JPS61117456A - Reagent for diagnosing iga nephrosis - Google Patents

Abstract

PURPOSE:To make possible the diagnosis of the IgA nephrosis with high sensitivity by measuring an anti-M-alpha2MIgA antibody. CONSTITUTION:The anti-M-alpha2M, more particularly anti-M-alpha2MIgA antibody existing in a specimen by the M-alpha2M constituting a reagent is detected by an antigen-antibody reaction, by which the IgA nephrosis is diagnosed. The M-alpha2M refers to each one unit of the four sub-units constituting alpha2- macroglobulin. More specifically, the alpha2-macroglobulin is glycoprotein having 725,000mol.wt. and two pieces each of the four sub-units constitute a dimer crosslinked byan S-S bond. The two dimers are further connected by a non- covalent bond and the M-alpha2M is each one unit of such four sub-units and the mol.wt. thereof is about 185,000. There is a carrier, for example, synthetic resin tube which can carry the M-alpha2M as the carrier. The M-alpha2M in the reagent is thus capable of diagnosing the IgA nephrosis with high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a reagent for diagnosing IgA nephropathy that is simple in procedure, has good sensitivity, and has excellent industrial productivity.

[Prior art]

Most cases of chronic nephritis are IgA nephropathy, and if left untreated, some will progress to uremia, so it is necessary to detect 1gA nephropathy early and take countermeasures. However,
Currently, diagnosis of 1gA nephropathy is made by renal biopsy, which is complicated and causes pain to the patient. Therefore, there is a long-awaited need for a method or reagent for diagnosing IgA syndrome that is simple and painless for patients.

[Problem that the invention seeks to solve]

Therefore, the purpose of the present invention is to easily [gA
The purpose of the present invention is to provide an IgA nephropathy diagnostic reagent that is capable of diagnosing nephropathy and is easy to manufacture.

[Means for solving problems]

In order to achieve the above object, the present inventors have conducted intensive research and found that the α2-macroglobulin subunit (M-72
It was discovered that the antigen M) is an antigen that induces IgA nephropathy. In other words, the onset mechanism of IgAl disease is
A specific antibody mainly composed of IgA is produced against an unknown antigen, and a primary immune complex is formed. Then, a specific antibody characterized by IgG is produced as a rheumatoid factor against this primary immune complex, and a secondary immune complex is produced. Immune complexes are formed, and these immune complexes are deposited mainly in the mesangium and partially in the peripheral snare wall, forming various glomerular lesions.

Under these circumstances, the inventors of the present invention have conducted various studies and have found that
It was found that at least M-α2M was involved in this unknown antigen. Therefore, IgA
Anti-M-α2M antibodies present in the serum of nephropathy patients, especially anti-M
It has been found that IgA nephropathy can be diagnosed by measuring -α2 MIgA antibodies.

The present invention was completed based on such new findings, and relates to a reagent for diagnosing IgA nephropathy containing M-α2M.

The measurement reagent of the present invention can be used to improve the anti-M-α2M antibodies present in the sample due to the M-α2M constituting the reagent, particularly anti-M-α2M.
Detecting the α2MIgA antibody by an antigen-antibody reaction,
This is used to diagnose IgA nephropathy.

M-cr2M is R, P, Swenson et
Al, The Jour-nal of Bio
logical (:hemisLry, Vol.
, 254+ No,], 1+4452~4456 (
It refers to each of the four subunits constituting α2-macroglobulin disclosed in ``78).

That is, α2-macroglobulin has a molecule fi of 72.5
10,000 glycoproteins, two of each of the four subunits are S
- constitutes a dimer cross-linked with 3 bonds, and furthermore, these 2
M-α2M is composed of two dimers connected by non-coexisting bonds, and M-α2M is one unit of each of these four subunits, and its molecular weight is approximately 185,000.

M-α2M can be prepared from α2-macroglobulin by methods known per se, such as J. M. Jones et al.
8io-che+m, J, Vol, 127.18
7-197 ('72) or a method similar thereto. Also, M-α2M
is a pH of 0.1M to 2M from α2-macroglobulin.
3.5 to 2.5 glinone buffer (preferably 4
℃ for 16 hours), and a fraction with a molecular weight of about 185,000 is collected by gel filtration.

α? - Macroglobulin can be purified by a method known per se, for example, the method described in J.M. Jones et al., or a method analogous thereto. In addition, α2-macroglobulin can be obtained from α2-macroglobulin-containing substances such as human serum and human placenta (
Placenta, etc., is subjected to ammonium sulfate fractionation (for example, a combination of fractionation with 30% saturated ammonium sulfate followed by 50% saturated ammonium sulfate, etc.), and then fractionated using a DEAE column. It can be obtained by subjecting it to affinity chromatography using an anti-α2-macroglobulin antibody as a ligand to obtain purified α2-macroglobulin, and then separating the fraction with a molecular weight of approximately 725,000 by gel filtration. can.

The diagnostic reagent of the present invention can take the form of any of the conventionally known serological reagents based on antigen-antibody reactions, for example, reagents for precipitation reactions, reagents for agglutination reactions, etc. A particularly preferred embodiment is M-α
This is an embodiment of a reagent for measuring anti-M-α2M antibodies by binding anti-M-α2M antibodies in a specimen to a carrier bound to 2M.

As the carrier, any carrier can be suitably used as long as it can support M-α2M and achieve the object of the present invention. Specifically, examples include synthetic resin tubes, synthetic resin particles, kaolin, bentonite, latex, and red blood cells.

When binding M-α2M to a carrier, it may be carried out according to a known method.

Next, the measuring method according to the present invention was applied to a synthetic resin tube.
-A reagent to which α2M is bound will be explained as an example.

The reagent is preferably prepared as a kit, and its components are, for example, as follows.

■M-α2M adsorption tube ■Enzyme-labeled anti-1gA ■Enzyme coloring substrate The above M-α2H4% & adsorption tube is prepared by known means! ! ! can be sent. For example, M-α2M is diluted with a buffer solution (e.g., about pl+9, preferably pnq, z carbonate buffer), placed in a tube and left for an appropriate period of time, then the diluted solution is removed and the solution is added to an appropriate washing solution. Prepared by washing. Note that the unadsorbed portion is, for example, 1% human albumin at a pH of about 9 (preferably ρl(9)).
, 2) is preferable.

Examples of the enzyme for the enzyme-labeled anti-1gA antibody include peroxidase, alkaline phosphatase, and β-D galactosodase.

The enzyme-labeled anti-IgA antibody can be prepared by diluting the enzyme-labeled anti-IgA antibody (for example, 10%
00 times dilution).

As the enzyme coloring substrate, for example, those corresponding to the enzyme of the enzyme-labeled anti-IgA antibody are used. For example, for peroxidase, ortho-phenylene diamine, para-hydroxyphenylene acetic acid 1,5-aminosa+/thylic acid are used. Specifically, add O-phenylenediamine to 50a+g/d1 in a 0.1M citric acid solution (pH 4°5), and add hydrogen peroxide to a concentration of 0.1% just before use. Examples include:
Also, for other enzymes, corresponding known enzyme coloring substrates can be used.

The outline of the measurement method using the above kit is as follows.

Add a phosphate buffer solution (e.g. 1
% human albumin-containing phosphate buffer) (e.g., 20-fold dilution, 40-fold dilution), and incubate at an appropriate temperature (e.g., 4 to 8°C) for an appropriate time. Leave to stand (for example, 4 hours). After washing this with a phosphate buffer etc., a peroxodase-labeled anti-1gA antibody is added and the mixture is incubated at an appropriate temperature (e.g. 37°C) for an appropriate time (e.g. 3
0 minutes) Leave it alone. After washing this with a phosphate buffer or the like, an enzyme coloring substrate is added, and the OD is measured at a specific wavelength.

[Action/Effect]

M-tr 2 in the reagent for diagnosing IgA nephropathy of the present invention
M easily reacts with anti-M-α2M antibodies, especially anti-M-α2M+gA antibodies, which are present in the serum of IgA nephropathy patients in larger amounts than in the serum of normal subjects or other nephropathy patients, by an antigen-antibody reaction. The reagent for diagnosing IgA nephropathy of the present invention can be used to diagnose IgA nephropathy with high sensitivity. Also, M-α2M
Since it is easy to manufacture, the reagent for diagnosing IgA nephropathy of the present invention has excellent industrial productivity.

[Example/Experimental Example/Reference Example j Example I M-α2M was adjusted to 20 μg/ml with 0.1 M carbonate buffer at pH 9.2, and 0.5 ml of it was added to a polystyrene dipstick (Eiken tube No. 1 (manufactured by Eiken Kagaku Co., Ltd.) and left at 37°C for 1 hour, then at 4°C overnight. This is washed with 0.1 M carbonate buffer at pH 9.2. pH 9.2 with 1% human albumin added separately
Prepare a 0.1M carbonate buffer, put it into the above tube, leave it at room temperature for 2 hours,
After washing with M phosphate buffered saline, a reagent consisting of an M-α2M adsorbed polystyrene tube was obtained.

Experimental Example 1 To 740.025 ml of sample blood (add EDTAo, 05 ml and allow to stand at 37°C for 30 minutes), 1% human albumin 11 was added to 0.05 M phosphate buffered physiological solution with ρ++ 7.2. Add 1+++I of saline solution. This was added to the reagent obtained in Example 1 (M-α2M adsorbed polystyrene tube) and left at 37°C for 1 hour and then at 4°C overnight.

This was washed with 0.05M phosphate 1M &i physiological saline, pH 7.2, and the 6-berooquinodase-labeled anti-IgA
(manufactured by DAKOPATTS) in 0.05M phosphate buffered saline containing 1% human alphamin.
Add 0.5 ml of the 0x diluted solution, leave at 37°C for 1 hour, and wash with 0.05 M phosphate buffered saline, pH 7.2. Add 0.5 ml of substrate (500 μg/l 0-phenylenediamine + 0.1% hydrogen peroxide) and
Leave at 7°C for 30 minutes. INHCj! Add 1ml 4
The OD was measured at 92 nm and the results are shown in FIG.

In FIG. 1, A indicates IgA nephropathy patient blood l#, B indicates direct serum from patients with other kidney diseases, and C indicates normal human serum.

From the results shown in Figure 1, in the direct serum of patients with IgA nephropathy,
It was found that specific 1gA antibodies against M-α2M were present at a clearly higher rate than in serum from patients with other kidney diseases and serum from normal individuals, and it can be understood that the reagent of the present invention is superior. .

Reference example 1 Placenta was cut into small pieces, suspended in physiological saline, and homogenized (4℃
, 10,000 r, p, m, 20 min) and further centrifuged at 4°C, 10,000 r, p, m, 30 min, and acrinol was added to the supernatant to reduce the ntl1 degree to 0.28.
%. After stirring for 30 minutes at room temperature and leaving it for 1 hour,
Centrifugation (15℃, 3.00Or, p, m, 10 minutes)
do. Discard the supernatant, collect the precipitate, and add 5% Na to the precipitate.
Add C1, stir for 30 minutes, centrifuge # (15℃, 3,
000r, p, m, 10 minutes) and discard the precipitate.
Obtain the supernatant. Add ammonium sulfate to the top to make it 30% saturated ammonium sulfate,
Stir at room temperature for 2 hours. Centrifugation (4°C, s, oo.

Ammonium sulfate was further added to the supernatant to make it 50% saturated ammonium sulfate, and the mixture was stirred at room temperature for 2 hours.

Centrifugation (4℃, 8,000 r, 2.m, 20 minutes)
After solubilizing the precipitate with physiological saline, it was compressed to tM and concentrated (
After approximately 1 g/etl), thoroughly dialyzed with 0.04 M, pH 7,0 phosphate buffer (4°C), and developed on a DEAE column (3 cm x 20 cm):
Solution (0.04M, pH 7.0'J 7 acid buffer e)
After near-sufficient equilibration, fill the column and sample 3
ml, adsorb, and SL until unadsorbed protein is removed.
arting PFlow the heavy liquid.

■ Startingff solution + 0.04M NaC
1 is played.

(α2 macroglobulin-free) ■ Startingu solution + 0.16 M NaC
1 is played.

(Contains α2 macroglobulin) After concentrating the fraction (2), it was dialyzed against pH 7, 0, 0,01M phosphate buffer, and the crude fraction was obtained (approximately 0.8 g/d1 of protein
).

CNBr-activated Sepharose 4B gel +5+*1,
After adsorbing the T-globulin fraction 100+lIg of anti-human α2 macroglobulin rabbit serum, O, OIM, 1)) 1
7.4 phosphate buffered saline (ino, 02% Na
N5). Pack the anti-α2M adsorbed Sepharose 4B gel into a column of L (J Dispense 1 volume of the adsorbed protein using Green's buffer. After concentrating the eluted protein, adjust the pH to 7.
, 4 into 0.01 MIJ salt buffered saline (protein temperature approximately 500+*g/a7). Sepharose CL
Apply concentrated protein 2 field 1 to -6B column (lclIX80clI) and perform gel filtration. At this time, a 0.01 M, pH 7.4 phosphate buffer was used as the buffer. Collect and concentrate α2M peak (protein temperature approx. 500 mg
/d1) I did. By acrylamide electrophoresis, a Hayabusa band was observed at a molecular weight of approximately 725,000, and a single sedimentation line was obtained by the ophthalony method with an anti-α2-macroglobulin antibody. It was confirmed that α2-macroglobulin was purified.

Purified α2M was added to pH 3, 3, 1M glinone buffer (inQ
, dialysis against 9% NaCl (overnight at 4°C).

The above acid-treated α2M was applied to Sepharose CL-6B (lclIIX80 (J), and gel filtration was performed (
At that time, Ool M, pH 3,3 glycinate buffer in Na (J) was used as a buffer, and the molecular weight was approximately 1.
Collected 85,000 peaks, /81i! (Protein concentration 200
Cod／〃) した. A band was observed at a molecular weight of about 185,000 by gradient polyacrylamide electrophoresis, and a single dc dropout line was obtained by off-clony method using an anti-α2-macroglobulin antibody.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the effects of the reagent of the present invention. In the figure, A shows serum from patients with IgA nephropathy, B shows serum from patients with other kidney diseases, and C shows serum from normal people.

Claims (1)

[Claims] A reagent for diagnosing IgA nephropathy, comprising an α_2-macroglobulin subunit constituting α_2-macroglobulin.