JP2609858B2 - Enzyme immunoassay for collagenase inhibitors - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human collagenase in human bone, skin, dental pulp, amniotic fluid, blood, synovial fluid, articular chondrocytes, synovial fluid cells, various tissue-derived fibroblasts, and fibrosarcoma cell culture. The present invention relates to a method for quantifying an inhibitor with high sensitivity.

Collagenase inhibitor (Tissue inhibitor of metalloprotease: TIMP) is used in human and other animal bone, skin, dental pulp, amniotic fluid, blood, synovial fluid and articular chondrocytes, synovial fluid cells, various tissue-derived fibroblasts, A protein present in the fibrosarcoma cell culture extracellular fluid (Murphy
Biochem. J. 195, 167-170, 1981; Welgus et al., J. Bio.
l. Chem. 258, 12259-12264, 1983; Kishi et al., J. Biochem.
96, 395-404, 1984 et al.). TIMP is a specific protein and is a single protein-chemical substance.

The present invention provides a method for quantifying a human collagenase inhibitor by an enzyme immunoassay based on a sandwich method using a monoclonal antibody against a bovine collagenase inhibitor.

That is, the present invention provides a monoclonal antibody against bovine collagenase inhibitor, which is a combination of two types of monoclonal antibodies that specifically bind to different antigenic determinants of human collagenase inhibitor, and one of which is used as a solid phase antibody. Another object of the present invention is to provide a method for quantifying a human collagenase inhibitor present in a sample by an enzyme immunological method by using the other as an antibody for enzyme labeling by a sandwich method.

Conventionally, as a means for quantifying a collagenase inhibitor, a method based on measurement of its biological activity is known.
However, J. Lab. Clin. Med. 75 , 258-263 (1970)
Proteins in serum or plasma as described by Eisen et al interfere with measurement of collagenase inhibitor activity, for example, alpha ₂ - for macroglobulin is present, accurately measure the collagenase inhibitor activity in such a case Is virtually impossible to do. If a collagenase inhibitor can be quantified specifically, accurately, and
Measurement (monitoring) of fluctuations in the concentration of collagenase inhibitor in blood during the course of treatment of a disease by administration of collagenase inhibitor, analysis of the content of collagenase inhibitor in raw materials in the production of medical collagenase inhibitor, or the use of collagenase inhibitor in products This is extremely important for measuring purity and the like.

The present inventors specifically and precisely quantify a collagenase inhibitor with a small amount of a sample using a monoclonal antibody against a bovine collagenase inhibitor and performing an enzyme immunoassay (EIA) based on the San Deutschia method with a small amount of sample. Completed the way to.

That is, the present invention uses a monoclonal antibody that specifically binds to a different antigenic determinant of bovine collagenase inhibitor as an antibody to be bound to a solid support and an antibody to give an enzyme label, A method for quantifying a collagenase inhibitor and a human collagenase inhibitor is provided.

In carrying out the method of the present invention, various carriers such as polystyrene beads as solid-phase carriers, polycarbonate, polypropylene, polyvinyl microplates, sticks, and test tubes that passively adsorb antigens and antibodies well are used. Can be used. On the other hand, as an antibody to be labeled with an enzyme, after the ammonium sulfate fractionation of the antibody-containing substance, DEAE-
An IgG fraction purified by an anion exchange gel such as Sephacel, and a specific binding portion Fab ′ obtained by digesting with pepsin and then reducing the same can also be used.

According to the method of the present invention, the antibody specifically binding to different antigenic determinants of bovine collagenase inhibitor is used as an antibody to be bound to a solid support and an antibody to be labeled with an enzyme.
Using a combination of different types of monoclonal antibodies, bovine collagenase inhibitor and human collagenase inhibitor can be quantified by a solid phase enzyme immunoassay.

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

Example 1 Preparation of Anti-Bovine Collagenase Inhibitor Monoclonal Antibody (a) Preparation of Plateau-Bovine Collagenase Inhibitor The root pulp of bovine unerupted dentinal teeth was prepared according to the method of the present inventors described in J. Biochem. 96, 395-404 (1984). Eagle MEM
Collagenase inhibitor was purified from the culture outside solution cultured in a medium (manufactured by Nissui Pharmaceutical Co., Ltd.) using Con A-Sepharose, Ultrogel AcA44 and DE-52 cellulose columns. Purification inhibitors are described in J. Mol. Biol. 80 , 579-599 (19
73), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG) according to the method of Laemmli et al.
Inspection by E) showed a single band with a molecular weight of about 32,000 daltons (D).

(B) Preparation of antibody-producing cells Two 6-week-old Balb / c female mice are first immunized with the purified bovine collagenase inhibitor described in (a) above in Freund's complete adjuvant band. Mouse to each
48 μg of bovine collagenase inhibitor is administered intraperitoneally as a 0.4 ml solution. Further, on the 30th day, 84 μg of bovine collagenase inhibitor dissolved in physiological saline is additionally immunized. Intraperitoneal administration on day 58 (95 μg / 50
(0 μl physiological saline), and after 3 days, the mouse spleen is taken out and spleen cells are prepared.

(C) Cell fusion (1) The following materials and methods are used.

RPMI 1640 medium: RPMI No. 1640 (Difco Laboratories)
Sodium bicarbonate (12 mM), sodium pyruvate (1 mM), L-glutamine (2 mM), potassium penicillin G (50 U / ml), streptomycin sulfate (50 μg / ml),
And amikacin sulfate (100 μg / ml), adjust the pH to 7.2 with dry ice, and remove bacteria with a 0.2 μm Toyo membrane filter.

NS-1 medium: Fetal calf serum (MABioproducts), which has been sterilized in the above RPMI 1640 medium, is added to a concentration of 15% (v / v).

PEG 4,000 solution: 50% polyethylene glycol 4,000 (PEG 4,000, Merck & CO., Inc.) in RPMI 1640 medium (w /
w) Prepare serum-free solution.

8-Azaguanine resistant myeloma cells NS-1 (P3-NS
Fusion with 1-1) is selected method in Cellular Immun
ology (ed.BBMishell and SMShiigi), WHFreema
n and Company (1980), 351-372, with minor modifications.

(2) The nucleated spleen cells prepared in the above (b) (viable cell rate 10
0%) and myeloma cells (100% viable cell rate) are fused at a ratio of 5: 1. The spleen cells and myeloma cells are separately washed in the above-mentioned RPMI 1640 medium. It is then suspended in the same medium and mixed in the above proportions for fusion. Using a 50 ml conical styrene resin test tube (Iwaki Glass), centrifuge in 40 ml of RPMI 1640 medium at 400 × g for 10 minutes, and completely aspirate the supernatant. 1.3 ml of a PEG 4,000 solution heated at 37 ° C. is added dropwise to the precipitated cells for 1 minute with gentle stirring, and the mixture is further stirred for 1 minute to resuspend and disperse the cells. Then 37 ℃
1.3 ml of warm RPMI 1640 medium is added dropwise over 1 minute. After repeating this operation once more, 9 ml of the same medium is added dropwise with stirring for 2 to 3 minutes to disperse the cells. 400x
g, centrifuge for 10 minutes and completely aspirate the supernatant. Next, 12.9 ml of NS-1 medium warmed at 37 ° C. is immediately added to the precipitated cells, and the large clumps of cells are carefully pipetted and dispersed using a 10 ml pipet. 26 ml of the same medium
And dilute, and add 6.0 × 10 ⁵ cells / 0.1 ml of cells per well to a polystyrene 96-well microwell (Iwaki Glass). The 96-well microwell to be used at this time is added with 0.2 ml of NS-1 medium as a pretreatment, kept in a carbon dioxide incubator (37 ° C.) overnight, and aspirated to remove the medium before use. The microwells containing the cells are cultured in 7% carbon dioxide / 93% air at a temperature of 37 ° C. and a humidity of 100%.

(D) Selective growth of hybridoma by selection medium (1) The medium used is as follows.

HAT medium: In addition to the NS-1 medium described in (c) above, hypoxanthine (100 μM) and aminopterin (0.4 μM)
M), and thymidine (16 μM).

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

(2) On the next day (day 1) after the start of the culture in (c), 2 drops (about 0.1 ml) of HAT medium are added to the cells using Pasteur pipette. On days 2, 3, 5, 8, and 11, half (0.1 ml) of the medium is replaced with fresh HAT medium, and on day 14, half of the medium is replaced with fresh HT medium. Thereafter, every three to four days, half of the medium is replaced with fresh HT medium. Usually, sufficient hybridoma growth is observed in 2 to 3 weeks. Positive wells are checked by the solid phase-antibody binding test (ELISA) described in the following section (e) for all wells in which hybridomas have grown. Next HT medium 1 containing 10 ⁷ mouse thymocytes as Fuida
The whole contents of each of the positive hybridomas detected above are transferred using a solution obtained by adding ml to a polystyrene 24-well cell well (Iwaki Glass). This is cultured for about 1 week at 37 ° C. in the presence of 7% carbon dioxide as in (c) above. During that time, 0.5 ml of the supernatant of each well is replaced with 0.5 ml of fresh HT medium once or twice. When the hybridomas have grown sufficiently, the positivity is reconfirmed by ELISA, and each is cloned by the limiting dilution method described in the following section (f). The residual solution used for cloning is transferred to a 25 cm ³ tissue culture flask (Iwaki Glass) made of polystyrene to prepare a sample for cryopreservation.

(E) Search for hybridoma producing anti-bovine collagenase inhibitor antibody by solid phase-antibody binding test (ELISA) Rennard described in Anal. Biochem. 104, 205-214 (1980).
A slightly modified method is used. This method is suitable for detecting a hybridoma antibody. Insert a 96-well microtitration plate (Flow Laboratories, Inc.)
Coated with 5 to 1.0 μg of bovine collagenase inhibitor, and then the uncoated part was 1% bovine serum albumin (BS
Block with A). A part of the supernatant of the hybridoma growth well obtained in the above (d) was added thereto, and the mixture was added at room temperature for about 1 hour.
Incubate for hours. Horseradish peroxidase-labeled goat anti-mouse immunoglobulin (ca
ppel Lab.) and incubate at room temperature for about 1 hour. Next, hydrogen peroxide and o-phenylenediamine as a substrate are added to determine the degree of brown color produced qualitatively with the naked eye, or using a corona two-wavelength microplate photometer (MTP-22, Corona Electric). And measure the absorbance at 500 nm.

(F) Cloning After the operation (d), since there is a possibility that two or more types of hybridomas may grow in each well, cloning is performed by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma. The cloning medium containing 10 ⁷ mouse thymocytes were prepared as NS-1 medium 1ml per Fuida, 36 wells of a 96-well micro-well, five wells per 36 well and 24-well, one and 0.5 hybridomas Add. On days 5 and 12, about 0.1 ml of NS-1 medium is added to all wells. After 14 to 15 days from the start of cloning, sufficient hybridoma growth is observed, and ELISA is performed on a group in which the number of colony formation negative wells is 50% or more. If all the tested wells are not positive, the number of colonies in the antibody-positive well is confirmed, and 4 to 6 wells in which one colony is confirmed in the wells are selected and recloned. Finally, 17 monoclonal antibody-producing hybridomas against bovine collagenase inhibitor were obtained.

(G) In Vitro Propagation and In Vivo Propagation of Monoclonal Antibody Propagation of the monoclonal antibody is carried out by a conventional method. That is,
Monoclonal antibody is used for each hybridoma obtained by NS
-1 cultured in an appropriate culture medium such as medium (in vitro propagation)
It can be obtained from the culture supernatant (monoclonal antibody concentration is 10-100 μg / ml). On the other hand, in order to obtain a large amount of antibodies, tumor-promoting agents pristane (2,6,10,14-tetramethylpentadecane, Aldrich Che) were used in animals (Balb / c, mouse) syngeneic to spleen cells and myeloma cells.
mical) was intraperitoneally administered to each mouse at 0.5 ml.
One week later, 1 × 10 ⁷ hybridomas were similarly administered intraperitoneally in vivo, and after 1-2 weeks,
Ascites with a monoclonal antibody concentration of 4-7 mg / ml can be obtained.

(H) Isotype of monoclonal antibody Each ascites obtained in the above (g) is first bound to a microtitration plate coated with a bovine collagenase inhibitor according to the ELISA method described above. After washing with PBS, isotype-specific rabbit anti-mouse Ig
Add antibody (Zymed Laboratories). After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG
(H + L) antibody was added and detected using 2,2'-azino-di (3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide as substrates. The results are summarized in the first section below.
It is shown in the table. Of the resulting monoclonal antibodies against bovine collagenase inhibitor, 15 had immunoglobulin chains γ1 / κ, one had γ2a / κ, and one had γ2b / κ.

(I) Purification of monoclonal antibody Each ascites fluid obtained in (g) above was subjected to ammonium sulfate fractionation (40% saturation)
After that, 40 mM phosphate buffer containing 0.06 M sodium chloride, pH
The non-adsorbed fraction of DEAE-Sephacel (Pharmacia) equilibrated in 8.0 was collected, and the IgG fraction was further separated in Sephacr equilibrated with 50 mM phosphate buffer containing 0.42 M sodium chloride, pH 7.4.
The gel was passed through a yl S-300 Superfine (Pharmacia) column to separate FCS and mouse-derived proteins in the medium.
Removed.

Example 2 Cross-reactivity between bovine dental pulp collagenase inhibitor and monoclonal antibody (a) Method for preparing enzyme-labeled monoclonal antibody (Fab'-POD complex) (1) Preparation of Fab 'fraction Obtained in Example 1 (i) The IgG fraction was dissolved in 0.1 M acetate buffer (pH 4.2) containing 0.1 M sodium chloride, and the solution was digested with pepsin as described below. That is, 2% (w / w) of pepsin was added to IgG in the above fraction, and digestion was performed at 37 ° C. for 24 hours. Further, the digestion reaction was stopped by adding 2 M Tris solution to the digest to adjust the pH to 7.0, and using an Ultrogel AcA 44 column (manufactured by LKB) equilibrated with 0.1 M phosphate buffer (pH 7.0). The F (ab ') ₂ fraction was collected by gel filtration.

Next, the F (ab ') ₂ fraction was dialyzed in a 0.1 M phosphate buffer (pH 6.0) containing ethylenediaminetetraacetic acid (EDTA), and aminoethanethiol (ME) was adjusted to a final concentration of 10 mM.
A) and reduce at 37 ° C. for 1.5 hours.
Ultrogel AcA4 equilibrated with M phosphate buffer (pH 6.0)
The gel was passed through four columns, and the Fab 'fraction was collected.

(2) Preparation of Maleimide-Labeled POD Fraction Apart from the above operation (1), horseradish-derived peroxidase (POD) was labeled with maleimide as described below. That is, POD is dissolved in a 0.1 M phosphate buffer (pH 7.0) in an amount of 10 mg / ml, and a 25-fold molar amount of N- (ε-maleimidocaproyloxy) succinimide ( EMCS) as a dimethylformamide solution
The reaction was performed at 30 ° C. for 30 minutes. Add this to a 0.1M phosphate buffer (pH
The gel was passed through a Sephadex G-50 column equilibrated in 6.0), and a maleimide-labeled POD fraction was collected.

(3) Preparation of Fab′-POD complex fraction The Fab ′ in the fraction prepared as in (1) above is equimolar as the maleimide-labeled POD in the fraction obtained in (2) above. And mix both fractions, then add Fa
It was diluted with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA so that the final concentrations of b 'and the maleimide-labeled POD were 100 µM. After the mixture was reacted at 4 ° C. for 20 hours, unreacted thiol groups were blocked with N-ethylmaleimide in a 10-fold molar amount of Fab ′. This was passed through an Ultrogel AcA44 column equilibrated with 0.1 M phosphate buffer (pH 6.5), and Fab '
-After fractionating the POD complex fraction, 0.1% bovine serum albumin (BS
A) and 0.005% thimerosal were added and stored at 4 ° C.

(B) Western blotting The bovine dental pulp collagenase inhibitor purified in Example 1 (a) was subjected to SDS-PAGE, and then obtained from a commercially available POD-labeled goat anti-mouse immunoglobulin and Example 2 (a) above. Cell Engineering 1 & 2, 1061 using Fab'-POD complex
Western blotting was performed according to the method of Tabe described in 1068 (1983) to obtain a pattern of immunostaining using an enzyme-labeled antibody. This is shown in FIG. In FIG. 1, A and B show nitrocellulose membranes after Western blotting, respectively, of Fab ′ obtained in Example 2 (a).
FIG. 4 shows the results of immunostaining with (clone 7-3F1) -POD complex and Fab ′ (clone 7-21B12) -POD complex.

In addition, 1 to 16 are obtained by immersing the nitrocellulose membrane after Western blotting in a solution of each of the following monoclonal antibodies (all IgG types), and then re-coating each nitrocellulose membrane with a POD-labeled goat anti-mouse immunoglobulin (Cap
pel Laboratories).

1: Clone 7-3F1, 2: Clone 7-4F2, 3: Clone 7-5A1, 4: Clone 7-6C1, 5: Clone 7-7F11, 6:
Clone 7-8B2, 7: Clone 7-9B4, 8: Clone 7-
10E11, 9: clone 7-11A5, 10: clone 7-12B6, 1
1: Clone 7-15E8, 12: Clone 7-18F3, 13: Clone 7-19F6, 14: Clone 7-20C2, 15: Clone 7-21
B12, 16: Clone 7-23G9 As is evident from FIG. 1, all of the above monoclonal antibodies were found to cross the bovine dental pulp collagenase inhibitor.

Example 3 Enzyme-linked immunosorbent assay of San de Germani (a) Preparation of monoclonal antibody-bound ball Monoclonal obtained in Example 1 (i) according to the method of Ishikawa et al. Described in J. Immunoassay 4 , 209-327 (1983). Antibody was added to 0.1 M phosphate buffer containing 0.1% sodium azide (p
H7.5), and adjusted to a concentration of 100 μg / ml (A ₂₈₀ = 0.15). Then, the monoclonal antibody solution was added to a polystyrene ball (6.5 mm in diameter, manufactured by Precision Plastic Ball).
Was immersed and left at 4 ° C. for 24 hours. Next, after removing the monoclonal antibody solution, a 10 mM phosphate buffer solution (pH: 0.1% BSA, 0.1 M sodium chloride and 0.1% sodium azide) containing
7.0) (hereinafter abbreviated as buffer A) 5 times,
It was immersed in buffer A and stored at 4 ° C.

(B) San Deergenti assay A purified collagenase inhibitor solution or a sample solution containing a collagenase inhibitor was diluted with buffer A containing 1% BSA, and 300 µl was added to each test tube. Next, the antibody-binding ball prepared in the above section (a) was added, and the mixture was heated by shaking at 37 ° C. for 1 hour (first reaction), and each was added with 3 ml of 10 mM phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. The test tube was washed three times. Next, the Fab'-POD complex prepared in Example 2 (a) was diluted with a 10 mM phosphate buffer (pH 7.0) containing 0.1% BSA and 0.1 M sodium chloride so as to have a concentration of 20 ng / test tube.
The mixture was heated with shaking at ℃ for 1 hour (second reaction). After the reaction,
Washing was performed in the same manner as at the end of the first reaction. Next, 0.3 ml of a POD substrate dissolved in 0.1 M acetate buffer (pH 5.5), that is, 0.0134% tetramethylbenzidine (TMBZ) is added, and 0.1 ml of 0.01% hydrogen peroxide is further added, followed by shaking at 30 ° C. for 1 hour. Heating (3rd
After the reaction, the reaction was stopped by adding 0.6 ml of 1.33N sulfuric acid. As the A ₄₅₀ value of the reaction mixture was measured with a spectrophotometer to determine the collagenase inhibitor content of the sample from the standard line.

(C) Selection of monoclonal antibody for determination of San de Germanti Example 1 (i) for the purpose of searching for a combination of monoclonal antibodies capable of quantifying a collagenase inhibitor
Clones 7-3F1, 7-6C1, 7-19F6 prepared by the method of
Fab'-POD from the monoclonal antibodies 7-21B12 and 7-21B12
The conjugate was prepared. On the other hand, clones 7-3F1, 7-4F2,
7-5A1, 7-6C1, 7-7F11, 7-8B2, 7-9B4, 7-
10E11, 7-11A5, 7-12B6, 7-15E8, 7-18F3, 7
Using the monoclonal antibodies -19F6, 7-20C2, 7-21B12, and 7-23G9 as solid phases, 1 ng per test tube of purified bovine dental pulp collagenase inhibitor was used to determine the amount of Sangenti by the method of Example 3 (b). I went. The _A450 values obtained are shown in Table 2 (below).

Incidentally, A ₄₅₀ value in Table 2 is a numerical value obtained by subtracting the value when no addition of collagenase inhibitor from the value of the sample 1ng added. In the case of using any of the above four Fab'-POD complexes, 7-4F2, 7-11A
5, 7-12B6, 7-18F3, 7-20C2, and 6 of 7-23G9
_A450 when two kinds of antibodies were used showed a value of 2 or more. Next, for these 24 combinations, the amount of the bovine dental pulp collagenase inhibitor was changed, and the amount of San Deutsch was determined. Fab ′ (clone 7-3F1) -POD as a complex,
FIG. 2 shows the results obtained when the clone 7-4F2 antibody was used as a solid phase. As seen in Figure 2, a linear relationship is established in the between bovine dental pulp collagenase inhibitor quantity and A ₄₅₀ addition, quantitative sensitivity about per tube 0.6pg (19amo
l) The linear relationship described above was observed for combinations other than the above, and it was found that the San Germanti quantification was possible for all combinations.

Example 4 Identification of Collagenase Inhibitor in Serum or Plasma (a) Preparation of Affinity Column Axn et al. And Proc. Natl. Acad. Sci. USA, 61 , 636 described in Nature 214 , 1302-1304 (1967). 643 (1968), the purified monoclonal antibody obtained in Example 1 (i) was immobilized as a ligand to the carrier Sepharose 4B via cyanogen bromide according to the method of Cuatrecasas et al. Next, 0.3 ml of antibody-bound Sepharose 4B gel was filled into a glass tube,
The buffer was used after equilibrating with 30 mM Tris-HCl buffer (pH 8.0) containing 0.1 M sodium chloride and 5 mM calcium chloride.

(B) Affinity purification of collagenase inhibitor Normal human serum, plasma of pemphigus patients, and bovine serum
1 ml containing 0.1 M sodium chloride and 5 mM calcium chloride 30
After dialysis against mM Tris-HCl buffer (pH 8.0)
Clone 7-prepared according to the method described in the above (a)
Apply to a 21B12 antibody-conjugated Sepharose 4B column, wash with the above buffer (non-adsorbed fraction), and then wash the column with a 30 mM Tris-HCl buffer (pH 7.5) containing 2 M sodium chloride and 0.2 M glycine containing 0.5 M sodium chloride. -Sequentially washed with sodium hydroxide buffer (pH 11.0) (wash fraction), and finally the protein adsorbed on the column was washed with 0.2M glycine-HCl buffer (pH 2.
0) (elution fraction). As shown in Table 3 below, 2.0, 1.8 and 7.1 μm were used for the elution fractions when using healthy human serum (human serum), pemphigus patient plasma and bovine serum, respectively.
g of protein were obtained. Note that these eluted fractions were
When subjected to -PAGE, a number of bands were observed. Of these bands, Western blotting was performed to confirm the band corresponding to the collagenase inhibitor. FIG. 3 shows the results. As shown in FIG. 3, after elution fractions obtained using A: healthy human serum, B: pemphigus patient plasma and C: bovine serum were subjected to SDS-PAGE, 1: clone 7-3F1 , 2: clones 7-6C1, 3: 7-19F6 and 4: 7
Fab'-PO prepared from each monoclonal antibody of -21B12
As a result of Western blotting using the D complex, it was found that a collagenase inhibitor reacting with a monoclonal antibody against bovine dental pulp collagenase inhibitor was present in both serum and plasma.
Moreover, it was found that their molecular weight was 32,000 D, which is the same as that of the bovine dental pulp collagenase inhibitor obtained in Example 1 (a).

(B) Quantification of Collagenase Inhibitor in Bovine Serum and Human Serum by the San Deutsche Assay Method The amount of collagenase inhibitor in bovine serum and human serum was determined by performing the San Deutsche quantification on the combination of the monoclonal antibodies shown in Example 3 (c). Was measured. The results are shown in Table 4 (later).

In this determination of San Deutsch, a standard straight line was prepared using bovine dental pulp collagenase inhibitor purified in Example 1 (a) as an antigen, and a serum A was measured using buffer A containing 1% BSA. 1000 times, 2000 times, 4
After diluting 000-fold and 8000-fold, respectively, the San Germanti quantification was performed, and their average values are shown in Table 4. In the case of bovine serum, the collagenase inhibitor in the serum could be quantified for any of the combinations used for the measurement, and their average value was 0.28 μg / ml of serum. On the other hand, in the case of human serum, but has failed detected at all A ₄₅₀ in most combinations were subjected to the measurement, the clone as a solid phase antibody 7-23G9
When Fab '(clone 7-6C1) -POD was used as the antibody and the complex, it was found that the amount of quantification in San Deutsch could be determined.

Next, using the above-mentioned combination, healthy human serum (human serum)
The collagenase inhibitor present in 5 samples, 2 samples of liver cancer patient serum and 2 samples of pemphigus patient plasma was quantified by San Deutsch. The results are shown in Table 5 (later). Fifth
As shown in the table, the amount of collagenase inhibitor present in 1 ml of healthy human serum was 0.29 μg on average, which was almost the same as that in bovine serum. In addition, the amount of collagenase inhibitor in the plasma of pemphigus patients was found to be clearly smaller than that in the serum of a healthy individual, about 1/3.

[Brief description of the drawings]

FIG. 1 shows that bovine dental pulp collagenase inhibitor was converted to SDS-PAG.
FIG. 2 shows Western blotting patterns when various monoclonal antibodies were used after subjected to E. FIG. 2 shows solid-phase 7-4F2 antibody-complex Fab '(7-3F1) -POD.
FIG. 3 shows a standard straight line of bovine dental pulp collagenase inhibitor in the measurement system. FIG. 3 shows (A) human serum (healthy human), (B) pemphigus patient plasma, and (C) bovine serum, respectively, as a monoclonal antibody (7). FIG. 21B is a diagram showing a western blotting pattern of an eluted fraction from a bound Sepharose 4B column.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Taro Hayakawa 1355, Hirabari, Embashi, Tenpaku-cho, Tempaku-ku, Nagoya

Claims (1)

(57) [Claims] 1. A monoclonal antibody against bovine collagenase inhibitor, wherein a combination of two types of monoclonal antibodies that specifically bind to different antigenic determinants of human collagenase inhibitor is used, and one of them is used as a solid phase antibody; A method in which the other is used as an enzyme-labeling antibody, and a human collagenase inhibitor present in a sample is quantified by an enzyme immunological method by a sandwich method.