JPH0534353A - Immunological method for determination of human 92kda gelatinase - Google Patents

Abstract

PURPOSE:To determine human 92kDa gelatinase (human MMP-9) in a sample to be inspected, precisely, simply and quickly, by using a monoclonal antibody which is combined specifically with the human MMP-9. CONSTITUTION:Two kinds of monoclonal antibodies which are combined specifically with human MMP-9 are used as antibodies to be combined with a solid- phase carrier or antibodies giving tags thereto, and measurement is executed immunologically by a sandwich method. On the occasion when an antigen- antibody reaction is executed, polyoxyethylene lauryl alcohol ether is added to a reaction liquid preferably in a concentration range of 0.02 to 0.1% (W/V) generally. Thereby nonspecific combination and interference in the antigen- antibody reaction by serum protein can be lessened and, therefore, the human MMP-9 in a specimen can be measured with increased accuracy.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological quantification method of human 92 kDa gelatinase used in the field of medicine and physiology.

More particularly, the present invention relates to human 92
Human 92kD using a monoclonal antibody that specifically binds to a specific amino acid sequence of kDa gelatinase
a relates to a method for immunologically quantifying gelatinase.

[0003]

BACKGROUND ART The extracellular matrix is composed of adhesive glycoproteins such as collagen, proteoglycan, elastin, fibronectin and laminin. Matrix metalloprotease (MMP) plays an important role in the decomposition of these matrix components.
The 92 kDa (kilodalton) gelatinase, designated MMP-9, is described by Wilhelm et al. (J. Biol. Che.
m. 29,1989,17213-17221), SV40-transformed human fetal lung fibroblasts, human lung macrophages, external nuclear leukocytes, monocytic leukemia U937 cells,
It has been reported that it is produced by fibrosarcoma HT1080 cells and keratinocytes and has the activity of degrading gelatin and type IV collagen.

As described above, human 92 kDa gelatinase (hereinafter referred to as human MMP-9) is an enzyme that decomposes gelatin and type IV collagen (type IV collagenase), but Bergmann et al. Human MMP-9 was prepared, and human MMP-9 was quantified by a competitive reaction or a sandwich method using the rabbit polyclonal antibody against the human MMP-9 (J. Clin. Chem. Clin. Bioche.
m. 27,1989,351-359). In this method, a type IV collagenase substrate (gelatin) is used as a solid phase carrier.
Is adsorbed, reacted with an antigen in a sample, and further reacted with a polyclonal antibody.

However, since this method uses a polyclonal antibody, it has drawbacks such as long reaction time and low accuracy as a quantification method.

[0006]

OBJECT OF THE INVENTION The object of the present invention is to provide a method for quantifying human MMP-9 in a test sample accurately, simply and quickly by using a monoclonal antibody which specifically binds to human MMP-9. To provide.

[0007]

DISCLOSURE OF THE INVENTION The present invention uses two types of monoclonal antibodies that specifically bind to human 92 kDa gelatinase as an antibody that binds to a solid phase carrier or an antibody that imparts a labeled substance, and is immunized by a sandwich method. The present invention provides a method for quantifying human 92 kDa gelatinase, which is characterized by performing a biological measurement.

In the method of the present invention, human MMP- is used as an antibody to be bound to a solid phase carrier or an antibody to which a label is attached.
It is characterized in that each of the monoclonal antibodies specifically binds to 9 different antigenic determinants.

In the method of the present invention, in carrying out the antigen-antibody reaction, an appropriate amount of polyoxyethylene lauryl alcohol ether is added to the reaction solution to reduce nonspecific binding and interference of the antigen-antibody reaction with serum proteins. Therefore, the amount of human MMP-9 in the sample can be more accurately measured. The amount of the polyoxyethylene lauryl alcohol ether used is preferably 0.02 in the concentration range.
~ 0.1% (w / v).

In the quantification method of the present invention, an immunological measurement method is used, and the solid phase carrier in that case is a ball made of polystyrene, polycarbonate, polypropylene, or polyvinyl that well adsorbs proteins such as antibodies. Various materials and forms such as microplates, sticks, microparticles or test tubes can be arbitrarily selected and used. On the other hand, as the antibody to which the labeled substance is added, DEAE-Sep is used after the antibody-containing substance is fractionated with ammonium sulfate.
Anion exchange gels such as hacel and IgG fractions as well as specific binding Fab 'obtained by digestion with pepsin followed by reduction can be used. Examples of labeled substances in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, radioisotopes and the like.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of anti-human MMP-9 monoclonal antibody (a) Preparation of human MMP-9 polypeptide Human MMP-9 polypeptide was prepared as described in J. Biol. Che
m. , 264, 1989, 17213-17221 and the amino acid sequence of Wilhelm et al. Human MMP-9 polypeptides shown in Table 1 (P-1 to P-3)
The peptide synthesizer 9600 (Milligen /
It was synthesized by biosearch. In addition, cysteine was introduced into the C-terminal of each peptide. Synthetic peptide purity is about 70%
The following is μBondashere (5μ, C18
-100Å, Waters) column and purified by high performance liquid chromatography.

(B) Preparation of complex of each polypeptide and bovine serum albumin or each polypeptide and keyhole limpet hemocyanin 2 mg bovine serum albumin (BSA) in 1 ml of 0.1M
One dissolved in phosphate buffer (pH 7.0), or one dissolved in 2 mg keyhole limpet hemocyanin (KLH) in 0.1 ml of 0.1 M phosphate buffer (pH 7.5) and 1.85 mg N- (ε A mixture of maleimidecaproyloxy) succinimide dissolved in 200 μl of dimethylformamide was mixed and incubated at 30 ° C. for 30 minutes. Next, the above mixture is applied to a PD-10 column (Sephadex G-25M, Pharmacia) equilibrated with 0.1 M phosphate buffer (pH 7.0) to bind BSA or maleimide bound with maleimide. KLH was collected and concentrated to 1.5 ml or less. A 50-fold molar amount of each human MMP-9 polypeptide synthesized in (a) above with respect to maleimide-conjugated BSA or maleimide-conjugated KLH was added to 1 ml of 0.1 ml.
It was mixed with that dissolved in M phosphate buffer (pH 7.0). Incubate at 4 ° C for 20 hours, and MMP-
9 polypeptide-BSA and MMP-9 polypeptide-KLH complexes were prepared respectively.

(C) Preparation of antibody-producing cells 250 μg of each complex prepared by the method of (b) above was combined with complete Freund's adjuvant for 8 weeks old Balb / c.
Each female mouse was intraperitoneally administered for the first immunization. 15
After 200 days, 200 μg of each complex dissolved in 0.1 M phosphate buffer (pH 6.0) was intraperitoneally administered to each mouse that had been initially immunized, and boosted. After 38 days, 70 μg of each complex was intravenously or
g was intraperitoneally administered for the final immunization. Three days after that, the spleen was removed to prepare a splenocyte suspension.

(D) Cell fusion (1) The following materials and methods were used. RPMI 1640 medium: RPMI 1640 (Flo
w Lab. ) To sodium bicarbonate (24 mM), sodium pyruvate (1 mM), potassium penicillin G (50 U / ml), streptomycin sulfate (50 μg)
/ Ml) and amikacin sulfate (100 μg / ml) were added, pH was adjusted to 7.2 with dry ice, and sterilization filtration was performed with a 0.2 μm Toyo Membrane filter.

NS-1 medium: Calf fetal bovine serum (MA Biopro) obtained by sterilizing and filtering the above RPMI 1640 medium.
Ducts) was added to a concentration of 15% (v / v).

PEG 4,000 solution: RPMI 16
Polyethylene glycol 4,000 (PEG
4,000, Merck & Co. ) 50% (w /
w) to prepare a serum-free solution.

8-Azaguanine-resistant myeloma cell SP
2 (SP2 / 0-Ag14) fusion is Select
ed Method in Cellular Imm
unology (eds. BB Misshella
nd S. M. Shiigi), W.W. H. Freema
n and Company (1980), 351-3.
The method of Oi et al.

(2) Nucleated spleen cells (live cell ratio 100%) and myeloma cells (live cell ratio 1) prepared in the above (c)
00%) was fused at a ratio of 5: 1. Spleen cells and myeloma cells were washed separately in RPMI 1640 medium as described above, then suspended in the same medium and mixed at the above ratio for fusion. Using a polypropylene centrifuge tube (Iwaki Glass) with a capacity of 250 ml, 40 ml of RPMI 1640
The supernatant was completely aspirated by centrifugation at 400 xg for 10 minutes in the medium. To the precipitated cells, 6.0 ml of a PEG 4,000 solution heated at 37 ° C was added dropwise over 1 minute with gentle stirring, and further stirred for 1 minute to resuspend and disperse the cells. Then 37
6.0 ml of RPMI 1640 medium warmed at ℃ was added dropwise for 1 minute. After repeating this operation once more, the same medium 4
2.0 ml was added dropwise over 2-3 minutes with constant stirring to disperse the cells. This was centrifuged at 400 × g for 10 minutes, and the supernatant was completely removed by suction. Next, 3
60 ml of 7 ° C. warmed NS-1 medium was added rapidly and the large clumps of cells were dispersed by careful pipetting with a 10 ml pipette. Further, 120 ml of the same medium was added to dilute, and 6.0 × 10 ⁵ cells / 0.1 ml of cells were added to a 96-well microwell made of polystyrene (Iwaki Glass). 7% of the above microwell containing cells
The cells were cultured in carbon dioxide / 93% air at a temperature of 37 ° C. and a humidity of 100%.

(E) Selective growth of hybridoma by selective medium (1) The following mediums are used. HAT medium: In addition to the NS-1 medium described in (d) above, hypoxanthine (100 μM) aminopterin (0.4
μM) and thymidine (16 μM).

HT medium: It has the same composition as the above HAT medium except that aminopterin was removed.

(2) The following day (1) after the start of culture in (d) above
On the day), 2 drops (about 0.1 ml) of HAT medium was added to the cells with a Pasteur pipette. Half of the medium (0.1 ml) was replaced with fresh HAT medium on days 2, 3, 5, 8, 11 and half of the medium was replaced with fresh HT medium on day 14. Every 3-4 days thereafter, half of the medium was replaced with fresh HT medium. In about 2 weeks, sufficient growth of hybridoma is usually observed. For all wells grown with hybridoma, the solid phase-antibody binding test method (ELIS) described in (f) below.
Positive wells were checked according to A). Next, 1 ml of HT medium containing 10 ⁷ mouse thymocytes as a feeder
Was added to a polystyrene 24-well cell well (Iwaki Glass), and the entire contents of each positive hybridoma detected above were transferred. This was cultured in the presence of 7% carbon dioxide gas at 37 ° C. for about 1 week in the same manner as in (d) above. In the meantime, 0.5 ml of the supernatant in each well was replaced with 0.5 ml of fresh HT medium once or twice. When the hybridomas were sufficiently grown, the positiveness was confirmed again by the ELISA method, and each of them was cloned by the limiting dilution method described in the following item (g). The residual liquid used for cloning was transferred to a polystyrene 25 cm ² tissue culture flask (Iwaki Glass) to prepare a sample for cryopreservation.

(F) Anti-human MMP by ELISA method
-9 Search for antibody-producing hybridoma Anal. Biochem. 104, 205-214
The method of Rennard et al. Described in (1980) was slightly modified. This method is suitable for detecting hybridoma antibodies. 96-well microtitration plate (FlowLab.) With 100 ng of each human MM
Coated with P-9 polypeptide, then the uncoated portion was blocked with 1% BSA. A part of the supernatant of the hybridoma growing well obtained in (e) above was added thereto and incubated at room temperature for about 1 hour. Horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Cappel Lab.) Was added as a secondary antibody, and further added at room temperature to about 1
Incubated for hours. Next, add the substrate hydrogen peroxide and o-phenylenediamine, and measure the degree of brown produced by the microplate reader (MPR-A4, Toyo Soda).
Was used to measure and determine the absorbance at 492 nm.

(G) Cloning Since two or more hybridomas may grow in each well after the above operation (e), cloning is performed by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma. get. A cloning medium containing 10 ⁷ mouse thymocytes as a feeder per 1 ml of NS-1 medium was prepared, and 5 wells per well in 36 wells, 36 wells and 24 wells of 96-well microwells.
And 0.5 hybridomas were added. The fifth day,
On day 12, all wells were supplemented with about 0.1 ml of NS-1 medium. A sufficient amount of hybridoma growth was observed 14 to 15 days after the start of cloning, and an ELISA method was performed on a group in which the colony formation negative well was 50% or more. When all the tested wells are not positive, the number of colonies in the antibody-positive wells is confirmed, and 4 to 6 wells in which one colony is confirmed are selected and recloned.
Finally, as shown in Table 2, monoclonal antibody-producing hybridomas against human MMP-9 polypeptide were obtained.

(H) Proliferation of monoclonal antibody in vitro and in vivo Proliferation of monoclonal antibody is carried out by a conventional method. That is, each of the obtained hybridomas was cultured (in vitro growth) in an appropriate culture medium such as NS-1 medium, and 10 to 10
It was possible to obtain a monoclonal antibody at a concentration of 100 μg / ml. On the other hand, in order to obtain a large amount of antibody, an animal syngeneic with the origin of splenocytes and myeloma cells (Balb / c
To each mouse, 0.5 ml of a tumor formation promoter pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chem.) Was intraperitoneally administered to each mouse. After 1 to 3 weeks, 1 × 10 ^{7 of} each hybridoma was similarly intraperitoneally administered, and 1 to 2 weeks after that, ascites containing 4 to 7 mg / ml of the monoclonal antibody produced in vivo could be obtained. .

(I) Heavy chain and light chain of monoclonal antibody According to the above-mentioned ELISA method, the culture supernatant of each of the monoclones obtained in (g) above was placed on a microtitration plate coated with human MMP-9 polypeptide. Was added. Then, after washing with PBS, an isotype-specific rabbit anti-mouse Ig antibody (Zymed Lab.)
Was added. After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) antibody was added,
Hydrogen peroxide and 2,2'-azinodi (3
-Ethylbenzothiazoline sulfate) was used to determine each heavy and light chain. The results are summarized in Table 2.

(J) Purification of Monoclonal Antibodies Each ascites fluid obtained in (h) above was fractionated with 40% saturated ammonium sulfate, and then the IgG1 class antibody was diluted with 0.
The protein A Affigel (Bio-Rad) column equilibrated with 1.5 M glycine-NaOH buffer (pH 8.9) containing 5 M sodium chloride was adsorbed, washed with the above washing solution, and then 0.1 M citrate buffer (pH 5. Purified by eluting with 0).

Example 2 Preparation of Human MMP-9 (a) Cell Culture Human fibrosarcoma HT-1080 (purchased from American Type Culture Collection) in NS-1 medium, 37
Culturing was performed in the presence of 5% carbon dioxide at 3 ° C for 3 to 4 days. The culture supernatant was discarded, and an appropriate amount of 10 mM phosphate buffer solution (pH 7.4) containing 0.1 M sodium chloride (hereinafter referred to as PBS) was added and gently washed with shaking. Next, PBS containing 0.125% trypsin and 0.01% sodium ethylenediaminetetraacetate (hereinafter referred to as EDTA) was added, and gently shaken to detach the cells from the culture flask (within 1 minute).
After that, an appropriate amount of NS-1 medium was added. After centrifugation, discard the supernatant,
After adding an appropriate amount of NS-1 medium and suspending the cells, about 2 × 10 ⁵ cells / ml of cells were added to a new culture flask, and 3
The cells were cultured at 7 ° C in the presence of 5% carbon dioxide for 3 to 4 days.

(B) Cell stimulation The tumor necrosis factor (TNF-α) is allowed to act on the cultured cells. The culture broth cultured in the previous section (a) was discarded, about 100 ml of RPMI 1640 medium was added, and lactalbumin hydrolyzate (Gibco) and TNF-α (Gen) were added to a final concentration of 0.2% and 100 U / ml.
each). The culture supernatant was allowed to stand at 37 ° C in the presence of 5% carbon dioxide for 7 to 10 days, and the final concentration of the culture supernatant was 10
EDTA was added so that the concentration would be mM, which was used as a material for preparing human MMP-9.

(C) Preparation of human MMP-9 Ammonium sulfate was added to the culture supernatant prepared in the previous section (b) so that the final concentration was 80%, and an appropriate amount of PBS was added to the precipitate after centrifugation to dissolve it. It was dialyzed against PBS. M
In order to remove MP-2, the solution after dialysis was treated with anti- (MMP-
2) It was applied to an antibody (clone No. 43-3F9) binding column, and the flow-through fraction was collected. In addition, anti (MMP
-9) It is applied to an antibody (clone No. 57-13D8) binding column, and the adsorbed protein is subjected to 0.1M phosphate buffer (p
H6.0) followed by 0.1 M acetate buffer (pH 4.0)
Eluted at. Fractions eluted at pH 6 contained MMP-9
And was purified to almost a single component on SDS electrophoresis. On the other hand, SDS was added to the fraction eluted at pH 4.
Electrophoresis, MMP-9 and Tissue inhibi
tor of metalloproteinases
(Hereinafter referred to as TIMP) is present, and it is considered that a complex of MMP-9 and TIMP was obtained. MMP-9 eluted at pH 6 was used as a standard antigen for enzyme immunoassay (hereinafter referred to as EIA) to be described later.

Example 3 Preparation of enzyme labeled antibody (a) Preparation of IgG-POD complex 1) Preparation of SH group labeled IgG Immunoassay 4,209-327,1
Mouse anti-human MMP-9 IgG-POD conjugates were prepared according to the method of Ishikawa et al. Monoclonal antibody (IgG; clone Nos. 56-2A4) that was recognized to react with human MMP-9.
And 57-13D8) in 0.1 M phosphate buffer (pH
It was dialyzed against 6.5), 100 times mol of S-acetylmercaptosuccinic anhydride was added as a dimethylformamide solution to IgG contained in the solution, and the mixture was added at 30 ° C. for 3 times.
Incubated for 0 minutes. Next, 100 μl of 0.1 M Tris-hydrochloric acid buffer solution (pH 7.0), 0.1 M E
Add 10 μl of DTA solution (pH 6.0) and 100 μl of 1M hydroxylamine solution (pH 7.0), and add 30 ° C.
After standing for 5 minutes, Sephadex G equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA
Gel filtration at -25, SH group labeled mouse anti-human MMP-
9 IgG was obtained.

2) Preparation of maleimide-labeled peroxidase (POD) POD was dissolved in 0.1M phosphate buffer (pH 7.0) to a concentration of 10 mg / ml, and the molar amount was 25 times the POD amount. N- (ε-maleimidocaproyloxy) succinimide (EMCS) was added as a dimethylformamide solution, and the mixture was reacted at 30 ° C. for 30 minutes. This reaction solution was subjected to gel filtration with a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer (pH 6.0) to collect a maleimide-labeled POD fraction.

3) Preparation of IgG-POD Complex To about 1 mol of the SH group-labeled IgG prepared in 1) above, about 5 mol of the maleimide-labeled POD obtained in 2) above was added,
It was left standing at 4 ° C for 20 hours. Ultrogel equilibrated with 0.1 M phosphate buffer (pH 6.5).
Gel filtration with AcA 44 column, mouse anti-human MMP
The -9 IgG-POD complex fraction was collected. BSA and thimerosal were added to 0.1% and 0.005%, respectively, and stored at 4 ° C.

(B) Preparation of Fab'-POD complex 1) Preparation of Fab 'Mouse anti-human MMP-9 IgG (clone No. 56)
-2A4) in 0.1 M citrate buffer (pH 3.5)
It was dialyzed into 1%, and 1% (w / w) pepsin was added to the amount of IgG, and digested at 37 ° C. for 18 hours. 2 in that digestive juice
The digestion reaction was stopped by adding M Tris solution to adjust the pH to 7.0, and 0.1 M phosphate buffer (pH
The F (ab ') ₂ fraction was collected by gel filtration on an Ultrogel AcA54 column equilibrated with 7.0). Next, the F (ab) ₂ fraction was dialyzed against 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA, and aminoethanethiol (MEA) was added thereto so that the final concentration was 10 μM, and the mixture was incubated at 37 ° C. for 1 hour. Reduced for 5 hours. After that, 0.1 mM phosphate buffer containing 5 mM EDTA (pH
Gel filtration was performed using an Ultrogel AcA54 column equilibrated with 6.0) to collect the Fab ′ fraction.

2) Preparation of Fab'-POD complex For Fab 'prepared in 1) above, the above (a)-
2) Maleimide-labeled PO prepared according to the method described in the above item
D was added in an equimolar amount, and 5 mM ED was added so that the final concentration of Fab ′ and maleimide-labeled POD was 100 μM.
It was diluted with 0.1 M phosphate buffer containing TA (pH 6.0). After leaving this mixture at 4 ° C. for 20 hours, unreacted thiol groups were blocked with a 10-fold molar amount of N-ethylmaleimide with respect to Fab ′. This was Ultrogel AcA equilibrated with 0.1 M phosphate buffer (pH 6.5).
After gel filtration with a 54 column and collection of Fab′-POD complex fractions, BSA and thimerosal were each added to 0.1%.
And 0.005% were added and the mixture was stored at 4 ° C.

Example 4 Quantitative method of human MMP-9 (a) Preparation method of monoclonal antibody binding carrier Immunoassay 4,209-327 (1
983) according to the method of Ishikawa et al.
Mouse anti-human MMP-9 IgG (clone Nos. 5
6-6D1, 56-2A4, and 57-13D8) were each dissolved in 0.1M phosphate buffer (pH 7.0) containing 0.1% sodium azide to obtain 100 μg / ml (A _280).
= 0.15). The monoclonal antibody solution was added to a 96-well microplate at 100μ per well.
Polyethylene balls (φ6.5 mm, ICHIKO) were immersed in the antibody solution or left standing at 4 ° C. for 24 hours. Next, the monoclonal antibody solution was removed, washed twice with each physiological saline solution, immersed in a 10 mM phosphate buffer solution (pH 7.0) containing 1% BSA-0.1 M sodium chloride, and stored at 4 ° C.

(B) One-step sandwich EIA method 1% BSA, 0.1 M sodium chloride and 10 mM
Purified human MMP-9 diluted with EDTA-containing 30 mM phosphate buffer (pH 7.0) (hereinafter referred to as buffer A) or a sample containing human MMP-9 was added to a 96-well vinyl plate (Falcon) or a plastic tube (1).
10 μl or 20 μl was added to 2 × 75 mm). Next, IgG (clone No. 56-2A4) -POD or Fab '(clone No. 56-2A4) -P prepared in Example 3- (a) or (b) above.
Buffer A so that OD complex becomes 3000 ng / ml
And diluted to 100 μl or 300 μl, respectively, and mixed into the vinyl plate or plastic tube. 100 μl of this mixed solution was added to the antibody-bound plate prepared in the above (a) or an antibody-bound ball was added to this mixed solution and allowed to react at room temperature for 1 hour to prepare a physiological saline solution or 10 mM containing 0.1 M sodium chloride. 3 with phosphate buffer (pH 7.0) (hereinafter referred to as buffer B)
Washed twice. Next, 0.1M containing 0.02% hydrogen peroxide
4m dissolved in citric acid-phosphate buffer (pH 4.9)
100 g / ml o-phenylenediamine per well
Add μl or 300 μl per tube, and mix at room temperature for 3
After reacting for 0 minutes, 100 μl of 2N sulfuric acid or 1 ml was added to stop the reaction. A _{492 of} this reaction mixture is a microplate reader (MPR-A4 Toyo Soda)
Alternatively, Micro Flow Spectrophotometer U
V730 (Shimadzu) was used for measurement, and the amount of human MMP-9 in the sample was determined from the calibration curve.

Table 3 shows the above operation method. By the plate method described in Table 3, the clone No. IgG-POD conjugate from 56-2A4 and 3 prepared in (a) above
It was examined whether a standard curve can be drawn using the antibody binding carriers of various species (Table 4). Clone No. I from 56-2A4
When gG was used, since it recognized the same site as the enzyme-labeled antibody, no increase in absorbance was observed even on the high concentration side of the standard antigen (negative control).
Clone No. In the case of 56-6D1, clone No. 5
Since 6-2A4 and the immunogen are the same, it seems that they recognize almost the same site in the antigen. Clone No. 57-1
When IgG from 3D8 was used, absorbance dependent on the concentration of standard antigen was obtained. In the calibration curve, linearity was observed up to the concentration of standard antigen 37 ng / ml. Therefore, in the present invention, clone Nos. Antibodies from 57-13D8 and 56-2A4 were used in the immunoassays below. However, the present invention is not limited to this.

(C) Two-step sandwich EIA method buffer A or 1% BSA, 0.125M sodium chloride, 10 mM EDTA and 0.05% bridge-
35-containing 30 mM phosphate buffer (pH 7.0) (hereinafter,
Purified human MMP-9 diluted with buffer solution C) or the specimen is added to a vinyl plate or tube at 20 μm each.
1 and then 100 μl or 300 μl of buffer solution A or C were added and mixed. 100 μl of this mixture
Was added to the antibody-bound plate prepared in the above (a) or to this mixed solution, and the mixture was allowed to react at room temperature for 1 hour. After the reaction, it was washed with physiological saline or buffer B, and IgG (clone No. 56-2A4) -POD or Fab '(clone No. 56- prepared in Example 3- (a) or (b)) was used. 2A4) -POD
The complex was diluted with buffer solution A to 1500 ng / ml, and 100 μl or 300 μl of each was added.
After reacting at room temperature for 1 hour, the following operation is performed as described in (b) above.
The procedure was performed in the same manner as in Section. The above operating method is shown in Table 5.

(D) Measurement of human MMP-9 in serum by sandwich assay method Purified human MM was prepared by the plate one-step method and plate two-step method described in the above (b) and (c).
P-9 (standard antigen) and serum of a healthy subject were measured as samples (Tables 6-1 and 6-2). When using the IgG-POD complex, the absorbance of the standard step was higher in the 1-step method, and the linearity of the calibration curve was that of the standard antigen 31-1000n.
Although it was observed in the range of g / ml, almost no antigen was detected in the serum. In the two-step method, the absorbance is low, but the linearity of the calibration curve is 16-1000 ng / standard antigen.
It was observed within the range of ml, and the antigen in serum could also be detected. When Fab′-POD complex was used, Ig
Compared to the case of using the G-POD complex, the absorbance is lower and the standard antigen is within the range of 31-1000 ng / ml in the 1-step method, and the standard antigen is in the range of 16-1000 ng / ml in the 2-step method.
Linearity was observed in the calibration curve within the range of ml.

Since the 1-step method could not detect the antigen in the serum, only the 2-step method was performed by the ball method (Table 6-3). When using the IgG-POD complex,
The linearity of the calibration curve was observed in the range of standard antigen 94-3000 ng / ml, but when Fab'-POD complex was used, the absorbance was low even with standard antigen 3000 ng / ml.

Among the standard antigens, the plate method had a higher absorbance, and the linear minimum minimum concentration in the calibration curve was also lower in the plate method. It was also considered that the detection efficiency was high for the antigen in serum. In the plate two-step method, the absorbance of the standard antigen 0 ng / ml was measured 6 times, the average value (x) and the standard deviation (SD) were calculated, and x + 2S
When the standard antigen concentration corresponding to D was used as the sensitivity, the sensitivity was about 15 ng / ml.

Example 5 Effect of Addition of Polyoxyethylene Lauryl Alcohol Ether Antibody-binding carrier and standard antigen solution or sample (serum of healthy person)
At the time of the reaction of the medium antigen, the reaction solution was 0.05% polyoxyethylene lauryl alcohol ether
Buffer C containing 35) was used to examine the effect of serum protein on interference. Buffer A was used as a control. A known amount of standard antigen was added to the serum, and measurement was performed by the two-step sandwich EIA method, and the recovery rate of the added standard antigen was examined (Table 7). When the buffer solution A is used, the average recovery rate of the standard antigen is 84%, and when the buffer solution C is used,
Although a decrease in absorbance due to Bridge-35 was observed, almost 100% of the added standard antigen amount was recovered. Therefore, it was confirmed that when the buffer solution C was used, the antigen amount in 20 μl of serum could be read as an accurate value.

Example 6 Substrate capt
ure immunoassay method (a) Method for preparing gelatin-bound carrier Gelatin (derived from pig skin, calf skin, derived from bovine bone, etc.) was adjusted to 1 mg / ml with 0.1 M phosphate buffer (pH).
It was dissolved in 7.0), 100 μl was added to each well of a 96-well microplate, and the plate was allowed to stand at 4 ° C. for 24 hours. Next, the gelatin solution was removed, and after washing twice with physiological saline,
300 μl of 30 mM phosphate buffer (pH 7.0) containing 1% BSA and 0.1 M sodium chloride was added, and the mixture was stored at 4 ° C.

(B) Substrate captu
Re immunoassay method 1) 1-step assay method The procedure was as described in Example 4- (b). 10 μl or 20 μl of the standard antigen or sample diluted with buffer solution A was added to a 96-well vinyl plate. Next, IgG (clone Nos. 56 prepared in Example 3- (a)).
-2A4 or 57-13D8) -POD complex was diluted with buffer A to 500 ng / ml or 3000 ng / ml, and 100 μl was added to the vinyl plate.
1 by 1 and mixed. Each 100 μl of this mixed solution was added to the gelatin-bonded plate prepared in the above (a), reacted for 1 hour at room temperature, and washed 3 times with physiological saline or buffer solution B. Next, 100 μl of 4 mg / ml o-phenylenediamine dissolved in 0.1 M citric acid-phosphate buffer (pH 4.9) containing 0.02% hydrogen peroxide was added to each well, and the mixture was reacted at room temperature for 30 minutes, The reaction was stopped by adding 100 μl of 2N sulfuric acid. The A ₄₉₂ value of this reaction mixture was measured.

2) Two-step assay method In accordance with the method described in Example 4- (c), 10 μl or 20 μl of the standard antigen or sample was placed on a vinyl plate.
Then, 100 μl of buffer solution A was added and mixed. 100 μl of this mixed solution was added to the gelatin binding plate prepared in the above (a), and the mixture was reacted at room temperature for 1 hour. After the reaction, the wells were washed, and the IgG prepared in Example 3- (a) was used.
(Clone Nos. 56-2A4 or 57-13D
8) -POD complex, 500 ng / ml or 3 each
It was diluted with the buffer solution A to 000 ng / ml, 100 μl was added to each well, and the mixture was reacted at room temperature for 1 hour. The following operations were performed in the same manner as in the above item (b).

(D) Substrate captu
Measurement of Human MMP-9 in Serum by Re Immunoassay IgG that recognizes the C-terminal or N-terminal amino acid sequence of human MMP-9 (respectively clone Nos. 56-2A4
Alternatively, 57-13D8) -POD complex was used, and a sample (serum of healthy person) was measured by the one-step assay method or the two-step assay method according to the method described in (b) above. The results are shown in Tables 8-1 and 8-2. In the 1-step method, the absorbance of the standard antigen was high, but the antigen in serum could not be detected. On the other hand, in the two-step method, although the absorbance of the standard antigen was low, the antigen in serum could be quantified. In the one-step method, clone Nos. 56-2A4 or 57-
When the IgG-POD complex from 13D8 was used, linearity of the calibration curve was observed in both cases within the range of standard antigen 31-1000 ng / ml, and in the two-step method, clone No. When the IgG-POD complex from 56-2A4 was used, within the range of standard antigen 63-2000 ng / ml, while clone no. When the IgG-POD complex from 57-13D8 was used, the standard antigen 125
Linearity was observed in the calibration curve within the range of -2000 ng / ml.

The sensitivity or the range of linearity of the calibration curve can be changed at any time depending on the solid phase antibody concentration, standard antibody concentration, etc. used.

[0049]

[Table 1]

[0050]

[Table 2]

[0051]

[Table 3]

[0052]

[Table 4]

[0053]

[Table 5]

[0054]

[Table 6]

[0055]

[Table 7]

[0056]

[Table 8]

[0057]

[Table 9]

[0058]

[Table 10]

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Takashi Shintani             14 No. 1297 Nomura, Takaoka City, Toyama Prefecture (72) Inventor Kazushi Iwata             190 Igarito-cho, Takaoka City, Toyama Prefecture

Claims (3)

[Claims] 1. An immunological assay by a sandwich method using two types of monoclonal antibodies that specifically bind to human 92 kDa gelatinase as an antibody that binds to a solid phase carrier or an antibody that gives a labeled substance. A method for quantifying human 92 kDa gelatinase, which comprises: 2. A sample is obtained by adding an appropriate amount of polyoxyethylene lauryl alcohol ether to the reaction solution in carrying out an antigen-antibody reaction between a monoclonal antibody bound to a solid phase carrier and human 92 kDa gelatinase in the sample. Human 92kDa gelatinase in human is measured.
Quantitative method for gelatinase. 3. Human 9 is prepared by binding gelatin to a solid phase carrier.
The method for quantifying human 92 kDa gelatinase according to claim 1, wherein a labeled substance is added to a monoclonal antibody that specifically binds to 2 kDa gelatinase, and both gelatin and the labeled monoclonal antibody are used.