Patent Description:
It is reported that leucine-rich α2 glycoprotein (hereinafter, sometimes simply referred to as LRG) is one of serum proteins, is a glycoprotein of about <NUM> kDa and is secreted from neutrophils (Non-Patent Document <NUM>, <NPL>).

Moreover, it is disclosed that LRG is useful as a biomarker for testing autoimmune diseases such as Behcet's disease (Patent Document <NUM>, <CIT>).

Patent Document <NUM> shows that detection and quantitative determination of LRG have been conducted by proteome analysis and immunological methods and that the immunological methods include ELISA, Western blotting and the like using an anti-LRG monoclonal antibody.

As an assay reagent for the ELISA, for example, Human LRG Assay Kit-IBL (IBL Co, Ltd. ) is commercially available. The ELISA reagent for measuring human LRG enables the quantitative determination of the LRG concentration of a sample by forming an immune complex through antigen-antibody reaction between an antibody which binds to LRG and LRG and measuring the amount of the label in the immune complex.

In general, the concentration of a target of measurement which is suitable for measurement by ELISA is in the range of pg/mL to ng/mL. Therefore, when the substance to be measured exists at a relatively high concentration of the order of µg/mL in a biological sample, pretreatment has been required for diluting the sample to a concentration at which the measurement reagent can exert the capability.

Moreover, it is necessary to dilute the sample by multiple separate stages to prevent an error due to high-rate dilution, and the complexity of the steps has been a problem. Specifically, with the commercial reagent above, it has been necessary to dilute the sample so that the LRG concentration of the sample falls in the constant range of <NUM> to <NUM> ng/mL. The dilution means that dilution at <NUM> times is required, for example, when the maximum value of the concentration in human serum that can be possible in case of ulcerative colitis is <NUM>µg/mL.

Furthermore, it has been necessary to react the sample to be measured and the solid phase antibody for a long time with the conventional ELISA reagents for LRG measurement. For example, the commercial kit above requires one night for the primary reaction for bringing the solid phase antibody and the sample into contact with each other, and there has been no reagent which can measure LRG in a sample in a short time so far. <CIT> relates to diagnostic agents for sepsis. <CIT> relates to a diagnostic techique of neonatal infection or perinatal infection. <CIT> relates to an insulin measurement method.

An object of the invention is to provide a measurement method and use of a reagent for measurement which can measure LRG in a biological sample simply in a short time.

[-> page 4a] The present invention relates to an immunological measurement method for leucine-rich α2 glycoprotein (LRG) in a biologically derived sample comprising:
bringing the sample into contact 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,.

Further aspects of the invention are defined in the claims.

According to the invention, a measurement method and a reagent for measurement of LRG can provide results more simply in a short time.

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

Methods for measuring LRG by LTIA can be roughly classified into methods in which latex particles on which monoclonal antibodies to LRG are immobilized and LRG as the antigen are reacted to form a sandwich-shaped immune complex and in which LRG is measured by the degree of agglutination of the latex particles resulting from the immune complex formation, and methods in which antigen-immobilized latex particles and LRG in a sample are competed to inhibit the formation of an immune complex of the latex particles and the antibody and in which LRG is measured by the degree of inhibition of agglutination of the latex particles resulting from the inhibition of the immune complex formation.

The sample of the invention is not particularly limited as long as the sample is a biologically derived sample but is typically a blood sample, and examples include whole blood, serum and plasma. Plasma includes heparin-plasma, EDTA-plasma and the like.

The substance to be measured in the invention is leucine-rich α2 glycoprotein (LRG).

In a measurement method using immunoagglutination represented by LTIA, the substance to be tested can be measured by optically or electrochemically observing the degree of generated agglutination. An optical observation method includes a method for measuring the intensity of scattered light, the absorbance or the intensity of transmitted light with an optical device (endpoint method, rate method or the like). The concentration of LRG (quantitative value) contained in the sample is calculated by comparing the measured value of the absorbance or the like obtained by measuring the sample with the measured value of the absorbance or the like obtained by measuring a standard substance (a sample with a known LRG concentration). In this regard, the absorbance or the like of transmitted light, scattered light or the like may be measured by single wavelength measurement or dual-wavelength measurement (the difference or the ratio resulting from two wavelengths). The wavelength for the measurement is generally determined in the range of <NUM> to <NUM>.

LRG in the sample of the invention may be measured by a hand process or using an apparatus such as a measurement apparatus. The measurement apparatus may be a general-purpose automated analyzer or a single-purpose measurement apparatus (a special purpose apparatus). The measurement is preferably conducted by a method using multiple operational steps such as a two-step method (dual-reagent method).

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

The kind of animal used for immunization is not particularly limited, either, but is preferably a mammal. For example, mice, rats, cows, rabbits, goats, sheep and the like can be used, and mice can be used more preferably. An animal may be immunized according to a general method and can be immunized for example by injecting a solution of the antigen, preferably a mixture with an adjuvant, subcutaneously, intracutaneously, intravenously or intraperitoneally to the animal. Because the immune response generally differs with the kind and the line of the animal to be immunized, the immunization schedule is desirably set appropriately depending on the animal to be used. The administration of the antigen is preferably repeated several times after the first immunization.

When the monoclonal antibodies are obtained, the following procedures follow, but the method for producing the monoclonal antibodies themselves is not limited to the procedures and can be in accordance with the method described for example in<NPL>)).

After the final immunization, spleen cells or lymph node cells, which are antibody-producing cells, are taken out of the immunized animal, and hybridomas can be produced by cell fusion with myeloma cells having high growth potential. Cells with high antibody-producing potential (quality/amount) are preferably used for the cell fusion, and the myeloma cells preferably have compatibility with the animal from which the antibody-producing cells to be fused are derived. The cells can be fused according to a method known in the field, but for example, a polyethylene glycol method, a method using Sendai virus, a method using current and the like can be employed. The obtained hybridomas can be proliferated according to a known method, and a desired hybridoma can be selected while the properties of the produced antibody are examined. The hybridoma can be cloned by a known method such as limiting dilution analysis or soft agar analysis, for example.

The selection of hybridomas producing the first and second monoclonal antibodies is explained. The hybridomas can also be selected efficiently at the selection stage considering the conditions under which the produced antibodies are used for the actual measurement. For example, the hybridomas are obtained by selecting hybridomas producing an antibody that reacts with human LRG by ELISA, RIA, Biacore assay or the like. Specifically, monoclonal antibodies in a culture supernatant are first reacted with immobilized human LRG, and hybridomas producing monoclonal antibodies that have high reactivity with human LRG are then selected by antigen-immobilized ELISA in which a labelled anti-IgG antibody is reacted.

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

As the antibodies used in the methods and uses of the invention, functional fragments of antibodies having antigen-antibody reactivity can also be used in addition to the whole antibody molecules, and those obtained through the immunization step of an animal described above, those obtained using genetic recombination technique and chimeric antibodies can also be used. Examples of functional fragments of an antibody include F(ab')<NUM>, Fab' and the like, and such functional fragments can be produced by processing an antibody obtained as described above with a protease (for example, pepsin, papain or the like).

The insoluble carrier particles used in the invention are insoluble carrier particles which can carry an anti-LRG monoclonal antibody and which can measure LRG in a sample.

The average particle sizes and the critical coagulation concentrations of the insoluble carrier particles are appropriately determined considering the concentration of LRG in the sample, the detection sensitivity of the measurement device or the like.

The average particle sizes of the insoluble carrier particles are <NUM> to <NUM>, more preferably <NUM> to <NUM>.

Regarding the critical coagulation concentrations of the insoluble carrier particles, those having a critical coagulation concentration of <NUM> to <NUM>, more preferably <NUM> to <NUM>, are appropriately selected.

The latex particles, which are used in the invention, are not particularly limited as long as the latex particles are generally used as an immunological measurement reagent. The latex particles can be obtained by polymerization or co-polymerization of various monomers. Examples of the monomers here include polymerizable unsaturated aromatic compounds such as polymerizable monomers having a phenyl group including styrene, α-methylstyrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, <NUM>-vinylbenzoate, divinylbenzene, vinyltoluene and the like, polymerizable monomers having a phenyl group and a sulfonate including styrene sulfonate, divinyl benzene sulfonate, o-methyl styrene sulfonate, p-methyl styrene sulfonate and the like and polymerizable monomers having a naphthyl group including <NUM>-vinylnaphthalene, <NUM>-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 and fumaric acid, polymerizable unsaturated carboxylate esters such as methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, <NUM>-hydroxyethyl (meth)acrylate, glycidyl (meth)acrylate, ethylene glycol-di-(meth)acrylate ester and tribromophenyl (meth) acrylate, unsaturated carboxylic amides, polymerizable unsaturated nitriles, vinyl halides and.

conjugated dienes such as (meth) acrylonitrile, (meth) acrolein, (meth)acrylamide, N-methylol-(meth)acrylamide, methylenebis(meth)acrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride and vinyl bromide and the like. The monomers are appropriately selected by the required surface characteristics, the specific gravities and the like. A kind thereof can be used alone, or a mixture of two or more kinds thereof can be used.

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

Regarding the average particle sizes of the latex particles, those having average particle sizes of <NUM> to <NUM>, more preferably <NUM> to <NUM>, are appropriately selected. The reasons are as follows: when the average particle sizes are smaller than <NUM>, the sensitivity in the low concentration to middle concentration range decreases, and accurate measurement is difficult; and when the average particle sizes exceed <NUM>, the so-called hook effect is caused at a high concentration, which causes a phenomenon whereby a value lower than the actual concentration is obtained, although the sensitivity at a low concentration increases.

The critical coagulation concentration of latex particles means the maximum concentration of a salt at which latex does not agglutinate and means the salt concentration which is one step lower than the salt concentration at which latex particles self-agglutinate completely when a salt is stepwisely added to the latex particles that are not sensitized with an antibody. For example, aqueous sodium phosphate solutions (pH7. <NUM>) having concentrations that are different by <NUM> in the range of <NUM> to <NUM> are prepared, and latex particles in an amount which results in the final concentration of <NUM>% (W/V) are added to the aqueous sodium phosphate solutions with the different concentrations and stirred. The solutions are observed visually after one minute to determine whether or not the latex has self-agglutinated, and the concentration of the aqueous sodium phosphate solution that is one step lower than the concentration at which the latex has self-agglutinated completely is determined as the critical coagulation concentration (maximum non-coagulation concentration).

Regarding the critical coagulation concentrations (the concentrations of the aqueous sodium phosphate solutions) of the latex particles, those having critical coagulation concentrations of <NUM> to <NUM>, more preferably <NUM> to <NUM>, are appropriately selected. It is difficult to maintain the dispersion of the latex particles when the critical coagulation concentrations are smaller than <NUM>, while immune response is not caused easily, and the latex particles do not agglutinate when the critical coagulation concentrations are <NUM> or more.

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

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

Moreover, the concentrations of the latex particles during the agglutination reaction measurement in the invention are not particularly limited and can be appropriately set depending on the desired sensitivity and properties.

An antibody to LRG can be immobilized and carried on insoluble carrier particles such as latex particles by a known method such as a physical adsorption (hydrophobic bonding) method, a chemical bonding method or a combination thereof. The following explanation is given using latex as a insoluble carrier particles.

The physical adsorption method can be conducted according to a known method by mixing an antibody to LRG and latex particles in a solution such as a buffer and bringing the antibody and the latex particles into contact with each other or by bringing an antibody to LRG dissolved in a buffer or the like into contact with a carrier.

The chemical bonding method can be conducted according to a known method described in "<NPL>; "<NPL> or the like, by mixing and bringing a substance specifically binding to LRG and a carrier into contact with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, an imide ester or maleimide and reacting the amino groups, the carboxyl groups, the thiol groups, the aldehyde groups, the hydroxyl groups or the like of the substance specifically binding to LRG and the carrier with the divalent crosslinking reagent.

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

It is necessary that the first monoclonal antibody and the second monoclonal antibody are carried on different insoluble carrier particles.

In the invention, 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 depending on the desired sensitivity and properties as described above, but the insoluble carrier particles are preferably contained in same amounts in the reaction liquid phase.

Moreover, in the invention, the average particle sizes of the insoluble carrier particles carrying the first anti-LRG monoclonal antibody and the insoluble carrier particles carrying the second anti-LRG monoclonal antibody are each <NUM> to <NUM> as described above.

When treatment is necessary to inhibit spontaneous agglutination of the latex particles, non-specific reaction or the like, the carrier may be subjected to blocking (masking) by a known method such as contacting or covering the surface of the latex particles with a protein such as bovine serum albumin (BSA), casein, gelatin, egg albumin or salts thereof, a surfactant, defatted milk powder or the like.

That the sample containing LRG and the latex particles carrying the anti-LRG monoclonal antibodies are brought into contact with each other in a liquid phase typically means (<NUM>) to (<NUM>) below.

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

In particular, the reagent used for measurement of the invention is preferably a two-reagent type including a first reagent and a second reagent. For example, the first reagent of the two-reagent type contains a buffer for diluting the biologically derived sample containing LRG, and the second reagent is a reagent solution containing the latex particles carrying the first anti-LRG monoclonal antibody and the latex particles carrying the second anti-LRG monoclonal antibody.

When the reagent used for measurement of the invention is a three-reagents 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 the latex particles carrying the first anti-LRG monoclonal antibody, and the third reagent is a reagent solution containing the latex particles carrying the second anti-LRG monoclonal antibody.

The buffer may be a buffer that is generally used, and examples include tris-hydrochloric acid, boric acid, phosphoric acid, acetic acid, citric acid, succinic acid, phthalic acid, glutaric acid, maleic acid, glycine, a salt thereof and the like as well as Good's buffers such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES and HEPES and the like.

The concentration of the buffer component may be in a concentration range in which spontaneous agglutination of the insoluble carrier particles in the reagent is not caused and in which the desired immune response is caused, and the concentration in the reaction solution may be <NUM> or more and is preferably <NUM> or more, more preferably <NUM> or more, further more preferably <NUM> or more.

The measurement reagent used in the invention desirably further contains 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 in a concentration range in which spontaneous agglutination of the insoluble carrier particles in the reagent is not caused and in which the desired immune response is caused, and the lower limit of the concentration may be <NUM> or more in the reaction solution and is preferably <NUM> or more, more preferably <NUM> or more, further more preferably <NUM> or more. The upper limit of the concentration is preferably <NUM> or less in the reaction solution, more preferably <NUM> or less, further more preferably <NUM> or less, most preferably <NUM> or less.

The range of the concentration in the reaction solution is preferably <NUM> to <NUM>, more preferably <NUM> to <NUM>, further more preferably <NUM> to <NUM>, most preferably <NUM> to <NUM>.

The measurable range (measurement range) of the measurement reagent used in the invention may be around <NUM> to <NUM>µg/mL, desirably <NUM> to <NUM>µg/mL to measure LRG as a diagnostic marker.

The reagent kit used in the invention is characterized by including at least elements (<NUM>) and (<NUM>) below.

In the reagent kit used in the invention, (<NUM>) LRG as a standard antigen or a control can be included in addition to the measurement reagents above.

A sample pretreatment reagent for pretreating the sample may also be included. The sample pretreatment reagent can be contained in the first reagent of (<NUM>) containing a buffer or can also be included as an element other than (<NUM>) and (<NUM>).

The reagent kit can further include a user guide, a sample collection tool (a collection pipette, a syringe, a cotton swab, a filter and the like), a sample diluent and a sample extraction solution.

In the disclosure, the homogeneous method refers to a measurement method for specifically detecting the reaction that advances in the mixed solution of the sample and the reagent solution (reaction solution) without conducting B/F (bound/free) separation and means a measurement method named in comparison to a heterogeneous measurement method, in which the main reaction is advanced and detected after completely washing/removing the excess components which have not involved in the measurement reaction by a B/F separation operation. Therefore, that "the immunoagglutination assay is a method based on a homogeneous method" as in the disclosure means that step (<NUM>) is "a step of measuring the agglutination reaction of LRG and the insoluble carrier particles without a washing/separation step during step (<NUM>) or after step (<NUM>)" in typical steps (<NUM>) to (<NUM>) below.

The reagent used in the invention may contain a polymer such as polyethylene glycol, polyvinylpyrrolidone and a phospholipid polymer as a component that supplements the formation of agglutination of the insoluble carrier particles. Moreover, as a component that controls the formation of agglutination, a kind or a combination of generally used components such as proteins, amino acids, saccharides, metal salts, surfactants, reducing substances and chaotropic substances may also be contained.

The particle sizes of the insoluble carrier particles used for measuring LRG concentrations were changed, and whether or not a concentration-dependent change in absorbance would be observed and whether or not a calibration curve could be drawn were examined.

The reagent contains the components shown below. The pH was controlled to <NUM> to <NUM>.

The two kinds of anti-LRG monoclonal antibody-sensitized latex particle solution below were mixed in same amounts and diluted with <NUM> HEPES buffer (pH7. <NUM>) in a manner that the absorbance at a wavelength of <NUM> became <NUM> Abs. , and a second reagent was thus obtained.

To a <NUM>% polystyrene latex solution (<NUM> glycine buffer) having an average particle size of <NUM> and a critical coagulation concentration of <NUM>, the same amount of an anti-LRG monoclonal antibody (Kan10) solution which had been diluted with <NUM> glycine buffer to <NUM>/mL was added, and the mixture was stirred at <NUM> for two hours. Then, the same amount of a synthetic polymer (Blockmaster CE210 manufactured by JSR) was added, and the mixture was stirred at <NUM> for an hour. Subsequently, <NUM>/<NUM> amount of <NUM>% BSA solution dissolved in purified water was added, and the mixture was stirred at <NUM> for an hour. An anti-LRG monoclonal antibody (Kan10)-sensitized latex solution was thus produced.

An anti-LRG monoclonal antibody (Kan11)-sensitized latex particle solution was produced by the same method as that of (i) above using polystyrene latex having an average particle size of <NUM> and a critical coagulation concentration of <NUM>.

The latex particle sizes were analyzed with Laser diffraction/scattering particle size distribution analyzer LS13320 (manufactured by Beckman Coulter).

Aqueous sodium phosphate solutions (pH7. <NUM>) having concentrations that were different by <NUM> in the range of <NUM> to <NUM> were prepared, and latex particles in an amount which resulted in the final concentration of <NUM>% (W/V) were added to the aqueous sodium phosphate solutions with the different concentrations and stirred. Then, the solutions were observed visually after one minute to determine whether or not the latex self-agglutinated, and the concentration of the aqueous sodium phosphate solution that was one step lower than the concentration at which the latex self-agglutinated completely was determined as the critical coagulation concentration.

The same procedures were conducted except that the latex particles of Prescription Example <NUM> were changed as follows.

Prescription Example <NUM>: The average particle sizes were <NUM>, and the critical coagulation concentrations were <NUM>.

Purified LRG (manufactured by Bio Vendor Laboratory medicine) in amounts resulting in <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>µg/mL was added to physiological saline.

The first reagents and the second reagents of the Prescription Examples were each combined, and the samples containing LRG at known concentrations were measured using Hitachi <NUM> automated analyzer. Specifically, <NUM>µL of the first reagent was added to <NUM>µL of a sample, and the mixture was incubated at <NUM> for five minutes. Then, <NUM>µL of the second reagent was added, and the mixture was stirred. The change in absorbance resulting from the formation of agglutination was measured for five minutes after that at a dominant wavelength of <NUM> and a complementary wavelength of <NUM>, and the change in absorbance was measured. A calibration curve was drawn from the measured absorbances, and R<NUM> (coefficient of determination) was determined.

The parameter conditions of Hitachi <NUM> automated analyzer are shown below.

According to the results, in Prescription Example <NUM>, although the hook effect was observed at a high LRG concentration (<NUM>µg/ml) , the absorbance increased in a concentration-dependent manner at low to middle concentrations. In Prescription Examples <NUM> to <NUM>, the absorbances increased in a concentration-dependent manner at a low concentration to a high concentration, and the R<NUM> values were <NUM> or more. In particular, the R<NUM> values of Prescription Examples <NUM>, <NUM> and <NUM> were <NUM> or more, which are particularly excellent.

From the above results, it was found that accurate measurement is possible in the concentration range which covers the entire LRG concentration distribution in human serum when the average particle sizes of the latex particles are <NUM> to <NUM> and that, in particular, measurement with higher accuracy is possible when the average particle sizes are <NUM> to <NUM>.

It was also found that accurate measurement is possible in the concentration range which covers the entire LRG concentration distribution in human serum when the critical coagulation concentrations are <NUM> to <NUM> and that, in particular, measurement with higher accuracy is possible when the critical coagulation concentrations are <NUM> to <NUM>.

In order to examine whether or not specimens similar to an actual specimen could be measured, simulated LRG specimens with known concentrations were prepared by adding purified LRG to pooled serum, and the concentrations of LRG were measured.

The reagents of Prescription Examples <NUM> to <NUM> of Test Example <NUM> were used.

The test method and the measurement conditions are the same as those in Test Example <NUM>. Using the calibration curves drawn in Test Example <NUM>, the concentrations of the panel specimens were determined, and the relative ratios (%) to the known concentrations were calculated.

According to the results, it was found that simulated specimens which are similar to an actual specimen can be measured accurately using the reagents of Prescription Examples <NUM> to <NUM>. In particular, Prescription Examples <NUM> to <NUM> had extremely high accuracy of relative ratios to the known concentrations of <NUM>%.

From the above results it was found that accurate measurement is possible when the average particle sizes of the latex particles are <NUM> to <NUM> and that, in particular, measurement with higher accuracy is possible when the average particle sizes are <NUM> to <NUM>.

It was also found that accurate measurement is possible when the critical coagulation concentrations of latex are <NUM> to <NUM> and that, in particular, measurement with higher accuracy is possible when the critical coagulation concentrations are <NUM> to <NUM>.

It was confirmed that LRG can be measured as a marker by the measurement method using specimens of healthy individuals and specimens of patients.

The reagents of Prescription Example <NUM> of Test Example <NUM> were used.

Seven specimens purchased from PROMEDDEX.

Thirteen specimens purchased from Bioreclamtion.

Five specimens purchased from Bioreclamtion and Proteogenex.

The measurement method and the measurement conditions are the same as those in Test Example <NUM>.

According to the results, it was found that LRG in the specimens of healthy individuals and mild and severe ulcerative colitis patients can be measured and that measurement as a marker which can distinguish these states is possible.

Accordingly, the measurement reagent can play the role of a diagnostic agent.

According to the disclosure a measurement method and a measurement reagent for LRG in a biological sample which provide the results easily in a short time could be provided by bringing a biological sample containing LRG and insoluble carrier particles carrying anti-LRG monoclonal antibodies into contact with each other in a liquid phase and measuring the degree of agglutination. The measurement method and the measurement reagent of the disclosure can be applied also to a general-purpose automated analyzer, and thus easy and simultaneous measurement of a large number of LRG samples has become possible.

Claim 1:
An immunological measurement method for leucine-rich α2 glycoprotein (LRG) in a biologically derived sample comprising:
bringing the sample into contact 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,
wherein the insoluble carrier particles are latex particles having average particle sizes of <NUM> to <NUM> respectively, and
wherein the insoluble carrier particles are latex particles having critical coagulation concentrations of <NUM> to <NUM> respectively.