JPH0666805A - Sandwich immunoassay of beta-n-acetyl glucosaminidase and monoclonal antibody used therefor - Google Patents

Abstract

PURPOSE:To provide a highly sensitive measurement by sandwiching beta-N-acetyl glucosaminidase with respectively recognized solid phase monoclonal antibody and labelled monoclonal antibody. CONSTITUTION:A sample containing beta-N-acetyl glucosaminidase(NAG) and solid phase antibody is brought into reaction for about 0.5-24 hours at 4-40 deg.C to form a solidified antibody-NAG complex with which labelled antibody is brought into reaction at about 4-40 deg.C for about 0.5-24 hours to form a solid phase antibody-NAG-labelled antibody complex. Next, activity of a labelled substance of labelled antibody is detected to measure the NAG. Detection of the activity is appropriately performed in accordance with the labelled substance. A monoclonal antibody is used for both the solid phase antibody and labelled antibody.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sandwich immunoassay for β-N-acetylglucosaminidase (NAG) and a monoclonal antibody recognizing NAG used in the sandwich assay. More specifically, it relates to a sandwich immunoassay method for NAG isozyme B (and I) and a monoclonal antibody that recognizes NAG isozyme B (and I) used therefor.

[0002]

2. Description of the Related Art β-N-
Acetylglucosaminidase (NAG, another name: hexosaminidase) is a glycolytic enzyme having a molecular weight of about 120,000 contained in a lysosome. Although NAG is widely distributed in living tissues, high activity is recognized in the kidney, particularly in the proximal tubule, and it shows high activity in urine in various renal disorders. The assay system for urinary NAG activity (total NAG activity) developed by Noto et al. (Noto, A., et al., Clin. Chem. 29 , 1713, 1983) is simple, has high sensitivity, and has good reproducibility. Therefore, it is used as an index for diagnosis and follow-up of rejection after renal transplantation, drug-induced renal injury, and acute renal failure.

The existence of several isozymes in NAG is known. In normal humans, isozyme A accounts for 80 to 90% and isozyme B for 10 to 20% in the urine, and the existence of isozyme I is also known. The structure of the subunit of the protein is that A is αβ, B And I
Is considered to be ββ. These isozymes differ in isoelectric point, substrate specificity, thermal stability and the like.

The clinical significance of these isozymes has been a problem for a long time, and in particular, renal tubular disorders caused by aminoglycoside antibiotics (Gibey, R., et al., Clin.
Chim. Acta. 137 , 1, 1984), pyelonephritis (upper urinary tract infection) (Vigono, A., et al., Clin. Chim. Acta. 130 , 2
97, 1983), Kidney transplantation (Yuen, CT., Clin. Chim. Acta. 1
64 , 339, 1987) and the like, it has been reported that the proportions of B and I in NAG isozyme are increased in urine of patients. Therefore, if these isozymes can be specifically measured, a more sensitive diagnosis of renal damage can be made than by measuring total NAG.

To date, the fractionation of NAG isozymes has been carried out by ion exchange chromatography (Ellis, BGet al., Clin. Chi.
m. Acta. 64 , 195, 1975), electrophoresis (Pitkanen, E.
et al., Enzyme 28 , 14, 1982), heat treatment method (Oberkette
r, LV et al., Clin. Chim. Acta. 94 , 281, 1979)
Etc. are used, but both methods have low sensitivity,
It was not suitable for routine inspection because it had drawbacks such as complicated operation and low accuracy.

Recently, NAG using a monoclonal antibody
An enzyme immunoassay (EIA) for isozymes A and B has been reported (A. ISAKSSON, et al., Scand. J. Clin. L.
ab.Invest. 49 , 597-603, 1989). This method is NAG
This is not a so-called sandwich method of sandwiching the antibody with an antibody, but a method of reacting a solid phase anti-NAG monoclonal antibody with NAG and detecting the enzymatic activity of the reacted NAG as an index. However, since the anti-NAG monoclonal antibody used inhibits the enzyme activity of NAG, there is a problem in quantitative measurement of NAG isozyme.

[0007] Therefore, the present inventors conducted research for the purpose of establishing a highly sensitive and quantitative measurement system for isozymes B and I of NAG.

[0008]

Means for Solving the Problems As a result of intensive studies aimed at achieving the above object, the present inventors have found that a NAG that recognizes NAG and a labeled monoclonal antibody that recognizes NAG are used. A sandwich immunoassay method for NAG was established, which was characterized by sandwiching. Since the sandwich immunoassay method of the present invention does not use the enzyme activity of NAG as an index but a labeled product of a labeled monoclonal antibody as an index, NAG can be quantitatively measured. further,
The sandwich immunoassay method of the present invention has two different recognition sites.
Since NAG is sandwiched using two monoclonal antibodies, it is possible to detect the NAG isozyme more selectively than a method having only one anti-NAG antibody, particularly when detecting a specific isozyme having NAG. it can.

The sandwich immunoassay method of the present invention includes the following steps. (1) A solid phase immobilized monoclonal antibody that recognizes NAG and a labeled monoclonal antibody that recognizes NAG are reacted with a sample containing NAG to form a solid phase immobilized antibody-NAG-labeled antibody complex. (2) The activity of the radioisotope or enzyme of the labeled monoclonal antibody contained or not contained in the complex is detected.

In the above step (1), solid phase immobilized monochrome-
The reaction between the null antibody and the sample containing NAG and the reaction between the labeled monoclonal antibody and the sample containing NAG may be carried out simultaneously or separately.

The sandwich immunoassay method of the present invention may further include the following steps in addition to the above steps. (1) A monoclonal antibody that recognizes NAG is immobilized on a carrier to prepare a immobilized monoclonal antibody. (2) A labeled monoclonal antibody is prepared by labeling a monoclonal antibody that recognizes a site of NAG different from the immobilized monoclonal antibody with a radioisotope or an enzyme.

In the sandwich immunoassay of the present invention, NAG is recognized as a monoclonal antibody that recognizes NAG.
If a monoclonal antibody that recognizes isozyme B is used, NAG isozyme B can be detected quantitatively and selectively. Examples of the antibody that recognizes NAG isozyme B include the monoclonal antibody Hex31 and the hybrid antibody produced by hybridoma Hex31.
Monoclonal antibody He produced by Ma Hex32
x32 is mentioned. In the present invention, Hex31 is preferably used as the immobilized antibody and Hex32 is preferably used as the labeled antibody, but the reverse combination may also be used, or Hex31 or Hex32 and another NAG having different recognition sites from each other may be used. A monoclonal antibody or a polyclonal antibody that recognizes isozyme B may be combined.

Further, it was revealed that the sandwich immunoassay method of the present invention simultaneously measured the presence of isozyme I when NAG isozyme B was measured, as will be shown in the Examples below. Therefore, the sandwich immunoassay method of the present invention uses the NAG isozyme B.
When and are present, their total amount can be quantitatively measured.

The present invention further provides a monoclonal antibody that recognizes NAG, which can be used in the sandwich immunoassay method of the present invention. The present monoclonal antibody is preferably a monoclonal antibody that recognizes the NAG isozyme B described above. For example, the monoclonal antibody Hex31 produced by the hybridoma Hex31 and the monoclonal antibody produced by the hybridoma Hex32 described above. The antibody Hex32 may be mentioned. Hybridoma Hex3
1 and hybridoma Hex32 are both "Mouse hy" at the Institute of Microbial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry, located at 1-1-3 Higashi 1-1-3, Tsukuba, Ibaraki, Japan.
bridoma Hex 31 "Microtech Lab. No. 3858 (FERM BP-385
8) and "Mouse hybridoma Hex 32"
It has been deposited as 859 (FERMBP-3859) under the Budapest Treaty since May 20, 1992.

Further, as described above, the sandwich assay method of the present invention measures both forms of NAG isozymes B and I. Furthermore, the subunit structures of the B and I proteins are both ββ and B and I. Considering that the difference in the structure between the two is only in the sugar chain portion, the monoclonal antibody of the present invention recognizes both NAG azozymes B and I,
It can be said that the sugar chain portion of the protein is not recognized. Furthermore, since the monoclonal antibody of the present invention does not inhibit the enzyme activity of NAG at all, as shown in Examples below,
It recognizes a portion different from the recognition site of the monoclonal antibody of ISAKSSON et al.

The method for producing a monoclonal antibody and the sandwich immunoassay of the present invention will be briefly described below.

1. Production of Anti-NAG Monoclonal Antibody-Producing Hybridoma and Production of Antibody (1) Immunity / Cell Fusion NAG, preferably NAG isozyme B, is emulsified in a suitable adjuvant such as Freund's complete adjuvant.
Mice are repeatedly inoculated into the muscle several times every several months.
NAG isozyme B as an immunogen may be a crudely purified product or a partially purified product, but a monoclonal antibody against NAG isozyme B, preferably hybridoma He.
Those purified using the monoclonal antibody Hex33 produced from x33 can also be used. Hybridoma Hex33 is 1-1-3 East, Tsukuba City, Ibaraki Prefecture, Japan.
"Mouse hybridoma Hex 33", Micromachine Research Institute, Yoryo 3860 (FERM BP-3860, May 20, 1992) at the Budapest Treaty, Ministry of International Trade and Industry Has been deposited under

Three to five days after the final immunization, the spleen is removed and used as antibody-producing cells. At the same time, a myeloma cell line having an appropriate marker such as 8-azaguanine resistance is prepared as a parent cell to obtain a hybridoma by fusing with the antibody-producing cell, and the hybridoma is fused with the antibody-producing cell. Create. As a medium for preparing a hybridoma, a commonly used medium such as Eagle MEM, Dalbetzco modified medium and RPMI-1640 is used.

First, parent cells, myeloma and splenocytes, are prepared at a ratio of about 1: 5. It is said that the fusion rate is high when polyethylene glycol (PEG) is used at a concentration of 50% as the fusing agent. The fusion strain is selected by the HAT selection method. The resulting hybridoma is screened using the culture supernatant by a known method such as immunoassay,
A clone of the hybridoma secreting the immunoglobulin of interest is selected. The specific antibody-producing positive hybridoma thus obtained was subjected to repeated cloning several times by a known method such as a limiting dilution method to obtain a single hybridoma.
Get the ma.

(2) Production of Monoclonal Antibody Next, in order to produce the monoclonal antibody of the present invention,
The hybridoma obtained above was placed in a culture vessel (in vitro
o) or in vivo. When culturing in an in vitro system, the medium may be the normal medium described above with FCS added, and after culturing in this medium for 3 to 5 days,
Monoclonal antibody is obtained from the culture supernatant. For in vivo culture, the hybridoma is inoculated into the abdominal cavity of a mammal,
Ascitic fluid is collected from 1 to 3 weeks to obtain a monoclonal antibody. Culturing in an in vivo system is preferable because a much larger amount of antibody can be efficiently obtained as compared with culturing in an in vitro system.

Purification of the monoclonal antibody from the thus obtained culture supernatant or ascites can be carried out by appropriately combining known methods such as ammonium sulfate fractionation and protein G sepharose column.

2. NAG sandwich immunoassay (1) Preparation of immobilized antibody As a solid phase for immobilizing an antibody, a commercially available carrier for antigen-antibody reaction used in ordinary immunoassay, for example, glass or synthetic resin Granular materials (beads) or spherical materials (balls), tubes, plates and the like can be used. The above 1. Adsorb the antibody obtained in (2). Adsorption is usually in phosphate buffer, pH 6-1
It is carried out by leaving it at room temperature overnight at 0, preferably near neutrality. The antibody-adsorbed carrier is preferably stored in a cool place in the presence of a preservative such as sodium azide. Regarding the monoclonal antibody and the polyclonal antibody,
It can be bound to the carrier by a similar treatment.

(2) Preparation of labeled antibody As the labeled substance, for example, a radioisotope, an enzyme, a fluorescent substance and the like which are usually used in an immunoassay can be used by labeling the antibody with a usual method. However, in the present invention, preferably a radioisotope and an enzyme are used. When labeling with radioisotopes, chloramine T
The antibody may be labeled with ¹²⁵ I or the like according to a known method such as a method. When the enzyme is labeled, an enzyme usually used for EIA, such as horseradish peroxidase, β-D-galactosidase, and alkaline phosphatase, is labeled according to a known method such as maleimide method or hinge method.

(3) Measurement of NAG A sandwich immunoassay for NAG is carried out using the immobilized antibody prepared in (1) and (2) above and the labeled antibody. The method is as follows: (1) A sample containing immobilized antibody and NAG (for example, N
AG standard antigen solution, urine sample, blood sample, etc.)
C., preferably 20 to 30.degree. C., for 0.5 to 24 hours, preferably 1 to 3 hours to form a solid phased antibody-NAG complex, and (2) generated solid phased antibody-NAG A labeled antibody is added to the complex at 4 to 40 ° C., preferably 20 to 30 ° C.
And reacting for 0.5 to 24 hours, preferably 1 to 3 hours to form a solid phase antibody-NAG-labeled antibody complex,
(3) A two-step sandwich method in which NAG is measured by detecting the activity of a labeled antibody bound to or not bound to the complex is preferable, but (1) and (2)
Of course, a one-step method in which the above steps are simultaneously performed may be used. In addition, a sample containing a labeled antibody and NAG is reacted to form a labeled antibody-NAG complex, and then a solid-phased antibody is reacted with the labeled antibody-NAG complex to give a labeled antibody-NAG-solid. A phased antibody complex may be formed.

The activity of the labeled substance may be detected by an ordinary method, depending on the labeled substance. For example, in the case of a radioisotope, the radioactivity is measured using an appropriate measuring device such as γ-counter. In the case of an enzyme, the substrate is added to carry out an enzymatic reaction, and then the absorbance, fluorescence intensity, luminescence intensity or the like is measured.

The present invention further includes a solid-phased monoclonal antibody that recognizes NAG and a labeled monoclonal antibody that recognizes NAG.
A kit for NAG measurement, which comprises a null antibody, wherein the immobilized monoclonal antibody and the labeled monoclonal antibody can sandwich NAG.

In the NAG measurement kit of the present invention, N
If a monoclonal antibody that recognizes NAG isozyme B is used as a monoclonal antibody that recognizes AG, N
AG isozyme B can be detected quantitatively and selectively. As the antibody recognizing NAG isozyme B, the above-mentioned monoclonal antibody Hex31 and hybridoma He produced by hybridoma Hex31.
monoclonal antibody Hex32 produced by x32.
Is mentioned. In the kit of the present invention, Hex31
It is preferable to use Hex32 as the immobilized antibody and Hex32 as the labeled antibody, but of course the reverse combination may also be used, or Hex31 or Hex32 and another NAG isozyme B that recognizes different NAG isozyme B from both antibodies may be used. -A null antibody or a polyclonal antibody may be combined.

Furthermore, since the monoclonal antibody used in the kit of the present invention recognizes both NAG isozymes B and I as described above, the total amount of NAG isozymes B and I can be quantified using the kit of the present invention. Can be measured.

The present invention will be described in detail below with reference to examples.

[0030]

【Example】

1. NAG sandwich RIA method (1) Preparation of anti-NAG monoclonal antibody-producing hybridoma and production of antibody (a) Immunogen As a immunogen, human placenta-derived partially purified β-N- of Sigma
Balb / c mice (4 weeks old) were immunized with acetylglucosaminidase (isozyme B) by the following method.

Since the enzyme is commercially available as a suspension in 2.4 M ammonium sulfate, it was centrifuged at 1200 rpm for 5 minutes, the precipitate was dissolved in physiological saline and mixed with an equal volume of Freund's complete adjuvant. In the first immunization, a protein amount of 10 μg was intraperitoneally administered to each mouse. For the subsequent immunization, the same amount of antigen was intramuscularly administered three times about every two months.

(B) Cell fusion Four days after the final immunization, the antibody titer of the mouse antiserum was measured according to the method of (c) below, and the splenocytes of the mouse showing a significant increase in the antibody titer were found. Remove the cells and
The red blood cells were destroyed by placing them in an ammonium chloride solution for 5 minutes under ice cooling. The remaining cells were suspended in RPMI1640 medium to give splenic lymphocytes used for cell fusion. Then the same R
6.3 × 10 ⁷ 8-azaguanine-resistant myeloma cells (X63.653) suspended in PMI1640 medium and 3.
After centrifugation of 5 × 10 ⁸ splenic lymphocytes, the supernatant was removed. 5 dissolved in RPMI1640 medium in cell pellet
0.8 ml of 0% polyethylene glycol (molecular weight 4000, Merck) was added with stirring with a pipette tip for 1 minute, and further stirred for 1.5 minutes. afterwards,
2 ml of RPMI 1640 medium was added over 2 minutes, then similarly 2 ml over 1 minute with stirring. Further, 18 ml of RPMI1640 medium was gradually added dropwise with gentle stirring.

After centrifugation, the supernatant was removed and the precipitated cells were
00 ml of HAT medium (1 × 10 ⁻⁴ M hypoxanthine,
4x10 ^-7 M aminopterin, 1.6x10 ^-5 M thymidine, and RPMI1640 medium containing 20% fetal bovine serum), and 96-well cell culture plate (Falcon)
About 0.1 ml was dispensed into each of 12 wells. After that,
50 μl and 100 μ after 2 and 7 days, respectively
l of HAT medium was added to each well, and after 10 days, the culture supernatant was removed and screened by the following method (c). The obtained specific antibody-producing hybridomas were diluted by limiting dilution to 3 Antibody cloning strain H
ex31, Hex32 and Hex33 were obtained.

(C) Method for measuring antibody titer PBS containing 10 μg of rabbit anti-mouse IgG in each well of Microplate (Dynatech) (10 mM phosphate buffer containing 0.15 M sodium chloride, pH 7.
Add 100 μl of the solution of 4), leave it at 4 ° C. overnight, and
After washing once with PBS containing bovine serum albumin (BSA), 200 μl of the same solution was added and left at 37 ° C. for 1 hour to solid-phase rabbit anti-mouse IgG. Well to 0.05
The cells were washed three times with PBS containing 10% Tween 20, 100 μl of culture supernatant of antiserum or hybridoma was added, and the mixture was reacted at room temperature for 2 hours. After washing 3 times, 100 μl of PBS containing 0.5 mU of partially purified human placenta-derived NAG isozyme A or B (Sigma) and 0.1% BSA was added, and reacted at room temperature for 2 hours. After 3 washes, 1 mM 4
-Methylumbelliferyl-2-acetamido-2-deoxy-β-D-glucopyranoside-containing 50 mM citrate-phosphate buffer (pH 4.9) (100 μl) was added, and the enzyme reaction was performed at 37 ° C. for 2 hours. 100 μl of 0.5 M glycine-sodium hydroxide buffer pH 10.5
Stop the addition reaction. The released 4-methylumbelliferone was measured for fluorescence intensity in each well using a pre-trider (Corona, MTP-32) at an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm.

(D) Ascites hydration of hybridomas Pristane was pre-existed in the abdominal cavity of mice in an amount of 0.1.
2 ml was administered, and 21 days later, 2 × 10 ⁶ hybrids were
Ma Hex31 or Hex32 was administered intraperitoneally. 1 to
Collect ascites at 3 weeks and centrifuge the cells,
Stored at -80 ° C.

(E) Purification of monoclonal antibody and confirmation of antibody titer 20 ml of ascites fluid stored at −80 ° C. was thawed to obtain the same amount of PB.
S is added and diluted 2-fold, and 20 mM phosphate buffer (p
Prote equilibrated with H7.0) (hereinafter abbreviated as buffer A)
It was passed through a column (1.5 × 13 cm) of in G Sepharose 4 Fast Flow (Pharmacia). After washing the column with 60 ml of buffer A, 10 mM glycine-hydrochloric acid buffer (p
Mouse IgG was eluted with 60 ml of H2.7), neutralized with 1 M Tris solution, and treated with 50% ammonium sulfate at 0 ° C.
The precipitate obtained by centrifuging at 6000 rpm for 30 minutes is 10 times.
It was dissolved in ml of PBS and dialyzed against PBS at 4 ° C. overnight to obtain a purified antibody.

The antibody titer of the purified antibody was confirmed according to the method of (3) above. However, as a substrate, sodio-m-
Cresol sulfonephthalinyl-N-acetyl-β-
An enzymatic reaction was performed for 30 minutes using D-glucosamine, and the absorbance at 590 nm was measured. As a result, as shown in FIG. 1, a significant antibody titer was exhibited even at an antibody concentration of 0.1 μg / ml, and hybridomas Hex31 and Hex
Monoclonal antibody Hex31, H produced from 32
Both ex32 reacted well with B of NAG isozyme (graph of-●-in the figure), but showed a slight cross-reactivity with A (graph of-○-in the figure).

(F) Antibody Typing Typing of the immunoglobulin produced by the hybridoma was carried out by the enzyme antibody method using a monoclonal mouse immunoglobulin isotyping kit (Pharmigen). As a result, the anti-human NAG monoclonal antibodies Hex31 and Hex32 were both IgG ₁ . It was κ.

(G) Examination of NAG activity inhibitory effect of antibody NAG isozyme A or B was adjusted to 110 U / L using PBS containing 0.1% BSA, and anti-human NAG monoclonal antibodies Hex31 and Hex32 were each adjusted to 0.
The concentration was adjusted to 1 to 10000 ng / ml, and both were reacted at room temperature for 2 hours. The NAG activity after the reaction was measured using NAG test Shionogi (Shionogi & Co.). As a result, as shown in FIG. 2, even if the antibody was added in excess, NAG
The enzyme activity of A was not inhibited at all in A and B (A:-○-, B:-●-in the figure).

(2) NAG sandwich RIA (a) Radiolabeling of antibody 0.5M PB (pH 7.5) (hereinafter abbreviated as buffer B)
Dissolve 25 μg of Hex32 (purified antibody) in 50 μl,
25 μl of a buffer solution B containing 5 μl of 0.5 mCi of Na ¹²⁵ I and 50 μg of chloramine T was added thereto, and the mixture was allowed to stand at room temperature for 1 hour.
It was allowed to react for 5 seconds. After completion of the reaction, the reaction was stopped by adding 25 μl of a buffer solution B containing 100 μg of sodium pyrosulfite and 20 μl of an aqueous solution containing 2.5 mg of potassium iodide. The fraction containing the labeled antibody was collected by passing through a PD-10 column (Pharmacia) equilibrated with PBS containing 0.5% BSA and 0.1% sodium azide.

(B) Sandwich RIA method Hex was added to each well of the microplate (Dynatech).
31 100 μl of PBS containing 1 μg of purified antibody was added, and 4
After standing overnight at 0 ° C., the plate was washed once with PBS containing 1% BSA, 200 μl of the same solution was added, and left at 37 ° C. for 1 hour for blocking. Well with 0.05% Tween
Wash 3 times with PBS containing 20 and 75 μl of 0.2 M citrate phosphate buffer (pH 5.0) containing 0.1% BSA and 0.02% sodium azide, and then add standard antigen solution or urine sample. After 25 μl was added and mixed, the first reaction was performed at room temperature for 2 hours. After washing 3 times, ¹²⁵ I-labeled He
x32 (10 ng, about 10 ⁵ cpm), 0.1% BSA,
And 100 μl of 0.2 M citrate phosphate buffer (pH 7.0) containing 0.02% sodium azide were added, and the second reaction was performed at room temperature for 2 hours. After completion of the reaction, the plate was washed 3 times, and each well was cut off, and the γ-counter (ARC-6
00, Aloka).

A partially purified human placenta-derived N was used as a standard antigen solution.
An enzyme activity value of 0.5 to 20 U / L was prepared using AG isozyme A or B (Sigma).

The pH of the first reaction and the second reaction in the sandwich RIA method was 0.1% BS.
The procedure was as follows using 0.2 M citrate phosphate buffer (pH 4.5 to 8.0) containing A and 0.02% sodium azide.

First, the pH of the second reaction was set to 7.0 and the pH of the first reaction was changed. As a result, the reactivity of isozyme A with respect to isozyme B was 33% at pH 7.0, but As the pH is lowered, the reactivity of A is suppressed selectively, and the reactivity of A with B is 15% at pH 5.0.
(This 15% reactivity is not actually due to A as described later, but due to isozyme I mixed in the standard antigen solution of NAG isozyme A). However, at pH 4.5, neither A nor B showed reactivity. Next, the first reaction was set to pH 5.0 and the pH of the second reaction was changed, but the reactivity ratio of isozyme A to B was about 13% to 17%, which was almost unchanged. The influence of the pH of the first reaction and the second reaction on the reactivity of the isozyme is different from that of the solid-phase antibody He.
The properties of x31 and labeled antibody Hex32 were reflected. From the above, the first reaction was set to pH 5.0 to suppress the reactivity of A, and the second reaction was set to pH 7.0 to obtain a high count.

FIG. 3 shows a standard curve obtained by the sandwich RIA method when NAG isozyme B is used as a standard antigen. A standard curve with good linearity was obtained in the range of 0 to 10 U / L.

Table 1 shows the simultaneous reproducibility of this RIA method. When the standard antigen solution and urine specimens (8 cases) were measured in triplicate, good reproducibility of CV 10% or less was shown.

[0047]

[Table 1]

(3) N by anion exchange chromatography column
Analysis of AG isozyme The specificity of the NAG isozyme of this sandwich RIA method was examined by anion exchange chromatography. Fractionation of NAG isozyme was performed by FPLC system (Pharmacia) using Mono Q column (0.5 × 5.0 cm).
A 10 mM phosphate buffer (pH 6.0) was used as a solvent, and the elution was carried out by a linear concentration gradient method of sodium chloride at a flow rate of 1 ml / min. The amount of NAG in each fraction is the above-mentioned RIA.
Method and substrate method (by NAG test Shionogi (Shionogi & Co.) using Sodio-m-cresol-sulfonephthaleinyl-N-acetyl-β-D-glucosamine as substrate)
It was measured at.

As a sample for analysis of NAG isozyme, partially purified human placenta-derived NAG isozymes A and B (Sigma) or a urine sample (concentrated 5 to 10 times) was used, and a 10 mM phosphate buffer solution (pH 6.0) was used. ) To 4
After dialysis overnight at 0 ° C., 0.5 ml was injected (chromatography was performed by adding 0.1% BSA as a carrier protein).

As a result, as shown in FIG. 4 (A), when the human placenta-derived NAG isozyme A was used, the RI
The chromatographic pattern of method A (-●-graph in the figure) is different from the substrate method (graph of-○-in the figure) in the position of the peak, and the smaller peak () in the earlier elution position ( Fraction No. 16) was shown. This is considered to be a peak due to the presence of isozyme I because NAG isozyme A, which is an antigen, is a partially purified product. Because the RIA method sandwiches NAG using two monoclonal antibodies against NAG isozyme B, the selectivity for NAG isozyme B becomes very high, and the subunit structure of the protein reacts with A, which is completely different from B. However, the subunit structure of the protein of I is ββ, which is the same as that of B, and the difference is only in the sugar chain portion. Therefore, the monoclonal antibody used in this RIA is the same as B. This is because there is a good possibility that I also recognizes I.

On the other hand, as shown in FIG. 4 (B), the human placenta-derived NAG isozyme B also showed peaks (fraction Nos. 11 to 13) showing a mixture of I,
The chromatographic patterns of the substrate method and the RIA method were almost the same.
Next, as a result of comparing the chromatographic patterns of the NAG isozymes of the two urine specimens by the substrate method and the present RIA method, as shown in FIG. 5, NAG isozymes A (fraction No. 16) and B (fraction) were found by the substrate method. No. 2 to 4),
While the peaks corresponding to I (fraction No. 10 to 11) are shown, in the present RIA method, B (fraction N) is shown.
o. 2 to 4) and peaks corresponding to I (fraction No. 10 to 11) only, and peaks corresponding to A did not appear. From the above results, it was confirmed that this RIA method reflects the amounts of NAG isozymes B and I.

2. NAG sandwich EIA method (1) Purification of antigen NAG isozyme B from human placenta The main steps for purifying NAG isozyme B used as an antigen are 1) ammonium sulfate fraction of placental tissue extract, 2) concanavalin A ( Adsorption of glycoprotein by Con A) -Sepharose chromatography, 3) above 1.
(1) Antibody column chromatography using the monoclonal antibody Hex33 produced from the hybridoma Hex33 obtained in (b), Mono Q column (Pharmacia)
Isolation of Isozyme B from Diversely Present Isozymes by Chromatography (FPLC), 4) Seph
Separation and purification with acryl S-200 (Pharmacia).
The anti-NAG antibody column was prepared according to the method of Schneider et al. (J. Biol. Chem. 257 , 10766-10769, 1982). The activity of this enzyme is sodio-m-crezo-
NAG using rusulfonphthaleinyl-N-acetyl-β-D-glucosamine (MCP-GlcNAc) as a substrate
It measured using the test Shionogi (Shionogi Pharmaceutical). The specific method is described below (all operations were performed at 4 ° C.).

Twelve placenta from which the umbilical cord and the coating had been removed were washed with distilled water a few times, and after removing erythrocytes as much as possible, they were cut into small pieces with a knife, and about twice the amount of 25 mM sodium phosphate buffer (pH 6.0). ) Was added and treated with a blender. The obtained homogenate was centrifuged at 10,000 × g for 20 minutes, and the obtained precipitate was added with 50 M of 0.3 M sodium chloride.
It was dissolved in mM sodium phosphate buffer (pH 7.0, hereinafter abbreviated as buffer C), and dialyzed with the same buffer. Con A-Sepharose 400 ml total equilibrated with buffer C
Was added to the dialyzed sample and slowly stirred overnight, and then Con A-Sepharose was separated by filtration through a glass filter and packed in a column (2.6 × 46 cm). After washing the gel with 3 volumes of buffer C, the gel was eluted with buffer C containing 0.5M α-methylmannoside, and the active fraction was dialyzed against buffer C. Protein G-Seph equilibrated with buffer C
The dialyzed sample was passed through an arose (Pharmacia) column (1.5 × 12 cm), and then applied to an antibody column (2.8 × 3 cm) equilibrated with buffer C, washed with buffer C, and then washed with 0.2.
Elution was performed with 2M glycine hydrochloride buffer (pH 3.0).

The eluted fraction was immediately neutralized with 0.5M sodium phosphate buffer (pH 7.5) and dialyzed with 0.01M sodium phosphate buffer (pH 6.0, hereinafter abbreviated as buffer D). did. The dialyzed sample was concentrated with a Collodion bag (Sartorius) and a Mono Q column (0.5 x 5 cm) was used.
Chromatography was performed. The NAG isozyme B which was not adsorbed on the Mono Q column was recovered from the flow-through fraction, and the concentrated sample was used as a buffer solution D containing 0.1 M sodium chloride.
Sephacryl S-200 column (2.6 x 73c)
m) and collecting an active fraction to obtain about 2.3 mg of the target enzyme (NAG isozyme B). NAG isozyme A separated by Mono Q column was similarly purified by Sephacryl S-200, and finally about 3.5
mg was obtained. Purity was confirmed by Coomassie-Brilliant Bull-staining after electrophoresis using SDS-PAGE gradient gel (Daiichi Pure Chemicals).

As a result, under non-reducing conditions, NAG
56K (α chain) and 60K (β
Chain) and two bands of
Only the K (β chain) band was seen.

Under reducing conditions, isozymes A and B
Β-chain has been reduced to 30K (βa-chain) and 28K
Two bands of (βb chain) are shown. The α chain band of isozyme A did not change even under reducing conditions.

As a result of examining the β chain purity of NAG isozyme B under non-reducing conditions with a densitometer, it was confirmed that it could be purified to homogeneity.

(2) Preparation of enzyme labeled antibody A horseradish peroxidase was used as a labeling enzyme to prepare a labeled antibody by the hinge method.

The above 1. (1) The anti-NAG monoclonal antibody Hex32 (18 mg) purified in (e) was dissolved in 8.8 ml of 0.1 M citrate buffer (pH 4.1) 878.
Add μg of pepsin (Baehringer-Mannheim) and add 3
Digested at 7 ° C for 18 hours. The reaction mixture was equilibrated with 0.1 M citrate buffer (pH 6.0) Superose 12 (1.
5 × 50 cm, Pharmacia). Collected F (a
b ') The ₂ fractions were concentrated to 2.2 ml with Centricon 30 (Amicon) and dissolved in 0.1 M phosphate buffer (pH 6.0) containing 5 mM ethylenediaminotetraacetic acid (EDTA) in 0.1 ml.
1/10 volume of M2-mercaptoethylamine solution was added, and reduction was performed at 37 ° C for 90 minutes. The Fab ′ fraction was separated with Superose 12 (1.5 × 50 cm) equilibrated with the same buffer and then concentrated to 1 ml with Centricon 10.

On the other hand, 0.1M phosphate buffer (pH 7.0)
Peroxidase (Sigma) dissolved in 2.47 ml 1.
65 mg, N, N-dimethylformamide 164 μl
Ε-maleimidocaproyloxysuccinimide (EMCS) (Dojin) 13.1 mg dissolved in the
And reacted for 30 minutes. The precipitate formed is removed by centrifugation,
The supernatant was equilibrated with 0.1 M phosphate buffer (pH 6.0).
Low molecular weight substances were removed by PD-10 column. The maleimidated peroxidase thus obtained was reacted with the Fab ′ fragment at a molar ratio of 1: 1 at 4 ° C. for 15 hours. The peroxidase-labeled anti-NAG monoclonal antibody (Fab 'fraction) was 0.1 M phosphate buffer (pH 6.
The unreacted material was separated with Superose 12 (1.5 × 50 cm) equilibrated in 5). Finally 3.8 mg of labeled antibody was obtained.

(3) Method for preparing antibody solid phase plate An anti-NAG monoclonal antibody Hex31 solution (10 μg / ml) diluted with 10 mM PBS (pH 7.4) was used as an immunomodulator plate F-8H (Nunc). 200 μl was added to each well and solid phased at 4 ° C. for 24 hours.
Remove antibody solution from wells and add 0.05% Tween2
After washing 3 times with a 10 mM PBS (pH 7.4) solution containing 0, 300 μl of 10 mM PBS (pH 7.4) solution containing 10% saccharose and 1% bovine serum albumin (Sigma) was added per well. After standing at room temperature for 1 hour, the solution was removed from the wells and then dried under reduced pressure in a silica gel-containing desiccator overnight to give an antibody solid phase plate.

(4) Sandwich EIA The antibody solid phase plate prepared in the above (3) was added with 0.05% Tw.
een20, 0.15M sodium chloride and 0.06%
10 including Kason CG (Rom and Haas)
After washing once with 300 μl of mM phosphate buffer (pH 7.4), 150 μl of 0.4 M citrate buffer (pH 5.2) containing 0.1% BSA and Kason CG and standard antibody solution ( The purified NAG isozyme B obtained in 2. (1) above was used at a concentration of 0 to 80 ng / ml for 0.1
25 μl of 10 mM phosphate buffer (pH 6.0) containing 25% of BSA, 0.1% caseon CG and 0.15 M sodium chloride) or 25 μl of urine sample was added.
After stirring, the first reaction was performed at 30 ° C. for 2 hours, and then the washing operation was performed 4 times.

Next, 100 μl of the labeled antibody solution was added to each well (25 ng / ml of the labeled antibody prepared in 2. (2) above).
Of 10% phosphate buffer (pH 7.4) containing 0.1% BSA, 0.1% Caeson CG and 0.15M sodium chloride) at 30 ° C for 1 hour. The second reaction was performed. After washing 4 times, 10 in each well
0 μl of coloring solution (2 mM hydrogen peroxide and 4.3 mM
ABTS (10 mM citrate buffer containing 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), pH 5.0) was added, and the enzyme reaction was carried out at 30 ° C. for 15 minutes. Stop solution (0.05%
10 mM citrate buffer containing sodium azide, pH
5.0) was added to stop the reaction. Absorbance at 415 nm was measured with a microplate reader (Corona, MTP-32), and urinary NAG isozyme B was quantified from a standard curve.

(A) Standard curve and NAG isozyme A
Fig. 6 shows a reaction curve of the standard antigen (NAG isozyme B) (graph of-●-in the figure). 0-80 ng / ml
Showed good linearity. In addition, as a result of calculating the average + 3SD from the blank value of n = 20 as the detection limit,
It was 0.5 ng / ml. In addition, NA as a standard antigen
Sandwich EIA was similarly performed using G isozyme A or NAG derived from bovine kidney. As a result, NAG
The crossability with isozyme A (-○-graph in the figure) was 2-3%. Moreover, it did not show any cross-reactivity with NAG derived from bovine kidney.

(B) Reproducibility test Table 2 shows the results of reproducibility test of the sandwich EIA of the present invention using 3 urine samples having different concentrations. Simultaneous reproducibility (n = 8) is C.I. V. 2.5-5.4%, reproducibility between measurements (n = 14) was C.I. V. It was 6.2 to 9.1.

[0066]

[Table 2]

(C) Dilution linearity test FIG. 7 shows the results of examining the dilution linearity of three urine specimens with different concentrations using an antigen dilution solution. Good linearity was obtained for all urine samples.

(D) Addition recovery test The standard antigen was added to 3 urine samples of different concentrations and the recovery was calculated. As a result, as shown in Table 3, the recovery rate was 91.
% To 114%, which is a good result.

[0069]

[Table 3]

(E) Healthy person values Tables 4 and 5 show the measurement results of urinary NAG isozyme B by age and sex of healthy persons. The mean values of early morning urine (n = 137) and occasional urine (n = 177) were 4.2 μg and 3.3 μg / g creatinine, respectively. There was no significant change due to age or gender.

[0071]

[Table 4]

[0072]

[Table 5]

From the above results, the sandwich E of the present invention
The IA method was able to quantitatively and reproducibly measure NAG isozyme B as a protein amount. Further, compared with the conventional NAG isozyme analysis method (chromatography method, electrophoresis method, etc.), the required sample amount is as small as 25 μl, the operation is simple, and a large amount of samples can be processed. Further, the edge effect, which is a problem in the plate method, was not recognized in the EIA method. Therefore, in this way, the EIA method is
It can be sufficiently used as a chin test.

[0074]

The clinical significance of NAG isozymes is that the proportions of B and I in urine increase in renal disorders due to aminoglycoside antibiotics, interstitial disorders such as upper urinary tract infection, and renal transplantation. It has been known.
On the other hand, in blood, the proportion of I increases in hepatitis leukemia and the like.
However, the conventional NAG isozyme fractionation methods (chromatography, electrophoresis, and heat treatment) have
Even from the viewpoint of operation, there are many problems in applying it to a routine test for processing a large number of specimens. The sandwich immunoassay method of the present invention solves such problems and directly and specifically measures NAG isozymes B and I in urine and blood as an index of sensitive renal diseases, hepatitis and leukemia. can do.

[Brief description of drawings]

FIG. 1 (A) Monoclonal antibody Hex31 of the present invention.
NAG isozyme A (in the figure, −
It is a graph showing the measurement results of the antibody titer for (-) and B (-●-in the figure). (B) NAG isozyme A (-○-in the figure) at various concentrations of the monoclonal antibody Hex32 of the present invention,
It is the graph which showed the measurement result of the antibody titer with respect to B (-●-in the figure).

FIG. 2 (A) NAG isozyme A after reacting with various concentrations of the monoclonal antibody Hex31 of the invention.
(-○-in the figure) and B (-●-in the figure) are graphs showing the measurement results of the enzyme activity. (B) Various concentrations of the monoclonal antibody Hex of the present invention
NAG isozyme A after reacting with 32 (in the figure, −
It is a graph showing the measurement results of the enzyme activities of (-) and B (-●-in the figure).

FIG. 3 shows a standard curve according to the sandwich RIA method of the present invention when NAG Isozam B is used as a standard antigen.

[FIG. 4] (A) Fractionation of partially purified human placenta-derived NAG isozyme A by anion exchange chromatography was performed, and the amount of NAG isozyme in each fraction was determined by the sandwich RIA method of the present invention (-●-) and substrate. Method (-○ in the figure
It is a graph which shows the chromatographic pattern when it measures by-). (B) Fractionation of partially purified human placenta-derived NAG isozyme B by anion exchange chromatography was performed, and the amount of NAG isozyme in each fraction was determined by the sandwich R of the present invention.
It is a graph which shows a chromatographic pattern when it measures by IA method (-●-in a figure) and a substrate method (-○-in a figure).

FIG. 5 (A) and (B) fractionation of NAG isozymes in urine samples by anion exchange chromatography, and the amount of NAG isozymes in each fraction was determined by the sandwich RIA method (-●-in the figure) of the present invention. Substrate method (-○ in the figure)
It is a graph which shows the chromatographic pattern when it measures by-).

FIG. 6 shows NAG isozymes B (-●-in the figure) and A.
The standard curve by the sandwich EIA method of the present invention when (-○-in the figure) is used as a standard antigen is shown.

FIG. 7 shows a dilution curve when three urine samples having different concentrations were diluted with an antigen diluent.

Claims (20)

[Claims] 1. A solid-phased monoclonal antibody that recognizes β-N-acetylglucosaminidase (NAG) and NA.
A sandwich immunoassay method for NAG, which comprises sandwiching NAG with a labeled monoclonal antibody that recognizes G. 2. A NAG sandwich immunoassay method comprising the following steps. (1) A solid phase immobilized monoclonal antibody that recognizes NAG and a labeled monoclonal antibody that recognizes NAG are reacted with a sample containing NAG to form a solid phase immobilized antibody-NAG-labeled antibody complex. (2) The activity of the radioisotope or enzyme of the labeled monoclonal antibody contained or not contained in the complex is detected. 3. The sandwich immunoassay method according to claim 2, further comprising the following steps. (1) A monoclonal antibody that recognizes NAG is immobilized on a carrier to prepare a immobilized monoclonal antibody. (2) A labeled monoclonal antibody is prepared by labeling a monoclonal antibody that recognizes a site of NAG different from the immobilized monoclonal antibody with a radioisotope or an enzyme. 4. The reaction between the immobilized monoclonal antibody and a sample containing NAG and the reaction between the labeled monoclonal antibody and a sample containing NAG are carried out simultaneously or separately. The sandwich immunoassay described. 5. The sandwich immunoassay method according to claim 1, wherein the NAG is NAG isozyme B. 6. The sandwich immunoassay method according to claim 1, 2, 3 or 4, wherein the NAG is NAG isozymes B and I. 7. The hybridoma Hex31 (FE) comprising the immobilized monoclonal antibody and the labeled monoclonal antibody.
Monoclonal antibody He produced by RM BP-3858)
x31 and hybridoma Hex32 (FERM BP-385
The sandwich immunoassay method according to claim 1, which is obtained from the monoclonal antibody Hex32 produced by 9). 8. The sandwich immunoassay method according to claim 7, wherein the immobilized monoclonal antibody is an immobilized Hex31 monoclonal antibody produced by hybridoma Hex31 (FERM BP-3858). . 9. The sandwich immunoassay method according to claim 7, wherein the labeled monoclonal antibody is a labeled monoclonal antibody Hex32 produced by hybridoma Hex32 (FERM BP-3859). 10. A monoclonal antibody that recognizes NAG, which can be used in the sandwich immunoassay according to claim 1, 2, 3, 4, 5 or 6. 11. The monoclonal antibody according to claim 10, wherein the NAG is NAG isozyme B. 12. The monoclonal antibody according to claim 10, wherein the NAG is NAG isozymes B and I. 13. The monoclonal antibody according to claim 10, 11 or 12, which does not inhibit the enzymatic activity of NAG. 14. A hybridoma Hex31 (FERM BP-
3858) produced a monoclonal antibody Hex31
Alternatively, the monoclonal antibody Hex32 produced by hybridoma Hex32 (FERM BP-3859) is the monoclonal antibody according to claim 10. 15. A solid phase immobilized monoclonal antibody that recognizes NAG and a labeled monoclonal antibody that recognizes NAG, wherein the solid phase immobilized monoclonal antibody and the labeled monoclonal antibody can sandwich NAG. Is something
NAG measurement kit. 16. The kit according to claim 15, wherein the NAG is NAG isozyme B. 17. The kit according to claim 15, wherein the NAG is NAG isozymes B and I. 18. The hybridoma Hex31 comprising the immobilized monoclonal antibody and the labeled monoclonal antibody.
(FERM BP-3858) produced by the monoclonal antibody Hex31 and hybridoma Hex32 (FERM BP-
3859) produced a monoclonal antibody Hex32
The kit according to claim 15, 16 or 17, which is obtained from 19. The kit according to claim 18, wherein the immobilized monoclonal antibody is an immobilized monoclonal antibody Hex31 produced by hybridoma Hex31 (FERM BP-3858). 20. The kit according to claim 18, wherein the labeled monoclonal antibody is a labeled monoclonal antibody Hex32 produced by hybridoma Hex32 (FERM BP-3859).