JP2932837B2 - Method for measuring human podocalyxin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring human podocalyxin. The method of the present invention can be applied to the diagnosis of nephritis.

[0002]

2. Description of the Related Art Podocalyxin is a glycoprotein having a molecular weight of about 140,000 which is present in glomerular epithelial cells. Conventionally, as a method for detecting podocalyxin, a method by tissue staining using a polyclonal antibody obtained by immunizing goats or rabbits with podocalyxin is known.

[0003] However, in the conventional method, since cells and the like are stained, the fixing of tissues and preparation of sections are extremely complicated and require a lot of time, and it is difficult to quantify the amount of podocalyxin.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for measuring human podocalyxin which is simpler, requires less time, and can quantify human podocalyxin as compared with the conventional method. It is to be.

[0005]

According to the present invention, after reacting a sample with a first anti-human podocalyxin antibody bound to a solid phase, the first anti-human podocalyxin antibody reacts with a corresponding epitope. Reacting a second anti-human podocalyxin monoclonal antibody, and then measuring the second anti-human podocalyxin monoclonal antibody bound to a solid phase. Hereinafter, this may be referred to as a first invention).

Further, the present invention provides a method of reacting a first anti-human podocalyxin antibody bound to a solid phase with a specimen, and then reacting a lectin specific to a sugar chain of human podocalyxin,
A method for measuring human podocalyxin in a sample, comprising measuring the lectin bound to a solid phase (hereinafter referred to as a second method).
Of the present invention).

[0007] Furthermore, the present invention relates to a method comprising reacting a sample which may contain human podocalyxin with an anti-human podocalyxin monoclonal antibody bound to particles to generate an immunoagglutination reaction. A method for measuring human podocalyxin, which comprises measuring the above human podocalyxin by aggregation (hereinafter, this may be referred to as a third invention).

[0008] Furthermore, the present invention provides a method of reacting a sample which may contain human podocalyxin with an anti-human podocalyxin monoclonal antibody to cause an immunoagglutination reaction. Provided is a method for measuring human podocalyxin, which comprises measuring podocalyxin (hereinafter, this may be referred to as a fourth invention).

Hereinafter, the present invention will be described in detail.

As described above, the method of the first invention uses an anti-human podocalyxin antibody bound to a solid phase. As the solid phase, any of those used in conventional immunoassays can be used.For example, it is preferable to use wells of a plastic microtiter plate or magnetic particles or the like as described in Examples below. Can be.

The anti-human podocalyxin antibody immobilized on the solid phase may be a polyclonal antibody or a monoclonal antibody. When a monoclonal antibody is used, it is necessary to use a second monoclonal antibody having a corresponding epitope different from that of a second monoclonal antibody described later.

The binding of the anti-human podocalyxin antibody to the solid phase can be conventionally performed by a method well known in the art. For example, when binding to a well of a microtiter plate, about 1 μg / ml is used. This can be performed by adding an antibody solution having a concentration to a solid phase and allowing it to stand at 4 ° C. overnight. When the solid phase is a magnetic particle, the solid phase can be bound by a method described in detail in Examples below. After binding, blocking is performed with a protein such as BSA according to a conventional method to block nonspecific adsorption sites of the protein.

Next, the sample is reacted with the anti-human podocalyxin antibody bound to the solid phase in this way, and human podocalyxin in the sample is reacted with the anti-human podocalyxin bound to the solid phase by an antigen-antibody reaction. It is bound to the solid phase via the docalixin antibody. The sample can be any that may contain human podocalyxin. The sample may be, for example, urine. This antigen-antibody reaction can be carried out preferably at 4 ° C. to 45 ° C., more preferably at 25 to 38 ° C., and the reaction time is preferably 10 minutes to 1 minute.
8 hours, more preferably about 10 minutes to 1 hour.

Next, after washing, a second anti-human podocalyxin monoclonal antibody having a corresponding epitope different from that of the anti-human podocalyxin antibody bound to the solid phase is added to the human port in the sample bound to the solid phase. React with dokalyxin. Preferred reaction temperature and reaction time are the same as described above. Thereby, the second anti-human podocalyxin monoclonal antibody is bound to the solid phase via the human podocalyxin and the first anti-human podocalyxin antibody.

Next, after washing, the second anti-human podocalyxin monoclonal antibody bound to the solid phase is measured.
This can be performed by various methods commonly used in the field of immunoassay. For example, the second anti-human podocalyxin monoclonal antibody is labeled with an enzyme, fluorescence, biotin, a radiolabel, or the like, and the second anti-human podocalyxin bound to the solid phase is determined by measuring these labels. Monoclonal antibodies can be measured. Among these, labeling with an enzyme is preferable, and as the enzyme, peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or the like can be used. The detection of the label can be performed by reacting the corresponding substrate with the enzyme label, and measuring the dye, fluorescence, luminescence, and the like resulting from the reaction. Alternatively, when the second anti-human podocalyxin monoclonal antibody is not labeled, a labeled third antibody against the second anti-human podocalyxin monoclonal antibody is reacted,
By measuring the third antibody based on this label, the second anti-human podocalyxin monoclonal antibody can also be measured.

The first and second anti-human podocalyxin antibodies may be immunoglobulin fragments such as Fab and F (ab ') ₂ specific for human podocalyxin. . The term "antibody" in the claims is used to include these fragments specific to human podocalyxin (the same applies to the second to fourth inventions). The method for preparing these fragments is well known in this field, and is described in Examples below.

In the method of the second invention, human podocalyxin in a sample is bound to a solid phase via an anti-human podocalyxin antibody in the same manner as described above, and then specifically bound to the sugar chain of human podocalyxin. Lectin that reacts specifically, and after washing,
The lectin bound to the solid phase is measured. The reaction between human podocalyxin and lectin can be preferably carried out at the same reaction temperature and reaction time as described above. The measurement of the bound lectin can also be performed by using a labeled lectin to be reacted with human podocalyxin and measuring the lectin based on the label. The same label as described above can be used, and the detection can be performed in the same manner as described above.

The particles used in the method of the third invention preferably include latex having a diameter of 0.05 to 10 μm, gelatin particles having a diameter of 0.5 to 10 μm, and animal erythrocytes. Methods for binding antibodies to particles are well known in the art and can be readily accomplished by dispersing the particles in a solution of the antibody.

The particles in which the anti-human podocalyxin monoclonal antibody is bound on the particles and the sample are mixed on, for example, a black slide glass, and the presence or absence of the particles that aggregate and precipitate is observed. Human podocalyxin can be detected. It is also possible to quantify human podocalyxin by measuring absorbance.

In the method of the fourth invention, a solution containing an anti-human podocalyxin monoclonal antibody is mixed with a specimen. When human podocalyxin is present in the sample, the turbidity of the mixture increases, so that human podocalyxin in the sample can be detected by observing the change in turbidity of the mixture.

In the first to fourth aspects of the present invention, the anti-human podocalyxin monoclonal antibody is a biotechnology industrial technology laboratory obtained in the following example.
Hive deposited at the bureau with accession number FERM P-13024
A highly sensitive monoclonal antibody PCX-1C3 produced by lidoma PCX-1C3 can be preferably used.

Since human podocalyxin is present in higher amounts in nephritis patients than in healthy individuals, the method of the present invention can be applied to the diagnosis of nephritis.

[0023]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of anti-human podocalyxin rabbit IgG 2 ml of a human podocalyxin 1 mg / ml solution and 2 ml of Freund's complete adjuvant were sufficiently mixed to form an emulsion. This was placed in the back skin of a Japanese rabbit (2.5 kg) (10 kg).
0 μl / 1 place) 10 places and thigh (300 μl / 1 place)
Two places were immunized three times every other month. The serum of this rabbit was collected from the ear to obtain 20 ml of serum.

20 ml of this serum was precipitated with 40% saturated ammonium sulfate and centrifuged to obtain an IgG fraction. The IgG fraction was further purified using a Pharmacia Superose 12 column to obtain an IgG fraction excluding albumin.

Preparation of Anti-Human Podocalyxin Mouse Antibody A mouse was intraperitoneally immunized several times with 10 μg of human podocalyxin together with an adjuvant, and it was confirmed that the serum titer increased. On day 3 after the boost (intravenous), the spleen was removed to obtain splenocytes. This was fused with mouse myeloma cells in the presence of polyethylene glycol 2000 (2: 1 cells) to prepare hybridoma cells. The cells were cultured for one week at 37 ° C. under CO ₂ atmosphere, and the culture supernatant was examined for the presence of anti-human podocalyxin antibody. 4 in 96 wells
Antibody production was observed in two wells. The cells in the positive wells were diluted by the limiting dilution method and cultured for 2 weeks, and the culture supernatant was similarly examined for the presence of anti-human podocalyxin antibody. Strong antibody production was observed in 8 of the 96 wells. The cells in this well were again subjected to limiting dilution and the same culture was performed. Antibodies were produced in 29 of the 96 wells, which were cultured in flasks, a part of which was suspended in FCS containing 10% DMSO (5 × 10 ⁶ cells / ml) and stored in liquid N ₂ .

Next, using the supernatant of each well, the reactivity to human podocalyxin was examined. Human podocalyxin was sensitized at 50 ng / well to a titertech plate at 4 ° C. overnight and post-coated with 2% BSA / 0.02 M phosphate buffer. To this, 50 μl of the culture supernatant was added, and 37
℃ 1 hour. This was added to 20 mM phosphate buffered saline pH 7.4 (containing 0.5% Tween-20) (hereinafter referred to as PB
(Abbreviated as ST) 4 times, and 50 μl of anti-mouse IgG-POD-labeled antibody (1000-fold diluted by Tago) was added.
Heated at 37 ° C for 1 hour. After washing four times with the above-mentioned PBS-T, 200 µl of ABTS solution (1 mg / ml ABTS, 0.
01% H ₂ O _2, pH 5.5) was added and left at 4 ° C. overnight, and the absorbance was measured.

As a result, cells in 10 wells produced antibodies strongly. These cells were cultured in 25 ml flasks and further in 75 ml flasks. The cells were injected into the abdominal cavity of a pristane-treated mouse, and ascites was collected.
The ascites was purified and the anti-human podocalyxin mouse IgG was purified.
I got A hybridoma producing one of these monoclonal antibodies was named PCX-1C3, and was deposited at the Japan Institute of Fine Arts. The accession number is No. 13024 of the microbial laboratory.

Preparation of anti-human podocalyxin rabbit IgG-bound magnetic particles Carboxysilanated ferrite particles (particle size 0.3 μm,
1 mg of a water-soluble carbodiimide (manufactured by Nacalai Tesque) was added to 1 ml of a 1% suspended aqueous solution (manufactured by Nippon Paint Co., Ltd.) and stirred. The ferrite particles were allowed to react at room temperature for 10 minutes, and the ferrite particles were collected using a 5000 gauss permanent magnet. The supernatant was removed, and an anti-human podocalyxin IgG No. 1 solution (1 mg / m 2
l pH 4.5) and stirred once at room temperature with an end-over-end mixer. The suspension was treated with the above-described permanent magnet to collect the ferrite particles. After removing the supernatant, it was washed four times with PBS-T and stored in a Tris solution containing 2% BSA. Thus, anti-human podocalyxin rabbit IgG-bound ferrite particles were prepared.

Anti-human podocalyxin mouse IgG binding
Preparation of Lucali phosphatase 4 mg of anti-human podocalyxin mouse IgG (No. 4) was added to 2 ml of 0.1 M acetate buffer (pH 4.2, containing 1 mM EDTA), and 0.2 mg of pepsin was added.
Stirred for hours. Prepare this reaction solution in advance with 0.1 M phosphoric acid, pH
Gel filtration was performed on Ultrogel AcA44 which had been equilibrated in 6.3.
The protein fractions having a molecular weight of about 100,000 were pooled and concentrated to 1 ml by ultrafiltration. As a result, ₂ mg of anti-human podocalyxin mouse F (ab ') 2 was obtained.

(A) Preparation of GMB-Alkaline Phosphatase 5 mg of alkaline phosphatase was dissolved in 0.2 M phosphate buffer pH 7.0, and N- (γ-maleimidobutyloxy) succinimide 20 μg / 100 μl DM
The F solution was added and reacted at room temperature for 1 hour. Sephadex G-25 desalting column (Pharmacia) equilibrated with 0.1 M Tris-HCl, pH 7.0
The unreacted GMBS was separated by the above to obtain 5 mg of GMB-modified alkaline phosphatase.

(B) Preparation of anti-human podocalyxin mouse Fab-SH
b ') ₂ 2 mg / ml 1 ml of 0.2 M 2-mercaptoethylamine hydrochloride aqueous solution (50 μl) was added, and the mixture was heated at 37 ° C. for 2 hours, and the reaction solution was added to 0.1 M acetate buffer (1 mM ED).
The salt was desalted using a G-25 column equilibrated with TA (pH 7.0) to obtain 2 mg of Fab '.

Measurement of human podocalyxin in urine 10 μl of urine was added to 250 μl (0.02%) of anti-human podocalyxin rabbit IgG-bound ferrite particles, and 37 ° C.
For 10 minutes. The reaction tube was set on a 5000 gauss permanent magnet for 30 seconds to collect ferrite particles. The particles were washed with 20 mM Tris buffered saline (0.2% Tween-20, pH 7.2) TBS-T.
And washed 4 times. In this tube, human podocalyxin mouse IgG Fab binding-alkaline phosphatase 250
μl (1 μg / ml solution) was added, stirred well, and reacted at 37 ° C. for 10 minutes.

As described above, the ferrite particles are magnetized,
Washed twice with TBS-T. Add AMPPD solution (0.
02% AMPPD, 0.04% BDMQ, 0.1 mM M
gCl ₂ , 0.1 M diethanolamine, pH 9.9) 20
0 μl was added, the reaction was allowed to proceed at 37 ° C. for 5 minutes, and the luminescence was counted with an allo-carminometer for 2 seconds.

Table 1 shows the concentration of human podocalyxin and the amount of luminescence.

[Table 1]

Human podocalyxin in urine was measured using human urine as a sample in the same manner as described above. Table 2 shows the results.

[Table 2]

Example 2 Wheat Germ Agglutinin (WGA) -alkaline phosphatase
Preparation of conjugate 5 mg of WGA was dissolved in 1 ml of 50 mM carbonate buffer (pH 8.0), and 5 μl of 10 mM cuminothiolane (0.2 mM carbonate buffer 1 mM EDTA · 2Na, pH 8.0) was added.
The reaction was performed at 37 ° C. for 30 minutes. This reaction solution was previously
Unreacted iminothiolane was removed by passing through a G-25 desalting column equilibrated with M phosphate buffer pH 7.0.
5 mg of the iminothiolanated WGA was reacted with 5 mg of the GMB-alkaline phosphatase prepared in Example (a). After stirring at 37 ° C for 2 hours, 20 µl of 0.1 M 2-mercaptoethylamine was added, and the mixture was left at 37 ° C for 1 hour.
This reaction solution was subjected to Superose 12 by Pharmacia FPLC.
Fractionated by column. Fractions with a WGA-ALP ratio of 2: 1 were pooled and used as WGA-bound alkaline phosphatase.

Measurement of human podocalyxin in urine 10 μl of urine was added to 250 μl (0.02%) of anti-human podocalyxin mouse IgG-bound ferrite particles, and 37 ° C.
For 10 minutes. The reaction tube was set on a 5000 gauss permanent magnet for 30 seconds to collect ferrite particles. The particles were washed with 20 mM Tris buffered saline (0.2% Tween-20, pH 7.2) TBS-T.
And washed 4 times. To this tube was added 250 μl of WGA-bound alkaline phosphatase (1 μg / ml solution),
The mixture was stirred well and reacted at 37 ° C. for 10 minutes.

As described above, the ferrite particles are collected,
Washed with TBS-T once. Add AMPPD solution (0.02% AMPPD, 0.04% BDMQ, 0.1
mM MgCl ₂ 0.1 M diethanolamine, pH9.
9) 200 μl was added and reacted at 37 ° C. for 5 minutes, and the luminescence was counted for 2 seconds with an allo-carminometer.

Table 3 shows the concentration of human podocalyxin and the amount of luminescence.

[Table 3]

[0041]

As is clear from the above, according to the method of the present invention, human podocalyxin in a sample can be measured in a short time and with high accuracy by a simple operation.

Continuation of front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 33/53

Claims (7)

(57) [Claims] 1. After reacting a sample with a first anti-human podocalyxin antibody bound to a solid phase, a second anti-human podocalyxin antibody having a corresponding epitope different from that of the first anti-human podocalyxin antibody. A method for measuring human podocalyxin in a specimen, comprising reacting a docalixin monoclonal antibody and then measuring the second anti-human podocalyxin monoclonal antibody bound to a solid phase. 2. After reacting an anti-human podocalyxin antibody bound to a solid phase with a sample, a lectin specific to a sugar chain of human podocalyxin is reacted, and the lectin bound to the solid phase is measured. A method for measuring human podocalyxin in a sample, comprising: 3. After reacting a sample which may contain human podocalyxin with an anti-human podocalyxin monoclonal antibody bound to particles to generate an immunoagglutination reaction, the obtained agglutination is used to obtain the human A method for measuring human podocalyxin, comprising measuring podocalyxin. 4. The method according to claim 3, wherein the particles are selected from the group consisting of latex of 0.05 to 10 μm, gelatin particles of 0.5 to 10 μm in diameter and animal red blood cells. 5. A sample which may contain human podocalyxin is reacted with an anti-human podocalyxin monoclonal antibody to cause an immunoagglutination reaction, and then said human podocalyxin is measured from the obtained agglutination. A method for measuring human podocalyxin comprising: 6. The anti-human podocalyxin monoclonal antibody is obtained from FERM P-13024 by the Institute of Biotechnology and Industrial Technology.
Hybridoma PCX-1C3 deposited under accession number
The method according to any one of claims 1, 3, 4 and 5, which is a monoclonal antibody PCX-1C3 produced by the following method. 7. The anti-human podocalyxin antibody according to claim 7 ,
Deposited with the Engineering Research Institute under the accession number FERM P-13024.
Produced by hybridoma PCX-1C3
The method of claim 2 wherein the monoclonal antibody PCX-1C3 that.