 or more, which was particularly excellent.

From the above results, it was found that when the average particle diameter of the latex particles is from 100nm to 340nm, accurate measurement can be achieved within a concentration range covering the entire LRG concentration distribution in human serum, and particularly when the average particle diameter is from 150nm to 260nm, measurement with higher accuracy can be achieved.

It was also found that when the critical agglutination concentration is 65mM to 270mM, accurate measurement can be achieved within a concentration range covering the entire LRG concentration distribution in human serum, and particularly when the critical agglutination concentration is 150mM to 270mM, measurement of concentration with higher accuracy can be achieved.

[ test example 2]

To examine whether a sample similar to the actual sample can be determined, a simulated LRG sample of known concentration was prepared by adding purified LRG to the pooled serum, and the concentration of LRG was determined.

1. Reagent

The reagents of formulation examples 2 to 6 of test example 1 were used.

2. Sample to be tested

(1) The pooled sera were used as a panel sample (panel specimen) L.

(2) Purified LRG at 28.2 u g/mL added to the combined serum, and used as small group sample M.

(3) Purified LRG was added to pooled sera in an amount of 63.1. mu.g/mL and used as panel sample H.

3. Measurement method and measurement conditions

The test method and the measurement conditions were the same as those in test example 1. Using the calibration curve plotted in test example 1, the concentrations of the small group samples were determined, and the relative proportions (%) to the known concentrations were calculated.

4. Results and discussion of the assay

The results are shown in Table 1.

From the results, it was found that a simulated sample similar to the actual sample can be accurately measured using the reagents of prescription examples 2 to 6. In particular, formulation examples 3 to 5 have extremely high relative ratio accuracy with respect to the known concentration of 99%.

From the above results, it was found that accurate measurement can be achieved when the average particle diameter of the latex particles is from 100nm to 340nm, and particularly that measurement with higher accuracy can be achieved when the average particle diameter is from 150nm to 260 nm.

It has also been found that when the critical coagulation concentration of the latex is 65mM to 270mM, accurate measurement can be achieved, and particularly when the critical coagulation concentration is 150mM to 270mM, measurement with higher accuracy can be achieved.

[ Table 1]

LRG concentration is in μ g/mL, values in parentheses are relative proportions (%) to known concentrations.

[ test example 3]

It was confirmed that LRG as a marker can be measured by the measurement method of the present invention using a sample of a healthy individual and a sample of a patient.

1. Reagent

The reagent of formulation example 5 of test example 1 was used.

2. Sample to be tested

(1) Serum samples from healthy individuals

Seven samples were purchased from PROMEDDEX.

(2) Serum samples from patients with ulcerative colitis (mild (remission))

Thirteen samples were purchased from bioreclaintion.

(3) Serum samples from patients with ulcerative colitis (severe (active phase))

Five samples were purchased from bioreclaintion and Proteogenex.

3. Measurement method and measurement conditions

The measurement method and measurement conditions were the same as in test example 1.

4. Results and discussion of the assay

The results are shown in FIG. 7.

Based on this result, it was found that LRG can be determined in samples of healthy individuals as well as patients with mild and severe ulcerative colitis, and can be determined as a marker that can distinguish these states.

Thus, the assay reagent of the present invention can function as a diagnostic agent.

INDUSTRIAL APPLICABILITY

According to the present invention, by bringing a biological sample containing LRG and an insoluble carrier particle carrying an anti-LRG monoclonal antibody into contact with each other and measuring the degree of agglutination, it is possible to provide a method and a reagent for measuring LRG in a biological sample, which can easily obtain results in a short time. The measurement method and the measurement reagent of the present invention can be applied to a general-purpose automatic analyzer, and therefore, a large number of LRG samples can be easily and simultaneously measured.

Therefore, LRG as a biomarker can be easily quantified in a short time, and the activity of diseases such as inflammatory bowel disease can be easily evaluated.