JP2001337092A - Immunological measurement method and measurement reagent - Google Patents

Abstract

(57) [Summary] (With correction) [Solution] A sample containing an antigen to be measured and (A)
Insoluble carrier particles on which the antigen is immobilized, (B) a specific antibody in a free state that reacts with the antigen, and (C) a receptor having a site other than the binding site of (A) in the specific antibody as a ligand, An immunological measurement method characterized by optically measuring an immunological agglutination inhibition reaction caused by a free antigen to be measured in a sample, and a measurement reagent used for the method. [Effect] An antigen present in a sample can be specifically, simply and quickly measured by utilizing an immunological agglutination inhibition reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for simply, rapidly and specifically measuring an antigen in a sample by using an immunological particle aggregation inhibiting method.

[0002]

2. Description of the Related Art In hospitals, examination centers, etc., automation and simplification of clinical examinations and shortening of measurement time are being pursued with the reduction of personnel and costs. Among the immunological measurement methods based on the antigen-antibody reaction used here, the measurement method called immunoturbidimetry is the degree of immunological aggregation formed by reacting the antigen to be measured with a specific antibody against the antigen. Is a method of measuring optically. In particular, a method for causing an immunological particle agglutination or agglutination inhibition reaction by using insoluble carrier particles to qualitatively and quantitatively determine a test substance is a method capable of increasing sensitivity and requiring B / F separation. Compared to the ELISA method and the RIA method, the number of reaction steps is smaller, suitable for automation, and dedicated measuring devices based on various measuring principles have been commercialized from an early stage. Recently, measurement has been started using a general-purpose automatic analyzer together with other measurement items based on an enzyme reaction or the like.

[0003] In the immunological particle agglutination method, a specific antibody to an antigen to be analyzed is immobilized on insoluble carrier particles by physical adsorption or covalent bond, and a test sample such as serum or urine is mixed with the antigen to form a corresponding antibody. In a method of measuring an antigen in a test sample by generating an immunoagglutination reaction based on an antigen-antibody reaction, a wide range of components has been adopted as a measurement target. However, among these components to be measured, C-reactive protein (CRP) which is an inflammatory marker, serum amyloid A, and Ig which is an index of allergic symptoms
E. In the measurement of a component with a wide dynamic range, such as albumin in urine, which is an indicator of renal impairment, which is usually at a low concentration but fluctuates widely up to a very high concentration when abnormal, the measurement target in the test sample If the concentration of the antigen is much higher than the concentration range of the reagent, the excess measurement suppresses agglomeration, lowers the apparent measurement value, and erroneously measures the concentration to be within the normal range. May be given. This phenomenon is called a hook phenomenon or a prozone phenomenon, and has become a serious problem in a measurement system using the principle of the immunological particle aggregation method.

On the other hand, among the immunoturbidimetric methods, a measuring method known as immunological particle agglutination inhibition method comprises a test sample, insoluble carrier particles in which an antigen similar to the antigen to be measured is immobilized in advance, and specificity for the antigen. The antigen to be measured is mixed with the antibody, and the antigen to be measured in the free state present in the test sample competes with the antigen-antibody reaction between the immobilized antigen on the insoluble carrier particles and the specific antibody against the antigen to form an immunological aggregate. Is a method for measuring an antigen to be measured in a test sample due to inhibition of the antigen. The immunological particle agglutination inhibition method is based on the measurement principle, because even if the concentration of the antigen to be measured contained in the test sample greatly exceeds the upper limit of the measurement, aggregation formation inhibition is not attenuated,
It has the characteristic that a high antigen concentration does not give an erroneous measurement value as if it were a low value.

In addition, in an immunological particle agglutination method which essentially comprises particle agglomeration based on an antigen-antibody reaction between an antigen to be measured in a free state in a test sample and a specific antibody immobilized on carrier particles, Can not be measured depending on the type of the antigen to be measured, and there are various restrictions on the antibody used.However, in the immunological particle agglutination inhibition method, they can be applied without any hindrance. can do. That is, when the antigen to be measured is a low-molecular-weight monovalent antigen such as a hapten, the hapten in a free state cannot be aggregated because it is not cross-linked by the antibody, and the antigen in the test sample is immobilized on an antibody-immobilized carrier. The measurement cannot be performed by the immunological particle aggregation method in which particles are aggregated. In recent years, monoclonal antibodies having higher antigen specificity and uniform performance have been used for various immunological measurement reagents. Due to the nature of monoclonal antibodies that bind only to determinants,
Except for special antigens (antigens in which the same antigenic determinant is present on the molecule, for example, multivalent antigens such as CRP, ferritin and Lp (a)), a single type of monoclonal antibody cannot form aggregates. Because it is not applicable, it is usually used in an auxiliary sense to be used in combination with polyclonal antibodies, or used under conditions where the original advantages of monoclonal antibodies such as antigen specificity and homogeneity such as the use of multiple heterogeneous monoclonal antibodies are lost. Stays.

On the other hand, in the immunological particle agglutination inhibition method for measuring the antigen in a test sample, the formation of particle agglutination is prevented by the free antigen to be measured present in the test sample. The formation is caused by cross-linking between carrier particles by an immunoreaction between the antigen uniformly immobilized on the particle surface and the specific antibody, and aggregation elimination, which is the essence of the measurement method, is the antigen to be measured in the test sample. Inhibits the reaction between the immobilized antigen and the specific antibody, thereby reducing the formation of aggregates. Therefore, the presence or absence of the ability to form aggregates in the free state of the antigen and the specific antibody to be measured does not hinder the immunological particle aggregation inhibition method. As long as it can be applied as a measurement object, any of monoclonal antibodies and polyclonal antibodies can be used for the type of antibody. In fact, for the measurement of haptens such as drugs in blood, a measurement method based on an immunological particle aggregation inhibition method using a monoclonal antibody is widely used.

[0007] From the above facts, the measurement method based on the immunological particle agglutination inhibition method is not only suitable for measurement of an antigen having a wide dynamic range, but also applicable to various antigens. Since the monoclonal antibody alone can be easily used, it is a useful measurement method with higher specificity.

However, in the immunological particle agglutination inhibition method, as a prerequisite for application of the measurement, when the antigen-immobilized carrier particles and the free specific antibody are mixed, sufficient particle agglutination should not occur. There is a problem that it does not. If the particle agglomeration is insufficient, even when applied to the measurement, the change in sensitivity due to the antigen in the test sample is small, and an accurate measured value cannot be obtained. In particular, when a monoclonal antibody is used as a specific antibody, the tendency is more remarkable since aggregation is less likely to occur than a polyclonal antibody, and a large amount of a non-specific aggregating agent must be added.

For example, Japanese Patent Application Laid-Open No. 57-206859 proposes a particle agglutination inhibition method using a typical non-specific aggregating agent such as RF, Clq, mouse serum or ascites for measurement of hapten antigen. I have. The aggregating agent used here has low affinity with the free antigen-antibody complex,
Affinity becomes apparent only when the state of the antigen-immobilized carrier particle-antibody complex, that is, in the state where the immobilized antigen-antibody complex is densely distributed on the carrier particle, is characteristic of promoting aggregation formation. Therefore, it can be said that the action is not promotion of immunological aggregate formation but a non-specific action. Since such a non-specific aggregating agent acts synergistically on the formation of immunological agglutination, it facilitates discrimination between aggregated and non-aggregated carrier particles, and is measured, for example, by direct counting of non-aggregated carrier particles. The method can be used in a method for measuring a target antigen. However, in the turbidimetric immunoassay in which the antigen to be measured is optically measured based on the degree of immunological agglutination based on the antigen-antibody reaction, there is a high possibility that an erroneous measurement value is given, and such non-specific agglutination is likely to occur. No agents are available.

Japanese Unexamined Patent Publication (Kokai) No. 59-173760 proposes a measuring method for promoting aggregation formation by using a single type of monoclonal antibody used as a specific antibody carried on a carrier. However, Patent 2840852
As shown in the figure, when the monoclonal antibody is carried on a carrier, depending on the recognition site of the antibody on the antigen and the method of binding to the carrier, the antigen may be present in a free state for reasons such as steric hindrance. There is a problem that the specificity and reactivity with respect to may be lost or deteriorated, and it is difficult to use it simply.

[0011]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an assay method based on the immunological particle agglutination inhibition method, which does not have the above-mentioned problems and is simple, rapid and specific in a sample. It is to provide a method for measuring an antigen.

[0012]

Means for Solving the Problems In the immunological particle agglutination inhibition method, the present inventors use a specific receptor when forming an agglutination between an insoluble carrier particle on which an antigen is immobilized and a specific antibody. The present inventors have found that a sufficient agglutination reaction occurs, the sensitivity change due to the antigen in the sample is large, and the antigen can be accurately measured, and the present invention has been completed.

That is, the present invention provides a sample containing an antigen to be measured, (A) an insoluble carrier particle on which the antigen is immobilized,
(B) a free specific antibody that reacts with the antigen, and (C) in the specific antibody mixed with a receptor having a site other than the binding site of (A) as a ligand, depending on the free measurement target antigen in the sample It is intended to provide an immunological measurement method characterized by optically measuring the resulting immunological agglutination inhibition reaction.

The present invention also relates to a reagent used in combination with a sample to measure an immunological agglutination inhibition reaction caused by a free antigen to be measured in the sample, the reagent comprising (A) an insoluble antigen on which the antigen is immobilized. A carrier particle, (B) a free-form specific antibody that reacts with the antigen, and (C) an immunology comprising a receptor having a ligand other than a binding site to (A) in the specific antibody as a ligand. The present invention provides a reagent for quantitative measurement.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a sample containing an antigenic substance to be measured includes human or animal body fluid,
Examples include serum, saliva, urine and the like. The antigenic substances contained in these are not limited to the valence of the antigenic determinant,
It is also particularly useful for measuring monovalent haptens. Examples of the hapten include drugs and drug metabolites such as theophylline, barbitone, phenylhydantoin, digoxin, digitoxin, and gentamicin; intracellular transmitters such as cyclic-AMP, cyclic-GMP, prostaglandin, and prostaglandin metabolite Thyroid hormone releasing hormone, gonadotropin releasing hormone, somatostatin, melatonin, substance-P, pituitary hormone (oxytocin, vasopressin, etc.), thyroid hormone (thyroxine, triiodothyronine, etc.), gastrointestinal hormones (gastrin, secretin) , Cholecystokinin, serotonin, etc.), pancreatic hormones (glucagon, etc.),
Hormones such as steroid hormones (estradiol, progesterone, testosterone, aldosterone, cortisol, etc.); vitamins such as B ₁₂ , folic acid, vitamin D metabolites; haptens derived from bacteria or viruses such as glycolipids of leukemia virus; . In addition, because of having a wide dynamic range, an antigen that is likely to give erroneous measurement values in a normal immunological particle agglutination method,
For example, it is suitable for measurement of serum components such as CRP, serum amyloid A and IgE; urine components such as albumin.

The material of the insoluble carrier particles used in the present invention is not particularly limited, and any conventionally used materials may be used, and examples thereof include organic high molecular substances, inorganic substances, cell membranes, blood cells, and microorganisms. As organic polymer substances,
Latex particles are preferred, and latex particles in which one or two or more resin fine powders such as acrylic acid polymer, styrene polymer and methacrylic acid polymer are uniformly suspended are particularly preferred. As the inorganic substance, fine particles such as silica and alumina are preferable. These insoluble carrier particles preferably have an average particle size of 1.6 μm or less, particularly those having an average particle size of 0.05 to 1 μm, and more preferably 0.05 to 0.5 μm.
It is preferable for directly optically measuring immunoaggregates.

The antigen to be immobilized on the insoluble carrier particles in advance may be any as long as it has the same antigenicity as the antigen to be measured with respect to the specific antibody. Recombinant antigens obtained by recombination, fragments of antigens or substances having similar structures can be used. The method of supporting these antigens on the insoluble carrier particles is not particularly limited, and a known method such as physical adsorption or a chemical bonding method can be used. Particularly, the chemical bonding method is used to obtain a more stable reagent. Is preferred.

The (B) specific antibody used in the present invention forms an aggregate by an immunoreaction with the insoluble carrier particles on which the antigen (A) is immobilized. Such a specific antibody can specifically bind to the antigen to be measured, and may be in any free state. A monoclonal antibody, a polyclonal antibody, or the like can be used alone or in combination. In particular, if only a monoclonal antibody is used, one type may be used. Further, in both cases of monoclonal antibodies and polyclonal antibodies, those which do not form immunoaggregates when reacted with a free antigen are preferable. These can use a commercial item suitably.

The receptor (C) having a site other than the site for binding to (A) in the specific antibody used in the present invention as a ligand has an action of promoting the aggregation by (A) and (B). , (B).
The receptor is not particularly limited as long as it is in a free state, but preferably does not react with the ligand to form an aggregation, and polyclonal antibodies, monoclonal antibodies, protein A, and protein G are particularly preferred. Commercially available products can be used for these receptors,
Monoclonal antibodies can be prepared by a conventional method (G. Kohler and C. Milstein, Nature, 1975, 2
56, Yarmush M. et al., Proc. Natl. Acad. Sci. USA
77, 2899-, 1980).

That is, the monoclonal antibody used as a receptor is as follows: (1) When the antibody against the antigenic substance is a polyclonal antibody, spleen cells of a mouse immunized with immunoglobulin of an immunized animal derived from the polyclonal antibody and myeloma cells of the mouse Can be prepared from the hybridoma obtained. (2) When the antibody against the antigenic substance is a mouse-derived monoclonal antibody, a hybridoma obtained by fusing rat spleen cells immunized with mouse immunoglobulin with rat myeloma cells, or a rat or rabbit immunized with mouse immunoglobulin is used. It can be prepared from a heterohybridoma obtained by fusing animal spleen cells and mouse myeloma cells. (3) When the antibody to the antigenic substance is a monoclonal antibody derived from a heterohybridoma such as a rat or a rabbit, it can be prepared in the same manner as in (1). Among the obtained hybridomas, those producing the desired antibody are cloned to establish cells. Large-scale preparation of monoclonal antibodies can be performed by collecting the culture supernatant of the hybridoma, or inoculating the hybridoma into the intraperitoneal cavity of the animal and growing it, collecting the ascites, and salting out the monoclonal antibody contained in the ascites. Can be concentrated and purified by an adsorption method or an adsorption chromatography method. In addition, when inoculating intraperitoneally into an animal, it is preferable to use a nude mouse or a nude rat as a heterohybridoma.

In the present invention, the suspension of the insoluble carrier, the diluent of the test sample, and the like are not particularly limited, and commonly used buffers such as acetic acid, citric acid, phosphoric acid, Tris, glycine, and boric acid , Carbonic acid, Good's buffer and the like.
H6 to 9 are preferred.

The measuring method of the present invention comprises the above (A), (B) and
The antigen in the sample can be measured by preventing the antigen to be measured in the sample from forming an aggregate in (C), and the degree of aggregation is measured optically. The method for optically measuring aggregates is not particularly limited as long as it is a commonly used method, and a general-purpose spectrophotometer, an automatic biochemical analyzer based on the measurement principle of spectrophotometry, An optical measuring device such as a device having a measurement wavelength, a device having a measuring principle of integrating sphere turbidity, and a device for measuring scattered light intensity can be used.

In the present invention, the reaction between the test sample and the specific antibody is performed by using either the antigen-immobilized carrier particles or the receptor,
Alternatively, the reaction may be performed in the co-presence of both, or after the reaction is performed in the absence of the antigen-immobilized carrier particles and the receptor, the two may be reacted.

The measuring reagent of the present invention comprises the above (A), (B) and
(C). The reagent can be composed of two or more components, and (C) may be added to either (A) or (B).

[0025]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 (1) Preparation of a solution of carrier particles immobilized on human serum albumin (hereinafter, HSA) -protein A: 200 μL of polystyrene latex (average particle size: 0.2 μm, manufactured by Sekisui Chemical Co., Ltd.)
And 800 μL of 20 mM MES buffer (pH 6.5)
, And diluted with HSA (manufactured by Sigma).
1 mL of a solution dissolved in the above MES buffer at a concentration of 5 mg / mL
Was added and reacted at 4 ° C. for 2 hours. Next, 2 mL of a 2 mg / mL bovine serum albumin (hereinafter, BSA) aqueous solution was added, and the mixture was reacted at 4 ° C. for 1 hour. In this solution, 1.5% polyethylene glycol 20000, 0.1% BSA
Was added and stirred to obtain an HSA-immobilized carrier particle solution. To this solution, protein A (manufactured by Sigma) was added to a final concentration of 10 μg / mL, and H
An SA-immobilized carrier particle-protein A solution was obtained.

(2) Production of anti-HSA monoclonal antibody: (a) Immunization; commercially available HSA
100 μg was used. The initial immunization used Freund's complete adjuvant, and the booster immunization used Freund's incomplete adjuvant. Specifically, 100 μL of HSA and 100 μL of Freund's adjuvant were mixed, and 200 μL of the obtained emulsion was used for one immunization for one animal.
ALB / C male mice were injected intraperitoneally and immunization was repeated four times at two week intervals. Blood was collected from the fundus vein of the mouse, the antibody titer was measured by ELISA, and a mouse with a high antibody titer was selected and used for cell fusion.

(B) Cell fusion; two weeks after the fourth immunization, 100 μg of HSA diluted in 200 μL of physiological saline
Was intraperitoneally injected into the mouse, and three days later, the spleen was removed from the mouse. The excised spleen was well loosened with tweezers and a polished portion of a slide glass in RPMI 1640 medium, and spleen cells were collected. This was centrifuged at 1500 rpm for 5 minutes to collect spleen cells, which were further washed and centrifuged with the same medium. Finally, 2 mL of the same medium containing 15% fetal calf serum (hereinafter, FCS) was added to prepare a spleen cell suspension. The number of living spleen cells was counted under a fluorescence microscope by mixing a suspension of acridine orange / ethidium bromide (0.1 mg each in 1 mL of PBS) and the suspension at a ratio of 1: 1. 10 ⁸ living spleen cells were mixed with 10 ⁷ mouse myeloma cells (myeloma cells) SP2 / 0-Ag14 in a logarithmic growth phase that had been cultured in advance, followed by centrifugation at 1500 rpm for 5 minutes. Remove the supernatant,
After dissolving the cells well, a GKN solution (NaCl: 8
g, KCl: 0.4 g, glucose: 2 g, Na ₂ HP
O ₄ : 1.41 g, NaH ₂ PO ₄ .2H ₂ O: 0.78 g
Was dissolved in 1 L of purified water) and centrifuged at 1500 rpm for 5 minutes to wash the cells. After repeating the washing, 50% (w / v) polyethylene glycol 1
0.5 mL of a GKN solution containing 540 was gradually added, followed by gentle stirring for 1 minute. The reaction was stopped by gradually and gently adding 10 mL of a GKN solution, and the mixture was centrifuged at 1500 rpm for 5 minutes. For the obtained cells, RPM containing 15% FCS
Suspended in 30 mL of I1640 medium, feeder cells 10
HAT medium containing ⁶ cells (10 ^-4 M hypoxanthine, 4 ×
RPMI 1640 medium containing 10 ^-7 M aminopterin, 1.5 × 10 ^-5 M thymidine and 15% FCS)
100 μL per well was dispensed into three 96-well microculture plates dispensed at 200 μL per well and cultured in a 5% CO 2 incubator at 37 ° C. After 10 days, the growth of the fused cells was confirmed in all the wells.

(C) Selection and cloning of anti-HSA antibody producing cells; ELISA for the presence of anti-HSA antibody in the culture supernatant
Confirmed by law. That is, screening was performed using a 96-well microplate on which HSA (2 μg / mL) was immobilized. Specifically, HSA was added to 0.72% NaCl.
13 mM phosphate buffer (hereinafter referred to as PBS pH7.
4) Dilute to 2 µg / mL with 9 µl at a rate of 50 µL / well.
The solution was dispensed into a 6-well microplate and left at 4 ° C. overnight.
P with 1% BSA, 0.05% Tween 20
Washed three times with BS pH 7.4 (BSA-PBS).
The control plate was 1% BSA, 0.05% Tween.
The plate was washed three times with PBS pH 7.4 containing n20 (BSA-PBS). 50 μL of culture supernatant in each well of the plate
Was added and kept at 37 ° C. for 1 hour. Then, after washing three times with PBS, 50 μL of a peroxidase-labeled anti-mouse IgG antibody (specific to Fc site, derived from goat: manufactured by Daiichi Kagaku) diluted 1000-fold with BSA-PBS was added, and
It was kept at 7 ° C for 1 hour. After washing this with PBS 5 times,
50% citric acid-phosphate buffer pH 5.0 containing 0.2% orthophenylenediamine and 0.02% aqueous hydrogen peroxide
After adding μL / well and reacting at room temperature for 30 minutes, the reaction was stopped by adding 50 μL / well of 4.5 M sulfuric acid. 550nm
The wells with higher absorbance compared to the control plate were selected. Monocloning was performed by the limiting dilution method. That is, a 96-well microculture plates thymocytes from BALB / C mice were injected 10 ⁶ cells / 200 [mu] L increments min per well as feeder cells were diluted hybridoma specific antibodies in positive wells so as to be 10 cells / mL The solution was dispensed at 100 μL. The medium was cultured for 10 days in a 5% carbon dioxide incubator at 37 ° C. using an HT medium for the first time and an RPMI 1640 medium containing 15% FCS for the second and subsequent times. Selection of specific antibody-positive wells by ELISA and monoclonal cloning by limiting dilution were repeated three times each to obtain anti-HSA monoclonal antibody-producing cells.

(D) Separation and Purification of Anti-HSA Monoclonal Antibody The anti-HSA monoclonal antibody-producing cells obtained by the method described in the preceding section were cultured in the mouse intraperitoneal cavity to produce a monoclonal antibody. As a pretreatment, 0.5 mL of pristane (2, 6, 10, 14) was intraperitoneally injected into an 8-week-old BALB / C mouse.
-Tetramethylpentadecane). Eight days later,
Cells 4-suspended in 0.5 mL of RPMI 1640 medium
15 × 10 ⁵ mice were intraperitoneally administered. From day 9 after administration, ascites was repeatedly collected and pooled. The collected ascites was centrifuged at 3,000 rpm for 10 minutes to remove insolubles such as cells. An equal amount of a saturated ammonium sulfate solution was added to the supernatant with stirring, and the mixture was allowed to stand overnight at 4 ° C., and the resulting precipitate was collected by centrifugation. 2 precipitates
It was dissolved in 0 mM Tris-HCl buffer (pH 8.0) and dialyzed. DEAE-Sephac equilibrated with the same buffer
After adsorbing the dialysis contents to the el column, the column was eluted with a NaCl concentration gradient of 0 to 0.3 M in the same buffer to obtain a purified antibody.

(3) Preparation of anti-HSA monoclonal antibody solution: The HSA monoclonal antibody (mouse IgG) produced in (2) was diluted with purified water to a concentration of 1 mg / mL. Then, so that the antibody concentration is 60 μg / mL,
1.5% polyethylene glycol 20000, 0.1
The mixture was diluted with an aqueous solution containing% BSA to obtain an anti-HSA monoclonal antibody solution.

(4) Sensitivity measuring method: Hitachi 717 of general-purpose type
The sensitivity was measured using a type 0 automatic analyzer. Specifically, the anti-HSA monoclonal antibody solution prepared in (3) was used as the first reagent.
After adding 5 μL of a sample solution containing 100 or 250 μg / mL and heating at 37 ° C. for 5 minutes, 100 μL of the HSA-immobilized carrier particle-protein A solution prepared in (1) as a second reagent
Was added and stirred. Main wavelength 340nm for 1-5 minutes thereafter
The change in absorbance at a subwavelength of 800 nm was measured as sensitivity.

(5) Measurement Results: FIG. 1 shows the change in sensitivity when measuring the HSA-containing sample, taking the sensitivity when using physiological saline as the sample as zero.

Comparative Example 1 (1) Preparation of HSA-Immobilized Carrier Particle Solution: Protein A
Was added in the same manner as in Example 1- (1) except that H was not added.
An SA-immobilized carrier particle solution was obtained.

(2) Sensitivity measuring method: The procedure was the same as in Example 1 except that the second reagent was changed to the HSA-immobilized carrier particle solution prepared in (1).

(3) Measurement result: FIG. 1 shows the change in sensitivity when the HSA-containing sample was measured, with the sensitivity when physiological saline was used as the sample as zero.

As shown in FIG. 1, in Example 1 where protein A was used as a receptor, the HSA concentration was 250
Sufficient sensitivity change depending on the concentration of HSA was observed up to μg / mL. On the other hand, in Comparative Example 1 in which no receptor was used, the change in sensitivity due to the measurement of the HSA-containing sample was small and the HS
It reached a plateau at an A concentration of 100 μg / mL.

Example 2 (1) Preparation of HSA-immobilized carrier particle solution: polystyrene latex (average particle size: 0.1 μm, manufactured by Sekisui Chemical Co., Ltd.)
In 200 μL, add 800 μL of 20 mM MES buffer (pH
After adding 6.5) and diluting, 1.0 mL of a solution of HSA (manufactured by Sigma) at a concentration of 3 mg / mL in the above MES buffer was added, and the mixture was reacted at 4 ° C. for 2 hours. Then
2.0 mL of a 2 mg / mL BSA aqueous solution was added, and reacted at 4 ° C. for 1 hour. An aqueous solution 116 containing 2% polyethylene glycol 20,000 and 0.1% BSA was added to this solution.
mL was added and stirred to obtain an HSA-immobilized carrier particle solution.

(2) Production of anti-mouse IgG rat monoclonal antibody: (a) Immunization: For immunization of rats, 100 μg of commercially available normal mouse IgG was used each time. The initial immunization used Freund's complete adjuvant, and the booster immunization used Freund's incomplete adjuvant. Specifically, 100 μL of commercially available normal mouse IgG and 100 μL of Freund's adjuvant were mixed, and the resulting emulsion 200 μL
Was injected intraperitoneally into one SD male rat per immunization, and the immunization was repeated four times at two week intervals. Blood was collected from the tail vein of the rat, and the antibody titer was measured by ELISA.
Rats with high antibody titers were selected and used for cell fusion.

(B) Cell fusion; two weeks after the fourth immunization, normal mouse I diluted in 200 μL of physiological saline
100 g of gG was injected into the abdominal cavity of the rat, and three days later, the spleen was removed from the rat. Removed spleen was RPMI 16
Tweezers and a polished portion of a slide glass were thoroughly loosened in 40 medium, and spleen cells were collected. This is 1500 rp
centrifuge for 5 minutes to collect spleen cells, wash with the same medium,
Centrifuge. Finally, 2 mL of the same medium containing 15% FCS was added to prepare a spleen cell suspension. The number of living spleen cells was counted under a fluorescence microscope by mixing a suspension of acridine orange / ethidium bromide (0.1 mg each in 1 mL of PBS) and the suspension at a ratio of 1: 1. 1 After mixing live splenocytes ⁸ in advance mouse myeloma cells (myeloma cells) in the logarithmic growth phase which had been cultured SP2 / O-Ag14 of 10 ⁷ to
Centrifuged at 500 rpm for 5 minutes. After removing the supernatant and dissolving the cells well, a GKN solution (NaCl: 8 g, KC
l: 0.4 g, glucose: 2 g, Na ₂ HPO ₄ : 1.
41 g, NaH ₂ PO ₄ .2H ₂ O: 0.78 g was dissolved in 1 L of purified water) and centrifuged at 1500 rpm for 5 minutes to wash the cells. After repeating the washing,
0.5 mL of a GKN solution containing 50% (w / v) polyethylene glycol 1540 was gradually added, followed by gentle stirring for 1 minute. The reaction was stopped by gradually and gently adding 10 mL of a GKN solution, and the mixture was centrifuged at 1500 rpm for 5 minutes. The obtained cells were transformed into 30 ml of RPMI 1640 medium containing 15% FCS.
HAT medium (10 ^-4 M hypoxanthine, 4.0 × 10 ^-7 M aminopterin, 1.5 × 10 ^-5 M thymidine and RPMI 1640 medium containing 15% FCS) suspended in L and containing 10 ⁶ feeder cells ) To 20 per well in advance
100 μL per well was dispensed into three 96-well microculture plates into which 0 μL was dispensed, and cultured in a 5% CO 2 incubator at 37 ° C. After 10 days, the growth of the fused cells was confirmed in all wells.

(C) Selection and cloning of anti-mouse IgG antibody-producing cells; The presence of anti-mouse IgG antibody in the culture supernatant was confirmed by ELISA. That is, mouse IgG
(2 μg / mL) was screened using a 96-well microplate immobilized thereon. Specifically, normal mouse IgG was prepared using PBS containing 0.72% NaCl.
The solution was diluted to 2 µg / mL with 7.4, dispensed into a 96-well microplate at a rate of 50 µL / well, and left overnight at 4 ° C. These were washed three times with PBS pH 7.4 containing 1% BSA and 0.05% Tween 20 (BSA-PBS). Add 50 μL of culture supernatant to each well of the plate,
It was kept at 37 ° C. for 1 hour. Then, after washing three times with PBS,
50 μL of a peroxidase-labeled anti-rat IgG antibody (specific to the Fc site, derived from goat: manufactured by Biosource) diluted 1000-fold with BSA-PBS was added, and the mixture was incubated at 37 ° C. for 1 hour. This was washed 5 times with PBS, 50 μL / well of a citrate-phosphate buffer pH 5.0 containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was reacted at room temperature for 30 minutes. 50 μL of 4.5 M sulfuric acid
The reaction was stopped by adding wells. Measured at a wavelength of 550 nm, wells with higher absorbance compared to control plates were selected. Monocloning was performed by the limiting dilution method. That is, thymocytes of BALB / C mice were dispensed as feeder cells at 10 ⁶ cells / 200 μL per well.
Hybridomas in the specific antibody-positive well diluted to 10 cells / mL were dispensed into a 6-well microculture plate in 100 μL portions. The culture medium is initially HT
The medium was changed to RPMI 1 containing 15% FCS after the second time.
640 medium in a 5% CO 2 incubator at 37 ° C.
Cultured for days. Selection of specific antibody-positive wells by ELISA and monoclonal cloning by limiting dilution were repeated three times each to obtain anti-mouse IgG monoclonal antibody-producing cells.

(D) Isolation and purification of anti-mouse IgG monoclonal antibody; anti-mouse IgG obtained by the method described in the preceding section.
G monoclonal antibody producing cells were cultured in nude rats intraperitoneally to produce monoclonal antibodies. As pretreatment, 2.5 mL of pristane (2,6,10,14-tetramethylpentadecane) was administered intraperitoneally to 4-week-old nude rats (F344 / njcl-rnu). 10 days later, 1.0
1 × 10 ⁶ cells suspended in mL of RPMI 1640 medium were administered intraperitoneally to the nude rat. From day 9 after administration, ascites was repeatedly collected and pooled. The collected ascites was centrifuged at 3,000 rpm for 10 minutes to remove insolubles such as cells. An equal amount of a saturated ammonium sulfate solution was added to the supernatant with stirring, and the mixture was allowed to stand overnight at 4 ° C., and the resulting precipitate was collected by centrifugation. Precipitate 20 mM T
It was dissolved in a ris-HCl buffer (pH 8.0) and dialyzed. DEAE-Sephacel equilibrated with the same buffer
After adsorbing the dialysis contents to the column, Na
Elution was performed with a concentration gradient of Cl 0 to 0.3 M to obtain a purified antibody.

(3) Preparation of Anti-HSA Monoclonal Antibody-Anti-Mouse IgG Monoclonal Antibody Mixed Solution: The anti-HSA monoclonal antibody (mouse IgG) produced in Example 1- (3) was purified to a concentration of 1 mg / mL. Diluted with water. Next, the anti-mouse IgG rat monoclonal antibody prepared in (2) was diluted with purified water to a concentration of 0.5 mg / mL, and the two were mixed at a liquid volume of 1: 1. Thereafter, 0.1% BSA and 2% polyethylene glycol 20000 were contained so that the mixed antibody concentration became 45 μg / mL (anti-HSA monoclonal antibody concentration 30 μg / mL, anti-mouse IgG rat monoclonal antibody concentration 15 μg / mL). Diluted with aqueous solution of
An anti-mouse IgG rat monoclonal antibody mixed solution was obtained.

(4) Sensitivity measurement method: General-purpose Hitachi 717
The sensitivity was measured using a type 0 automatic analyzer. Specifically, the mixed solution of the anti-HSA monoclonal antibody and the anti-mouse IgG monoclonal antibody prepared in (3) was used as the first reagent, and 100 μL of the mixed solution was 5 μL of a sample solution containing HSA.
After heating at 37 ° C. for 5 minutes, (1) was used as the second reagent.
100 μL of the HSA-immobilized carrier particle solution prepared in the above was added and stirred. Thereafter, a change in absorbance at a main wavelength of 340 nm and a sub-wavelength of 800 nm for 1 to 5 minutes was measured as sensitivity.

(5) Measurement result: HSA concentration (0, 10,
Calibration curves were prepared from the sensitivities of the standard samples (25, 50, 100, and 250 μg / mL), and are shown in FIG. Using this calibration curve, the albumin concentration of a urine sample containing a known concentration of human albun was measured. The results are shown in Table 1.

[0046]

[Table 1]

Comparative Example 2 (1) Preparation of Anti-HSA Monoclonal Antibody Solution: The anti-HSA monoclonal antibody (mouse IgG) produced in Example 1- (2) was diluted with purified water to a concentration of 1 mg / mL. . Then, so that the antibody concentration is 30 μg / mL,
2% polyethylene glycol 20000, 0.1% B
After dilution with an aqueous solution containing SA, an anti-HSA monoclonal antibody solution was obtained.

(2) Sensitivity measurement method: The same procedure as in Example 2 was carried out except that the first reagent was changed to the HSA monoclonal antibody solution prepared in (1).

(3) Measurement results: HSA concentration (0, 10,
FIG. 2 shows the sensitivities when the standard products (25, 50, 100, and 250 μg / mL) were measured.

Comparative Example 3 (1) Preparation of Anti-HSA Monoclonal Antibody-RF Mixed Solution: Purified water of the anti-HSA monoclonal antibody (mouse IgG) produced in Example 1- (2) to a concentration of 1 mg / mL. Diluted. Then, the RF concentration in the final solution was 1 I
Diluted with an aqueous solution containing 2% polyethylene glycol 20,000 and 0.1% BSA to which human rheumatoid patient serum (manufactured by Intergen) was added so as to be U / mL so that the antibody concentration was 30 μg / mL. Then, an anti-HSA monoclonal antibody-RF mixed solution was obtained.

(2) Sensitivity measurement method: The same procedure as in Example 2 was carried out except that the first reagent was changed to the mixed solution of anti-HSA monoclonal antibody and RF prepared in (1).

(3) Measurement results: HSA concentration (0, 10,
FIG. 2 shows the sensitivities when the standard products (25, 50, 100, and 250 μg / mL) were measured.

As shown in FIG. 2, in Example 2, a good calibration curve was obtained due to a high sensitivity and a change in sensitivity depending on the HSA concentration. Human albumin could be measured. On the other hand, in Comparative Example 2 in which no receptor was used, a calibration curve could not be obtained because the sensitivity and the change in sensitivity were small. In Comparative Example 3 using RF as the aggregating agent, sensitivity was obtained, but no change in sensitivity depending on the HSA concentration was observed, and no calibration curve was obtained.

[0054]

According to the present invention, an antigen in a test sample can be specifically, simply and rapidly measured using an immunological agglutination inhibition reaction.

[Brief description of the drawings]

FIG. 1 is a diagram showing a change in sensitivity when an HSA-containing sample is measured in Example 1 and Comparative Example 1.

FIG. 2 is a diagram showing the sensitivity when measuring an HSA-containing sample in Example 1, Comparative Examples 2 and 3.

Claims (6)

[Claims] 1. A sample containing an antigen to be measured, and (A)
Insoluble carrier particles on which the antigen is immobilized, (B) a specific antibody in a free state that reacts with the antigen, and (C) a receptor having a site other than the binding site of (A) in the specific antibody as a ligand, An immunoassay method comprising optically measuring an immunological agglutination inhibition reaction caused by a free antigen to be measured in a sample. 2. The method according to claim 1, wherein the specific antibody (B) is a monoclonal antibody. 3. The method according to claim 1, wherein the specific antibody (B) is a polyclonal antibody. 4. The immunoassay according to claim 1, wherein the receptor (C) does not aggregate by reacting with the ligand. 5. An insoluble carrier particle having an average particle size of 1.6 μm
The immunological measurement method according to any one of claims 1 to 4, which is as follows. 6. A reagent used in combination with a sample for measuring an immunological agglutination inhibition reaction caused by a free measurement target antigen in the sample, wherein (A) an insoluble carrier particle on which the antigen is immobilized, (B) a free-form specific antibody that reacts with the antigen, and (C) an immunoassay reagent comprising a receptor whose ligand is a site other than the binding site to (A) in the specific antibody. .