JP2000241431A - Immunological measuring reagent and measuring method for immunoglobulin a-fibronectin complex - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent for measuring an IgA-fibronectin complex in body fluid accurately and immunologically using an anti-fibronectin antibody and an anti-IgA antibody. SOLUTION: A measuring reagent for immunoglobulin A-fibronectin complex includes solid phase carriers conjugating an antihuman-fibronectine antibody with an antibody conjugating amount from 150 to 500 ng/cm2 and an antihuman- immunoglobulin A antibody. A method for measuring the immunoglobulin A- fibronectin complex uses this measuring reagent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunoglobulin A.
The present invention relates to a reagent for immunoassay of fibronectin complex, an immunoassay, and a method for diagnosing IgA nephropathy patients using the same. More specifically, the present invention provides an immunoglobulin A-fibronectin complex immunoassay reagent, an immunoassay method, and a method for immobilizing an anti-human-fibronectin antibody. The present invention relates to an immunological diagnosis method for IgA nephropathy patients to be used. Hereinafter, immunoglobulin A is referred to as IgA.

[0002]

2. Description of the Related Art IgA nephropathy occurs in about 30 patients with chronic nephritis.
It is said that about 30% of patients with IgA nephropathy will go to dialysis. The criteria for diagnosing this renal disease were set forth in 1995 by the "Research Group for the Study of Specific Disease Progressive Renal Disorders and the Joint Committee of the Japanese Society of Nephrology". The criteria are based on urinalysis (continuous microscopic hematuria, continuous or intermittent proteinuria, gross hematuria) and blood tests (serum IgA level of 350 mg / dL or more). Observing the glomerulus is the only way.

However, Cederholm et al. Reported that the “IgA-fibronectin complex” was specifically present in the body fluid of IgA nephropathy patients (Proc.
Natl. Acad. Sci. , Vol. 8
5, pp. 4865-4868, 1988), and based on this finding, Cederholm et al. Immunologically measured the IgA-fibronectin complex utilizing the affinity of the complex and determined the IgA kidney disease based on the results. Diagnostic kit for diagnosing the disease (Japanese Patent No. 259212)
No. 1).

The method is based on the affinity between the fibronectin portion of the IgA-fibronectin complex and collagen, fibrin or heparin, or an anti-fibronectin antibody, or the affinity between the IgA portion of the complex and an anti-IgA antibody or an IgA binding lectin. It is a method of utilizing.

[0005] The publication discloses, as an embodiment of the invention, I
Using a test sample (antiserum) of a patient with gA nephropathy, a fraction that binds to collagen I and reacts with an anti-human-IgA antibody is applied to jacalin (lectin) -Sepharose to isolate the complex (Example). 2). Alternatively, human serum was run on heparin-sepharose and then incubated in denatured collagen-coated microtiter wells to bind bound I
The gA antibody is detected by a specific antibody conjugate (Example 8).

In these descriptions, a sandwich assay method for immobilizing an anti-fibronectin antibody on a solid phase and reacting the complex bound to the antibody with an anti-IgA antibody to know the presence of the complex is described in detail. There is no public disclosure.

[0007]

An object of the present invention is to provide a reagent for immunologically measuring an IgA-fibronectin complex in a body fluid using an anti-fibronectin antibody and an anti-IgA antibody. The present invention provides an immunoassay reagent that is high and has sufficiently high measurement sensitivity, and this reagent can assist in the diagnosis of IgA nephropathy.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the amount of immobilization of the anti-fibronectin antibody on the solid phase is within a specific range. The inventors have found that the measurement of the complex can be performed with high accuracy, and have reached the present invention. That is, the present invention is a reagent for measuring an immunoglobulin-fibronectin complex comprising an anti-human-immunoglobulin A antibody and a solid-phase carrier that binds an anti-human-fibronectin antibody having an antibody binding amount of 150 to 500 ng / cm ² . The present invention also provides a sample which is allowed to act on a solid support to which an anti-human-fibronectin antibody having an antibody binding amount of 150 to 500 ng / cm ² has been bound,
Then, a labeled immunoglobulin A antibody is reacted, and a label bound to the solid support or a label not bound to the solid support is measured.
This is a method for measuring a fibronectin complex.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The embodiments of the present invention will be described below, but they do not limit the scope of the present invention. In the present invention, an anti-human-fibronectin antibody means an antibody derived from a non-human animal that specifically reacts with human fibronectin. The manufacturing method is, for example,
An immunoglobulin produced by injecting human plasma-derived fibronectin as an immunogen into animals such as rabbits, goats and sheep is prepared by applying a means known per se for purifying immunoglobulins. More specifically, an anti-human-fibronectin antibody can be recovered by applying affinity chromatography utilizing immunological affinity with fibronectin.

The degree of purification of the anti-human-fibronectin antibody as an immunoglobulin and the degree of purification for the specificity of fibronectin refer to those which have been purified to such an extent that there is no problem in preparing a solid-phase carrier used for measurement. More simply, various commercially available anti-human-fibronectin antibodies can be used. For example, anti-human-fibronectin antibody (rabbit) manufactured by DAKO, anti-human-fibronectin antibody manufactured by BINDING SITE
An example is a fibronectin antibody (sheep).

In the present invention, immobilization means immobilizing an anti-human-fibronectin antibody by immobilizing it on an insoluble carrier (solid carrier). Examples of the carrier include microtiter plates, beads, test tubes, paper and the like made of synthetic resins, for example, polystyrene and nylon, and microtiter plates and beads made of glass. Particularly, a microtiter plate made of polystyrene is preferable. As means for binding the antibody to the solid phase carrier, means known per se can be applied depending on the properties of the solid phase carrier. In the present invention, a method utilizing a physical mode (physical adsorption) between a protein and a solid phase carrier was adopted as an optimal means.

For example, a 0.1 mol / L carbonate buffer (pH 9.
The antibody in 7) is immobilized on a solid support by a method of allowing the antibody in step 7 to stand or shake at a certain temperature for a certain time.

In the present invention, the amount of the antibody bound to the solid-phase carrier can be measured by means of immunoassay for the IgA-fibronectin complex while maintaining substantially high measurement sensitivity and immobilizing the antibody on the solid-phase carrier. This is an amount that substantially suppresses the nonspecific reaction of the portion, and the amount is determined based on the results of comparison of the amount of IgA-fibronectin complex in the serum of healthy humans and IgA nephropathy patients. Determined by the cutoff value established by the difference in absorbance. Specifically, it is determined by applying the means described in the embodiment,
It is more convenient.

Further, the amount of the anti-human-fibronectin antibody immobilized on the solid support is determined by the initial peak of the amount of the antibody that can be immobilized, which is achieved by the selected solid-phase means for the antibody and the solid support. The amount can be determined on the basis of the amount and in consideration of the economics of the amount of the immobilized antibody.

The immobilized amount of the anti-human-fibronectin antibody specified by the means disclosed in Examples of the present invention is as follows:
About 150 about 500 ng / cm ^2, more preferably from about 200 to about 460 ng / cm ^2. When the binding amount is less than about 150 ng / cm ² , not only the measurement sensitivity is low, but also the non-specific reaction increases due to the blocking state of the unbound portion, and it becomes difficult to detect the specific reaction. Conversely, when the amount of antibody binding exceeds about 500 ng / cm ² , nonspecific reactions begin to be noticeable irrespective of blocking, and the measured value becomes high even in samples other than patients with the target disease. Therefore, there is a high possibility that the discrimination of patients with the target disease is adversely affected. In addition, when the amount of the anti-human-fibronectin antibody used for immobilizing the antibody is extremely large, the raw material cost in the production cost becomes too high.

Thus, the specified values are the ones that give the best results in the means exemplified in the embodiments, but also the values that bring about substantially the same effects as appropriate by changing the means and conditions. Can be selected.

In the present invention, an anti-human-IgA antibody means an antibody derived from a non-human animal that reacts with human IgA. For example, it can be obtained by highly purifying immunoglobulins produced by administering to humans such as rabbits, goats and sheep using IgA derived from human serum as an immunogen, by a purification method known per se. In the present invention, the anti-human
The IgA antibody is used as a secondary antibody (labeled antibody), and is used for contacting a test sample with an immobilized anti-human-fibronectin antibody.
After the reaction, it is subjected to a secondary reaction with the trapped (trapped) IgA-fibronectin complex.

As the label, an enzyme, a fluorescent substance, a radioisotope, a luminescent substance and the like are used. Enzymes include alkaline phosphatase, peroxidase, β-D-
Galactosidase and the like are used. Labeling of the anti-human-IgA antibody is performed by a known method.

The immunoglobulin A-fibronectin complex measurement reagent of the present invention contains a carrier on which the above-mentioned anti-fibronectin antibody is immobilized and a labeled anti-IgA antibody, and further contains a buffer, a washing solution and the like as necessary.

In the present invention, the antibody binding amount is 150 to 500 n.
The sample is allowed to act on a solid support to which an anti-human-fibronectin antibody of g / cm ² is bound, and then the labeled immunoglobulin A antibody is reacted, and the label is not bound to the solid support or bound to the solid support. This is a method for measuring an immunoglobulin A-fibronectin complex in which a label is measured. In the measurement method, the intensity of color, luminescence, fluorescence, or radiation is measured according to a standard method according to each label.

The measuring method in the enzyme immunoassay (EIA) system is as follows.
The detection is carried out by means known per se, and various detection methods such as a colorimetric method, a fluorescent method, a chemiluminescent method, and a bioluminescent method are selected by selecting various enzyme substrates. In the examples, alkaline phosphatase was selected as an enzyme for labeling a secondary antibody, and p-nitrophenyl phosphate (pNPP) was used as a substrate.
Was detected by a colorimetric method. That is, a method was employed in which a substrate was added, the enzyme adsorbed on the solid phase was allowed to react for a certain period of time, and the absorbance at 405 nm of the test solution that had developed color was measured. When peroxidase is selected as an enzyme for labeling the secondary antibody, o-phenylenediamine (OPD) or tetramethylbenzidine (TMB) is used as a substrate.
There is also a colorimetric method in which a color is added to add color. The detection method is selected according to the desired detection limit. When a lower detection limit is desired, a means such as a so-called chemiluminescence method is selected.

[0022]

【Example】

[Reference Example 1] Determination of the amount of solid-phase antibody An anti-human-fibronectin antibody (manufactured by DAKO) was diluted with a 0.1 mol / L carbonate buffer (pH 9.7).
An antibody solution having a concentration of 1.275 mg / mL to 1.594 μg / mL was prepared, dispensed into polystyrene microtiter plates (manufactured by Nunc, 96-wells, high-adsorption type) at 200 μL / well at 4 ° C. After standing for 18 hours or more, a 0.01 mol / L phosphate buffer (p
H7.4) three times. The amount of antibody adsorbed on the plate is determined using the BCA protein quantification kit (Pierce)
Quantification was performed using

The BCA reaction solution was dispensed at 300 μL / well, shaken at 37 ° C. for 1 hour, and cooled to room temperature.
Measure the absorbance at 550 nm using a microplate reader (Ty
pe-MTP-120; manufactured by Corona Co., Ltd.). The same operation was performed using only the carbonate buffer containing no antibody, and the absorbance of only the carbonate buffer was subtracted from the absorbance of the well on which the antibody was immobilized.

At the same time, a BCA
Bovine serum albumin standard solution (Pi
erce) in a microtiter plate
After dispensing, the BCA reaction solution was dispensed at 300 μL / well, and the mixture was shaken at 37 ° C. for 1 hour, and cooled to room temperature.
Measure the absorbance at 0 nm with a microplate reader (Type
-MTP-120; manufactured by Corona Co., Ltd.) and used as a calibration curve. FIG. 1 shows the results obtained by using this calibration curve to determine the amount of antibody bound to the plate.

From the results shown in FIG. 1, the amount of the solid phase antibody was 430 n
It peaks at g / cm ² and then remains constant.
However, when the amount of antibody added was further increased, the amount of solid phase antibody tended to gradually increase. Therefore, in order to stabilize the solid phase amount of the antibody, the amount of the antibody added was 10 μg / well.
It is preferable to make the above.

[0026]

Example 1 Relationship between antibody solid phase amount and patient specificity Anti-human-fibronectin antibody (BINDING SI
TE) (0.1 mol / L carbonate buffer (pH 9.
7) to prepare an antibody solution having a concentration of 1 mg / mL to 2.5 μg / mL, and dispensed 200 μL / well into a polystyrene microtiter plate (Nunc, 96-wells, high adsorption type). And at 4 ° C
After allowing to stand for at least 8 hours, the plate was washed three times with a 0.01 mol / L phosphate buffer (pH 7.4).

Bovine serum albumin (ORGANON TE)
KNIKA B. V. 0.01% containing 1%
/ L phosphate buffer (pH 7.4) with 300 μL / we
After dispensing for 1 hour at 25 ° C. for 2 hours, 0.01 mol /
Washed three times with L phosphate buffer (pH 7.4). Commercially available stabilizers (Stabil Coat, BSI cor)
300 μL / well was dispensed, left at 4 ° C. for 30 minutes, and the solution was removed and air-dried to obtain a microtiter plate for measurement. The plate was sealed and stored at 4 ° C.

As test samples, serum of a patient with IgA nephropathy and serum of a healthy subject were mixed with 0.01 mol / L phosphate buffer (pH
7.4), dilute 25-fold and add 200 μL / well (2
It was dispensed. After reaction at 25 ° C for 60 minutes, 0.05
% Tween-20, and washed three times with a 0.01 mol / L phosphate buffer (pH 7.4).
ol / L phosphate buffer (pH 7.4) diluted with alkaline phosphatase-labeled anti-human-IgA antibody (Sig
ma) (200 μL / well).

After reacting at 25 ° C. for 60 minutes, 0.05% Tw
After washing three times with a 0.01 mol / L phosphate buffer (pH 7.4) containing een-20, 1 mg / mL p-protein prepared with a 1 mol / L diethanolamine-hydrochloric acid buffer (pH 9.8). 200 μL / we using disodium nitrophenylphosphate (Sigma) as a coloring solution
11 and dispensed with a microplate reader (Type-MTP-120; manufactured by Corona Co.)
Was measured at 405 nm. The result is shown in FIG.

From the results shown in FIG. 2, it can be said that when the amount of the antibody added is small, the measurement sensitivity is low and the patient specificity is low. FIG.
Assuming that the cutoff of the measured value (△ OD) at 0.3 is 0.3, 150 ng / cm ²
The above amount of immobilized antibody was necessary. Therefore, in order to enhance patient specificity, the antibody binding amount should be about 150 to about 500 n.
A range of g / cm ² is suitable.

[0031]

Example 2 Anti-human-fibronectin antibody solid phase play
(FN) anti-human-fibronectin antibody (BINDING SI
TE) (manufactured by TE Co.) was diluted with a 0.1 mol / L carbonate buffer, and the microtiter plate (Nu
nc, 96-wells, high adsorption type)
Binding was performed at 4 ° C. for 18 hours or more at a density ranging from 0 to about 500 ng / cm ² , followed by washing three times with 0.01 mol / L phosphate buffer (pH 7.4).

Bovine serum albumin (ORGANON TE)
KNIKA B. V. 0.01% containing 1%
/ L phosphate buffer (pH 7.4) with 300 μL / we
After dispensing for 1 hour at 25 ° C. for 2 hours, 0.01 mol /
Washed three times with L phosphate buffer (pH 7.4). Commercially available stabilizers (Stabil Coat, BSI cor)
300 µL / well was dispensed, left at 4 ° C for 30 minutes, and the solution was removed and air-dried to obtain an anti-human-fibronectin antibody solid phase microtiter plate (FN) for measurement.

The test sample was a serum of an IgA nephropathy patient and a healthy person, and was used as a 0.01 mol / L phosphate buffer (pH
The solution diluted 25 times in 7.4) was added to each microtiter plate at 200 μL / well (duplicate measurement), reacted at 25 ° C. for 60 minutes, and then 0.05% T
Washing was performed three times with a 0.01 mol / L phosphate buffer (pH 7.4) containing ween-20, and 0.01 mol / L
200 μL / well of alkaline phosphatase-labeled anti-human-IgA antibody (manufactured by Sigma) diluted with L phosphate buffer (pH 7.4) was dispensed.

After reacting at 25 ° C. for 60 minutes, 0.05% Tw
After washing three times with a 0.01 mol / L phosphate buffer (pH 7.4) containing een-20, 1 mg / mL p-protein prepared with a 1 mol / L diethanolamine-hydrochloric acid buffer (pH 9.8). 200 μL / we using disodium nitrophenylphosphate (Sigma) as a coloring solution
11 and dispensed at 405 nm after 60 minutes at 25 ° C. using a microplate reader (Type-MTP-12).
0; manufactured by Corona Co., Ltd.) is shown in FIG.

As a result, the measured value of the IgA-fibronectin complex of the healthy subject is converged to a lower level, and the cutoff absorbance can be set lower.

[0036]

[Comparative Example] Denatured collagen solid-phase plate (CB) Similar to Cederholm et al., A study was conducted for comparison of binding of collagen modified with cyanogen bromide to a microtiter plate. Collagen (CB) treated with cyanogen bromide on a polystyrene microtiter plate (96-wells, high adsorption type, manufactured by Nunc)
6 + 7 fraction, manufactured by Wieslab AB).
The mixture was diluted with a 1 mol / L carbonate buffer (pH 9.7), left standing at 4 ° C. for 18 hours or more, and then washed three times with a 0.01 mol / L phosphate buffer (pH 7.4). Patent No. 2
As described in 592121, no blocking operation was performed. Commercially available stabilizer (Stabi
l Coat, manufactured by BSI corporation)
Was dispensed at 300 μL / well and left at 4 ° C. for 30 minutes.
The solution was removed and air-dried to obtain a denatured collagen solid-phased microtiter plate (CB) for measurement. The test samples used were the same serums of IgA nephropathy patients and healthy subjects as those used in Example 2, and were diluted 25-fold with 0.01 mol / L phosphate buffer (pH 7.4). After adding 200 μL / well (duplicate measurement) to the microtiter plate and reacting at 25 ° C. for 60 minutes, a 0.01 mol / L phosphate buffer (pH 7.4) containing 0.05% Tween-20 was added. Wash 3 times, 0.01mol
/ L alkaline phosphatase-labeled anti-human-IgA antibody (Sigma) diluted with 1 / L phosphate buffer (pH 7.4)
Co., Ltd.) was dispensed at 200 μL / well. 60 at 25 ° C
After the reaction, 0.0% containing Tween-20 of 0.05% was added.
After washing three times with 1 mol / L phosphate buffer (pH 7.4), 1 mg / mL disodium p-nitrophenyl phosphate (1 mol / L diethanolamine-hydrochloric acid buffer (pH 9.8)) was prepared. Sigma) was dispensed as a coloring solution at 200 μL / well,
The results obtained by measuring the absorbance at 405 nm after one minute using a microplate reader (Type-MTP-120; manufactured by Corona) are shown in FIG.

The results of this comparative example are based on the I
The measured values of the gA-fibronectin complex are widely dispersed, suggesting that there are many side reactions and nonspecific reactions unrelated to the disease. This is an unavoidable result due to the nature of collagen as an adhesion molecule, and confirmed that non-specific substances were not directly bound to the plate due to no blocking. As a result, when using a plate on which denatured collagen is immobilized, it is necessary to set the cutoff absorbance higher.

From the results shown in FIGS. 3 and 4, in order to specifically detect an IgA nephropathy patient with a low non-specific reaction and a low cut-off setting, an anti-human-fibronectin antibody was bound. Microtiter plate (F
It was found that the ELISA method using N) was superior to the method using denatured collagen (CB).

[0039]

According to the immunoassay for immunoglobulin A-fibronectin complex provided by the present invention, an anti-human antibody having an antibody binding amount of 150 to 500 ng / cm ² is used.
By allowing a sample to act on a solid phase carrier to which a fibronectin antibody is bound, the reaction specificity and the measurement sensitivity are kept high, and the diagnosis of IgA nephropathy can be assisted.

[Brief description of the drawings]

FIG. 1 Amount of anti-human-fibronectin antibody added when binding an antibody to a microtiter plate (horizontal axis)
5 is a graph showing the value (vertical axis) obtained by quantifying the amount of antibody bound to the plate surface by the BCA method and converting the amount to the amount bound per unit area.

FIG. 2A shows the amount of the anti-human-fibronectin antibody bound to the microtiter plate (horizontal axis), the serum of two patients with IgA nephropathy (IgA-N1, IgA-N2), the serum of two healthy subjects and the PBS buffer. FIG. 2B is a graph showing the results (vertical axis) of the IgA-fibronectin complex contained therein measured by an ELISA method, and B is an enlarged view of a part of FIG. 2A.

FIG. 3 is a graph showing the distribution of IgA-fibronectin complex measurement values on an anti-human-fibronectin antibody-immobilized plate (FN).

FIG. 4 is a diagram showing the distribution of measured values of IgA-fibronectin complex using a denatured collagen-immobilized plate (CB).

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[Procedure amendment]

[Submission date] November 4, 1999 (1999.11.
4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 1

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction contents]

As a result, the average measurement value of a healthy person was 0.24
1, the average measured value of IgA nephropathy patients is 1.595,
The ratio of the absorbance of both was 1: 6.618. Sand
In addition, the measured value of the IgA-fibronectin complex of a healthy human sample converges to a lower level, and the cutoff absorbance can be set lower.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0037

[Correction method] Change

[Correction contents]

As a result of this comparative example, the average measurement value of a healthy person was
0.612, average measurement for IgA nephropathy patients 1.736
And the ratio of the absorbance of both was 1: 2.838.
That is, the measured values of the IgA-fibronectin complex in the test sample of healthy subjects were widely dispersed, suggesting that there are many side reactions and nonspecific reactions unrelated to the disease. This is an unavoidable result due to the nature of collagen as an adhesion molecule, and confirmed that non-specific substances were not directly bound to the plate due to no blocking. As a result, when using a plate on which denatured collagen is immobilized, it is necessary to set the cutoff absorbance higher.

Claims (5)

[Claims] (1) an antibody binding amount of 150 to 500 ng / cm ^2;
A reagent for measuring an immunoglobulin-fibronectin complex comprising an anti-human-immunoglobulin A antibody and a solid-phase carrier that binds the anti-human-fibronectin antibody. 2. The solid support is a synthetic resin support.
The reagent for measuring the immunoglobulin A-fibronectin complex according to the above. 3. The immunoglobulin A according to claim 1, wherein the anti-human-fibronectin antibody is physically adsorbed on a solid support.
-Fibronectin complex measurement reagent. 4. The reagent for measuring an immunoglobulin A-fibronectin complex according to claim 1, wherein the anti-human-immunoglobulin A antibody is a labeled antibody. 5. An antibody binding amount of 150 to 500 ng / cm ^2.
The sample is allowed to act on the solid support to which the anti-human-fibronectin antibody is bound, and then the labeled immunoglobulin A antibody is reacted to measure the label bound to the solid support or the label not bound to the solid support. A method for measuring an immunoglobulin A-fibronectin complex.