JPH0772148A - Reagent for sandwich enzyme immunological determination of human type iv collagen - Google Patents

Abstract

PURPOSE:To precisely and quickly detect the concentration of human type IV collagen peptide and to simply diagnose a liver disease by using a reagent, for the sandwich enzyme immunological determination of human type IV collagen, which contains a specific essential constituent element. CONSTITUTION:The reagent for the sandwich enzyme immunological determination of human type IV collagen contains essential constituent elements indicated in (1) to (4). (1) an enzyme-labeled antibody which has enzyme-labeled a monoclonal antibody which cross-reacts with pepsin-soluble human type IV collagen. (2) A buffer solution. (3) An antigen-bonded solid-phase carrier in which another antipepsin-soluble human type IV collagen monoclonal antibody recognizing a part different from the monoclonal antibody, under (1), which cross-reacts with pepsin-soluble human type IV collagen is bonded to a solid- phase carrier. (4) A purified pepsin-soluble human type IV collagen standard sample. By using the reagent, the concentration of human type IV collagen peptide is measured.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an enzyme immunoassay reagent for human type IV collagen peptide useful for easily diagnosing liver diseases. More specifically, the present invention provides a reagent for quantifying human type IV collagen by an enzyme immunoassay based on a one-step sandwich method using a monoclonal antibody against human type IV collagen.

[0002]

Background Art Conventionally, human type III procollagen N in blood
Terminal peptide, human type IV collagen N-terminal peptide 7
It has been reported that radioimmunoassay was performed using each polyclonal antibody of -S domain and C-terminal peptide NC1 domain (Rohde et al., Eu).
r. J. Clin. Invest. , 9 , 451-45
9, 1979, Hoegemann et al., Clin. Ch
im. Acta, 144 1~10,1984, Schu
ppane et al. Clin. Invest. , 78 ,
244-248, 1986).

However, a method for actually quantifying human type IV collagen in blood in a rapid and accurate manner and a reagent used for such a method have not yet been provided. The present inventors have conducted various studies on a method for specifically quantifying human type IV collagen, and as a result, according to the present invention, a monoclonal antibody against pepsin-solubilized human type IV collagen was used, and an enzyme immunological method based on a one-step sandwich method was used. By the quantification method, we have succeeded in providing a quantification method of human type IV collagen peptide that can be rapidly measured with good accuracy and a small amount of sample, and a reagent used for the quantification.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a reagent for quantifying human type IV collagen in human blood by an enzyme immunoassay based on the one-step sandwich method as described in detail below. The reagent for quantifying human type IV collagen of the present invention is characterized by containing the following (1), (2), (3), and (4) as essential constituent components. (1) An enzyme-labeled antibody obtained by adding an enzyme label to a monoclonal antibody that cross-reacts with pepsin-solubilized human type IV collagen. (2) Buffer solution. (3) Antibody binding in which another anti-pepsin-solubilized human type IV collagen monoclonal antibody that recognizes a site different from the monoclonal antibody of (1) above that cross-reacts with pepsin-solubilized human type IV collagen is bound to a solid phase carrier Solid phase carrier. (4) Purified pepsin-solubilized human type IV collagen standard sample.

In order to quantify human type IV collagen in a sample to be measured using the above-mentioned elements (1), (2), (3) and (4), the enzyme-labeled antibody of (1) is used. Human IV present in the sample to be measured is dissolved in the buffer solution of (2), the sample to be measured is diluted in the solution, and the antibody-bound solid phase carrier of (3) is mixed in the diluted solution. After performing an immune reaction between the type I collagen and the above-mentioned enzyme-labeled antibody and the antibody bound to the above-mentioned solid phase carrier,
The solid phase carrier is fractionated, and the enzyme activity of the solid phase carrier is measured by an ordinary method to quantify. The purified pepsin-solubilized human type IV collagen of (4) is a standard sample for preparing a calibration curve, and a calibration curve is prepared in advance by the same enzyme activity measuring method using this standard sample.

In the quantification reagent of the present invention, as the antibody to which the enzyme label is attached, the antibody-containing material is subjected to ammonium sulfate fractionation and then DE
IgG fractions purified by AE-Sephacel and protein A affinity columns are used. The monoclonal antibody used in the present invention includes an embodiment in which the specific binding portion F (ab ′) _{2 of} these antibodies or Fab ′ itself is used.

As shown in the attached FIGS. 2 and 5, the measurement value of the concentration of human type IV collagen peptide in the serum of liver disease patients measured using the reagent for quantification of the present invention is the same as that in the serum of healthy persons. It was observed that the level of the human type IV collagen peptide in blood was measured using the reagent for quantification of the present invention, and thus, liver disease without carrying out biopsy that burdens the patient, In particular, it can contribute to the prediction of liver fibrosis, liver cancer, and gastrointestinal liver metastasis cancer.

ZTT (zinc sulfate turbidity reaction), GOT (glutamine oxaloacetate transaminase), GPT (glutamine pyruvate transaminase), ALP (alkaline phosphatase), LDH (lactate dehydrogenation) which have been used as conventional liver function determination methods. Enzyme) and γ
-GTP (γ-glutamyl transpeptidase) and other measurements do not provide the accuracy required to determine liver tissue fibrosis, liver cancer, and gastrointestinal hepatic metastases.
It has been confirmed by the present inventors. Therefore, by combining the measurement of the human proline hydroxylase concentration in blood (see Japanese Patent Laid-Open No. 61-202162) previously reported by the present inventors with the quantification method using the quantification reagent of the present invention, blood The concentration of human type IV collagen peptide therein can be accurately measured, and early detection of the above-mentioned various diseases becomes possible.

The quantification reagent of the present invention enables diagnosis of liver tissue fibrosis, liver cancer and gastrointestinal hepatic metastasis cancer based on the measurement of the concentration of human type IV collagen peptide, and therefore the present invention has remarkable utility. It is a thing. Less than,
The present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

[0010]

【Example】

Example 1 Preparation of Anti-Human Type IV Collagen Peptide Monoclonal Antibody (a) Preparation of Antigen-Pepsin Solubilized Human Type IV Collagen Using human placenta as a material, Artery, 7, 262-28.
0 (1980) according to the method of Mayne et al.
Homogenize with 5N acetic acid and digest with pepsin (1 mg / m
After solubilizing the collagen in 1), sodium chloride was added so that the final concentration was 2 M to precipitate the collagen. This was dissolved in 0.5N acetic acid and dialyzed with 0.5M acetic acid solution containing 0.7M sodium chloride to precipitate type I and type III collagen, and the supernatant was treated with 0.5N acetic acid solution containing 1.2M sodium chloride. It dialyzed and the IV and V type | mold collagen was deposited. 0.5M of IV and V type collagen fractions
50 mM Tris-HCl buffer pH containing sodium chloride
Dissolved in 7.4) and containing 2.2M sodium chloride 50
Type IV collagen was precipitated with mM Tris-hydrochloric acid buffer (pH 7.4) and separated from type V collagen. The purity of the obtained Form IV was determined by Biochem. Biophys. Re
s. Commun. , 72, 1472 to 1480 (19
76) and according to the method of Sykes et al., Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-).
It was about 95% when examined by PAGE). At that time, 174KD, 135KD, 108KD, 92KD,
78KD, 68KD, 60KD, 56KD, 43KD,
Eleven different bands of 34 KD and 29.5 KD (in the presence of mercaptoethanol) were detected.

(B) Preparation of antibody-producing cells 100 μg of pepsin-solubilized human type IV collagen was intraperitoneally administered to two 8-week-old BALB / c female mice together with complete Freund's adjuvant. 0.5 after the second time
50 mM Tris-HCl buffer containing M sodium chloride (p
BALB / c female mice were boosted 2 to 4 times every 2 to 4 weeks with 100 μg of the antigen dissolved in H7.4). As the final immunization, 3 days before splenectomy, intravenous administration was performed to prepare splenocytes.

(C) Cell fusion The following materials and methods are used. RPMI 1640 medium: RPMI 1640 (Dif
co Laboratories) with sodium bicarbonate (12 mM), sodium pyruvate (1 mM), L
-Glutamine (2 mM), Penicillin G potassium (50
u / ml), streptomycin sulfate (50 μg / m
1) and amikacin sulfate (100 μg / ml) were added, the pH was adjusted to 7.2 with dry ice, and 0.2 μmTo was added.
Sterilize and filter with a yo membrane filter.

NS-1 medium: Calf fetal bovine serum (MA Biopro) obtained by sterilizing and filtering the above RPMI 1640 medium.
Ducts) is added to a concentration of 15% (v / v). HAT medium: Hypoxanthine (100 μM), aminopterin (0.4 μM), and thymidine (16 μM) are added to the above NS-1 medium. HT medium: HAT described above except that aminopterin was removed
It has the same composition as the medium. PEG 4,000 Solution: Polyethylene glycol 4,000 (PEG 4,000 in RPMI 1640 medium)
0, Merck & Co. Inc. Made) 50% (w /
w) Prepare serum-free solution.

8-azaguanine resistant myeloma cell NS
-1 (P3-NS1-1) fusion is Selected
Method in Cellular Immun
biology (ed. BB Michel and and
S. M. Shiigi), WH. Freeman an
d Company (1980), 351 to 372, and the method of Oi et al. The nucleated spleen cells (viable cell rate 95%) prepared in (b) above and myeloma cells (viable cell rate 95%) are fused at a ratio of 5 to 6: 1. The spleen cells and myeloma cells are separately prepared as described above for RPM.
Wash with I 1640 medium. Then suspend in the same medium and mix in the above proportions for fusion. 50m capacity
1 conical styrene resin test tube (Iwaki Gl
(manufactured by ASS) and centrifuged at 40.times.g for 10 minutes in 40 ml of RPMI 1640 medium to completely aspirate the supernatant. 1 ml of PEG4,000 solution heated to 37 ° C for the precipitated cells
Is gently added with stirring for 1 minute, and further stirred for 1 minute to resuspend and disperse the cells. Next, heat at 37 ℃ R
1 ml of PMI 1640 medium is added dropwise for 1 minute. After repeating this operation once more, 7 ml of the same medium is added dropwise over 2 to 3 minutes with constant stirring to disperse the cells. This is centrifuged at 400 × g for 10 minutes, and the supernatant is completely removed by suction. Next, the precipitated cells were added to NS-1 medium 1 heated at 37 ° C.
Immediately add 0 ml and add 10 ml of large clumps of cells.
Pipette carefully to disperse. Further, 20 ml of the same medium was added to dilute, and the 96-well microwell made of polystyrene (Iwaki Glass)
5.9 × 10 ⁵ cells / 0.1 ml per well. As a pretreatment for the 96-well microwell used at this time, 0.2 ml of NS-1 medium was added, and the mixture was kept warm in a carbon dioxide incubator (37 ° C.) overnight, and the medium was removed by suction at the time of use. 7% of microwells that have completed cell fusion
Incubate in carbon dioxide / 93% air at a temperature of 37 ° C. and a humidity of 100%.

(D) Selective growth of hybridoma by selective medium On the first day of culture, 2 drops (about 0.1 ml) of HAT medium are added with a Pasteur pipette. On days 2, 3, 5, 8, and 11 half of the medium (0.1 ml) is replaced with fresh HAT medium. The same operation is repeated every 3 to 4 days after switching to the HT medium on the 14th day. Sufficient hybridoma growth is usually observed within 2-3 weeks. For all wells grown with hybridoma, the solid phase-antibody binding test (ELIS) described in (e) below.
Check positive wells by method A). Next, 1 m of HT medium containing 10 ⁷ mouse thymocytes as a feeder
l is a polystyrene 24-well cell well (Iwaki G
(manufactured by Lass), and transfer the entire contents of each positive hybridoma detected above. This is incubated at 37 ° C. for about 1 week in the presence of 7% carbon dioxide as in (c) above. In the meantime, 0.5 ml of the supernatant in 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 the ELISA method, and each of them is cloned by the limiting dilution method described in the following item (f). The residual liquid after the cloning was transferred to a polystyrene 25 cm ² tissue culture flask (manufactured by Iwaki Glass),
Prepare samples for cryopreservation.

(E) Search for hybridoma producing anti-human type IV collagen peptide antibody by ELISA Anal. Biochem. 104, 205-214
The method of Rennard et al. Described in (1980) with some modifications is used. This method is suitable for detecting hybridoma antibodies. 96-well microtitration plate (Flow Laboratories, Inc.
0.5 to 1.0 μg of pepsin-solubilized human type IV collagen, and the others are coated with 1% bovine serum albumin (BSA) and blocked. Add a portion of the supernatant of the hybridoma growth well to this and add about 1 at room temperature.
Incubate for hours. As a secondary antibody, horseradish-derived peroxidase (POD) -labeled goat anti-mouse immunoglobulin (manufactured by CapelLab.) Is added, and the mixture is further incubated at room temperature for about 1 hour. Next, hydrogen peroxide and a substrate, o-phenylenediamine (OPD), were added, and the degree of brown produced was measured by a microplate reader (MPR-
A4, manufactured by Toyo Soda Co., Ltd.) is used to measure the absorbance at 492 nm.

(F) Cloning Since two or more hybridomas may grow in each well, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. A cloning medium containing 10 ⁷ mouse thymocytes as a feeder per ml of NS-1 medium was prepared, and 5 wells, 1 well and 0.5 hybridomas were added to 36 wells, 36 wells and 24 wells of 96-well microwells. Add. On day 5 and 12, add about 0.1 ml each of NS-1 medium. 14 to 1 after starting cloning
ELI was observed for a group in which sufficient hybridoma growth was observed in 5 days and colony formation negative wells were 50% or more.
Perform SA method. If all wells tested are not positive,
Check the number of colonies in the antibody-positive wells and
Select 4-6 colony wells and re-clon.
Finally, for pepsin-solubilized type IV collagen, 22
A clone of the strain was obtained.

(G) In Vitro and In Vivo Proliferation of Monoclonal Antibody Proliferation of the monoclonal antibody is carried out as follows. Each clone obtained in the above (f) is cultured (in vitro growth) in an appropriate culture medium such as NS-1 medium, and each monoclonal antibody can be obtained from the culture supernatant (monoclonal antibody protein concentration is 10 ~ 100 μg / ml). On the other hand, in order to obtain a large amount of antibody, the tumor formation promoter pristane (2, 6, 10, 14) was added to an animal (BALB / c, mouse) that is syngeneic with the origin of splenocytes and myeloma cells.
-Tetramethylpentadecane, Aldrich Che
0.5 ml / mouse) is intraperitoneally administered to each mouse. By similarly intraperitoneally administering 1 × 10 ⁷ hybridomas after 1 to 3 weeks, ascites having a monoclonal antibody protein concentration of 4 to 7 mg / ml can be obtained in vivo after 1 to 2 weeks.

(H) Isotypes of heavy and light chains of monoclonal antibody Each ascites fluid obtained in (g) above was first placed in each row of wells of a microtitration plate coated with pepsin-solubilized human type IV collagen. , ELIS mentioned above
According to method A, each monoclonal antibody in each ascites fluid is bound. After washing, isotype-specific rabbit anti-mouse Ig antibody (Zymed Laborato)
ries). After washing, a POD-labeled goat anti-rabbit IgG (H + L) antibody was added, and 2,2′-
Heavy chain and light chain isotypes were detected using azino-di (3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide. The results are shown in Table 1. Regarding each of the obtained monoclonal antibodies, 16 of them have immunoglobulin chains γ1 / κ, two have γ2b / κ, and one has α / κ, And 3 had μ / κ.

[0020]

[Table 1]

(I) Purification of Monoclonal Antibody Each ascites fluid obtained in (g) above was subjected to ammonium sulfate fractionation (40% saturation).
Then, DEAE-Sephac equilibrated with 40 mM phosphate buffer (pH 8.0) containing 0.06 M sodium chloride.
collecting the non-adsorbed fraction of EL (Pharmacia),
Fetal calf serum and mouse-derived protein in the medium were separated and removed. Furthermore, after adsorbing to a Protein A Cellulofine (Seikagaku Corporation) column equilibrated with a 50 mM Tris-hydrochloric acid buffer solution (pH 8.6) containing 0.15 M sodium chloride to remove the non-adsorbed fraction, 0.15 M chloride It was purified by eluting with sodium-containing 50 mM acetate buffer (pH 4.0). Immediately eluate 1.5
It was neutralized with M Tris-HCl buffer (pH 8.9).

Example 2 Quantification of type IV collagen peptide in human serum (a) Enzyme-labeled monoclonal antibody (Fab'-PO)
Preparation of D complex) (1) Preparation of Fab ′ fraction Each purified monoclonal antibody obtained in Example 1 (i) was added with 0.1M sodium chloride in 0.1M acetate buffer (pH).
4.2) and the solution was digested with pepsin as described below. That is, 2% (w / w) of pepsin was added to IgG in the fraction and digested at 37 ° C. for 24 hours. Further, the digestion reaction was stopped by adding 2M Tris solution to the digest to adjust the pH to 7.0, and the Ultrogel AcA44 column (manufactured by LKB) equilibrated with 0.1M phosphate buffer (pH 7.0). The (ab ') ₂ fraction was collected by gel filtration using. Next, this F (ab ') ₂ fraction was added with 0.1 mM phosphate buffer (pH 6.0) containing 5 mM ethylenediaminetetraacetic acid (EDTA).
After dialyzing in, adding aminoethanethiol (MEA) to a final concentration of 10 mM and reducing at 37 ° C. for 1.5 hours, 5 mM EDTA-containing 0.1 M phosphate buffer (pH
Gel filtration was performed using a Ultrogel AcA44 column equilibrated with 6.0) to collect the Fab ′ fraction.

(2) Preparation of Maleimide-Labeled POD Fraction Aside from the operation of (1) above, P
The OD was labeled with maleimide. That is, POD is 10
0.1 M phosphate buffer (pH 7.0) in the amount of mg / ml
Dissolved in water, and a 25-fold molar amount of N-
(Ε-maleimidocaproyloxy) succinimide (EMCS) was added as a dimethylformamide solution,
The reaction was carried out at 30 ° C for 30 minutes. Sephadex G-5 equilibrated with 0.1 M phosphate buffer (pH 6.0)
Gel filtration was performed with a 0 column to collect a maleimide-labeled POD fraction.

(3) Preparation of Fab′-POD complex fraction Fab ′ in the fraction prepared as in the above (1) was compared with maleimide-labeled POD in the fraction obtained in the above (2).
Both fractions were mixed in an equimolar ratio to each other, and the final concentration of Fab ′ and maleimide-labeled POD was 100.
It was diluted with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA so that the concentration became μM. This mixture at 4 ° C
After reacting for 20 hours, unreacted thiol groups were blocked with N-ethylmaleimide in a molar amount 10 times that of Fab '. This was subjected to gel filtration with a Ultrogel AcA44 column equilibrated with 0.1 M phosphate buffer (pH 6.5), and Fab′-
After collecting the POD complex fraction, 0.1% BSA and 0.0
05% thimerosal was added and stored at 4 ° C.

(B) Polystyrene balls (diameter 6.5 mm, Precision Plasti) as a solid phase carrier
One-step sandwich method using c Ball) Purified monoclonal antibody (clone N) against pepsin-solubilized human type IV collagen obtained in Example 1 (i)
o. 4H12) containing 0.1% sodium azide 0.1
It is dissolved in M phosphate buffer (pH 7.5) and its concentration is adjusted to 0.
Adjust to 1 mg / ml. A polystyrene ball as a solid phase carrier is immersed in this antibody solution to coat the polystyrene ball with the antibody. Next, the antibody immersion liquid is recovered, and the polystyrene balls are washed with 10 mM phosphate buffer (pH 7.0) containing 0.1% BSA, 0.1M sodium chloride and 0.1% sodium azide, and stored at 4 ° C. . At the time of use, antibody-bound polystyrene balls washed with 10 mM phosphate buffer (pH 7.0) containing 0.1 M sodium chloride are used.

As a standard sample, the purified pepsin-solubilized human type IV collagen obtained in Example 1 (a) was diluted with 1% BS.
A, 0.1 M sodium chloride, 1/50 (v / v) horse serum (MA Bioproducts) and 0.0
10 mM phosphate buffer containing 5% thimerosal (pH 7.
0 ng) was used to prepare a 40 ng / 20 μl solution, and 20 μl of each was taken as a measurement sample by serially diluting it.

On the other hand, as a serum sample, a healthy person (NOR) is used.
20 μl of the serum of each of the patients, liver cancer (HCC), liver cirrhosis (LC) and chronic active hepatitis (CAH). 0.8 μg / ml Fa for each of these samples
b '(clone No. 1D3) -POD complex, 1% B
It was dissolved in 300 μl of 10 mM phosphate buffer (pH 7.0) containing SA, 0.1 M sodium chloride, 1/50 (v / v) horse serum and 0.05% thimerosal. Monoclonal antibody-bound polystyrene balls were then added to each of these standard and serum samples and incubated at 37 ° C for 4 hours.
After incubating for 5 minutes (immune reaction), it is washed with 10 mM phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. Next, add 300 μl of POD substrate dissolved in 0.1 M acetate buffer (pH 5.5), that is, 0.0134% tetramethylbenzidine (TMBZ), and further add 100 μl of 0.01% hydrogen peroxide solution at 37 ° C. In 3
After incubating for 0 minutes (coloring reaction), the reaction is stopped by adding 600 μl of 1.33N sulfuric acid. After stopping the reaction, the absorbance at a wavelength of 450 nm is measured with a Shimadzu Microflow UV-Visible spectrophotometer (UV-730) using water as a control, and the absorbance difference between the blind test and the sample is determined. The amount of type IV collagen peptide corresponding to the absorbance of 20 μl of the sample was read from the calibration curve prepared from the standard sample, and the value was 50
The amount of type IV collagen per 1 ml of the sample was determined by doubling (see Table 2).

[0028]

[Table 2]

A calibration curve of pepsin-solubilized human type IV collagen is shown in FIG. As is clear from FIG. 1, the quantitative sensitivity of this sandwich method was 0.31 ng / test tube, and the quantitative range was 0.31-40 ng / test tube. Also,
The quantitative coefficient of variation (CV) was 2.6 to 9.5%. The reaction time by the bead method was 45 minutes for the immunoreaction and 30 minutes for the color development reaction, and it was possible to measure the reaction time in less than half the time of the conventional two-step sandwich method (see, for example, JP-A-61-202162).

NO was measured by this sandwich method.
As a result of measuring type IV collagen in the sera of patients with R, HCC, LC and CAH, M (average value) of NOR sera +2
When SD (standard deviation) was taken as the cutoff value, the positive rates of each liver disease were 97%, 80% and 80%, respectively (Fig. 2). In addition, 12 metastasis-free groups (M (-)) without gastric cancer patients, 13 liver metastasis groups (HM) confirmed by histological or image diagnosis, and lymphatic metastasis group (LM). The type IV collagen in serum of 10 cases was similarly measured by this sandwich method. As is clear from FIG. 3, the concentration of type IV collagen was HM, LM and M.
(−) Was 552 ± 300, 199 ± 81 and 104 ± 62 ng / ml (M ± SD), respectively. NO
When M + 2SD of R serum was used as the cutoff value, the positive rates of each disease were 100%, 80% and 25%, respectively.
Met.

(C) One-step sandwich method using polystyrene microplate (manufactured by Nunc) as a solid phase carrier 0.1% azide of the purified monoclonal antibody (clone No. 4H12) obtained in Example 1 (i) It is dissolved in 0.1 M phosphate buffer (pH 7.5) containing sodium chloride and the concentration is adjusted to 0.1 mg / ml. To coat this antibody solution on a polystyrene microplate as a solid phase carrier, 100 μl was added per well and stored at 4 ° C. When using, 300 μl of 10 mM phosphate buffer (pH 7.0) containing 1% BSA, 0.1 M sodium chloride and 0.1% sodium azide was used to block the trowel microplate, and then 0.1 M sodium chloride and 0.1% were added.
(V / v) A microplate washed with 10 mM phosphate buffer (pH 7.0) containing Tween 20 is used.

As a standard sample, the purified pepsin-solubilized human type IV collagen obtained in Example 1 (a) was dissolved in 1% BS.
A, 0.1 M sodium chloride, 1/50 (v / v) horse serum and 0.05 mM thimerosal-containing 10 mM phosphate buffer (pH 7.0) were prepared to 50 ng / 50 μl, which was serially diluted. 20 μl was used for each well. On the other hand, as a serum sample, a healthy person (NOR) or H
20 μl of each serum of each patient of CC, LC, CAH and chronic inactive hepatitis (CIH) was used per well. That is, 50 μl of each of these samples was 0.8 μg /
ml Fab '(clone No. 1D3) -POD complex, 1% BSA, 0.1 M sodium chloride, 1/50
(V / v) Horse serum and 0.05% thimerosal included 1
Dissolve in 200 μl of 0 mM phosphate buffer (pH 7.0), add 100 μl of sample solution per well to the monoclonal antibody-bonded microplate, and allow it to react at room temperature (10-3
After incubating (immune reaction) at 0 ° C for 1 hour, 0.1
M sodium chloride and 0.1% (v / v) Tween
The immune reaction was stopped by adding 100 μl per well of 10 mM phosphate buffer (pH 7.0) containing 20% sodium chloride and 0.1% (v / v).
10 mM phosphate buffer containing Tween 20 (pH 7.
Wash with 0). Next, 100 μl of a solution (pH 5.0) prepared by dissolving OPD.2 hydrochloride in a citric acid-phosphate buffer solution (pH 6.0) containing 0.02% hydrogen peroxide and adjusting its concentration to 4 mg / ml was prepared. After 15 minutes of incubation at room temperature (coloring reaction), 100 μl of 1.33N sulfuric acid is added to stop the reaction. After stopping the reaction, the absorbance at a wavelength of 492 nm is measured with a microplate reader, and the absorbance difference between the blind test and the sample is determined. The amount of type IV collagen corresponding to the absorbance of 20 μl of the sample is read from the calibration curve prepared from the standard sample and the value is multiplied by 50 to obtain 1 ml of the sample.
The amount of type IV collagen per unit was determined (see Table 3).

[0033]

[Table 3]

FIG. 4 shows a calibration curve of pepsin-solubilized human type IV collagen. As is clear from the figure, the quantitative sensitivity of this sandwich method is 0.16 ng / well,
The quantification range was 0.16-20 ng / well. The CV value of this quantitative system was 0.5 to 6.0%.
The reaction time in this plate method was 60 minutes for the immunoreaction and 15 minutes for the color development reaction, and it was possible to measure a large number of samples in less than half the time of the conventional two-step sandwich method. In addition, both the simultaneous reproducibility and the diurnal variation when the same sample was measured were CV values within 5%, which were very good results (see Table 4).

[0035]

[Table 4]

[0036]

[Table 5]

By quantifying blood collagen peptide using the quantification reagent of the present invention, HCC, LC, CA
A significant increase in the amount of collagen peptide in the serum of each H patient was observed. The positive rate of each liver disease is NOR
When M + 2SD of serum is used as the cutoff value, HCC9
2%, LC 87%, CAH 83%, CIH 39%
Was (Fig. 5). The increase in the amount of collagen peptide in the serum of liver disease patients based on such measured values showed the same tendency as in the measurement by the one-step sandwich method using polystyrene balls as the solid phase carrier.

Example 3 (a) Identification of antigen The pepsin-solubilized human type IV collagen purified in Example 1 (a) was subjected to SDS-PAGE, and then Example 2 was used.
Western blotting was performed according to the method of Tabe described in Cell Engineering 1 & 2 1061-1068 (1983) using the Fab′-POD complex obtained in (a).
A pattern of enzyme antibody staining was obtained. The pattern of protein stained with Coomassie brilliant blue after SDS-PAGE and the nitrocellulose membrane after western blotting were obtained respectively in Fab 'obtained in Example 2 (a).
(Clone No. 1D3) -POD complex immunostained, the pattern was 190 KD, 175 KD, 125
5 bands of KD, 94KD, and 86KD and 20
2 agglomerates (205 KD and 220 K or more)
The band of D) was recognized (in the presence of mercaptoethanol). Similarly Fab '(clone No. 4H12) -P
185K in the pattern of immunostained with OD complex
D, 170 KD, 155 KD, 120 KD, and 90
5 bands of KD and 2 aggregates above 200 KD (2
Bands of 00KD and 220KD) were observed (Fig. 6).

(B) Molecular Weight of Human Serum Immune Reactant The molecular weight size of the human serum immunoreactant supplemented by the human type IV collagen enzyme immunoassay described in Example 2 (b) and (c) was measured. did. That is, 0.05% Tween20 was added to serum of healthy subjects and patients with liver disease (HCC) (both containing 300 ng of human type IV collagen),
Sephacryl S- equilibrated with 20 mM phosphate buffer (pH 7.0) containing 0.15 M sodium chloride.
300 (1.6 x 90 cm, manufactured by Pharmacia)
After gel filtration, the immunoreactant of each fraction was quantified by the one-step sandwich method using the polystyrene balls of Example 2 (b). As shown in FIG. 7, the immunoreactants in the sera of healthy persons (-●-) and liver disease patients (-x-) had a single peak, and the pepsin-solubilized human type IV collagen peptide (300 ng, The molecular weight was slightly smaller than that of-○-). From the calibration curve using fibrinogen, immunoglobulin, bovine serum albumin and peroxidase, the molecular weight of the immune reaction product in serum was estimated to be 620 KD.

(C) Specificity Human I, III, IV prepared by the method described in the following note
(Acid extraction), V, VI type collagen and pepsin solubilized rat type IV collagen were quantified by the one-step sandwich method using the polystyrene balls described in Example 2 (b) to obtain a quantified value of pepsin solubilized human type IV collagen. Compared. As shown in Table 5, no cross-reactivity was observed with any other collagen except that a slight cross-reactivity (about 7%) was observed with type VI collagen.

Note: Using human placenta and neonatal rat as a raw material, human placenta was fractionally purified from human placenta in addition to pepsin-solubilized type IV collagen in addition to type IV collagen. In addition, pepsin-solubilized type IV collagen was similarly prepared from neonatal rats. Acid extraction I
V type collagen is prepared from human placenta as Eur. J. Bio
chem. , 84 , 43-52 (1978) and 9
5 , 255-263 (1979).

[0042]

[Table 6]

(C) Recovery rate type IV collagen 93.0 ng / ml and 234.0
31-50 for each of two human sera at a concentration of ng / ml
Pepsin-solubilized human IV collagen was added in the range of 0 ng / ml, and their recovery was examined by the one-step sandwich method using the polystyrene balls and polystyrene plates described in Examples 2 (b) and 2 (c). As shown in FIG. 8, the correlation coefficient and the recovery rate of the recovered type IV collagen to the added pepsin-solubilized type IV collagen peptide in the ball method and the plate method are γ = 0.999, respectively.
7, 99.4 ± 10.3% (M ± SD) and γ = 0.
It was 9993, 101.6 ± 7.1%.

(E) Antigenic determinant Example 2 as shown in the immunoblotting diagram of FIG.
The antigen recognition site by the solid phase antibody (clone No. 4H12) and the POD-labeled antibody (clone No. 1D3) used in the one-step sandwich method using the balls or plates described in (b) and 2 (c) is small. different. In order to clarify the antigen recognition sites of this solid phase antibody and the POD-labeled antibody, the antigen antibody inhibition rate was examined. That is, 1 ng of 10 ng pepsin-solubilized human type IV collagen and 0-10 μg of monoclonal antibody (clone Nos., 1D3, 3A9 and 4H12) were added per test tube.
Preincubation in SA, 10 mM phosphate buffer containing 0.1 M sodium chloride (pH 7.0) at 37 ° C. for 60 minutes, and then clone No. prepared according to the method of Example 2 (b). Monoclonal antibody-coated polystyrene balls from 4H12 and 40 ng Fab 'per test tube
(Clone No. 3A9) -POD complex and added 37
After reacting at 60 ° C. for 60 minutes, TMBZ and hydrogen peroxide solution were added, and the mixture was allowed to stand at 30 ° C. for 60 minutes, and then A ₄₅₀ was measured. Figure 9
The standard pepsin-solubilized human type IV collagen as shown in Clone No. No inhibition of the antigen-antibody reaction was observed when treated with the monoclonal antibody from 1D3 (-x-), but here the clone No. used for the solid phase antibody and the complex was used. 3A9 (-●-) and clone No. Four
Inhibition was observed when treated with the monoclonal antibody from H12 (-○-). From this result, the solid-phase antibody (clone No. 4H12) used in the one-step sandwich method disclosed in the present specification is pepsin-solubilized human type IV collagen with respect to the POD-labeled antibody (clone No. 1D3). It is clear that they specifically react with different antigenic determinants of.

From the above results (a) to (e), it is understood that the above-mentioned one-step sandwich enzyme immunoassay method is a measurement system that specifically recognizes type IV collagen molecule.

Example 4 (a) Preparation of Anti-Human Type IV Collagen Monoclonal Antibody According to the method described in Example 1, 17 monoclonal antibodies against human type IV collagen were prepared. Four of these clones have immunoglobulin chain γ1 / κ and the other 1
Three had μ / κ (Table 6).

[0047]

[Table 7]

(B) One-step sandwich method using polystyrene microplate (manufactured by Nunc) as solid phase carrier Purified monoclonal antibody against human type IV collagen obtained in (a) above (clone No. 31-8G9)
Was coated on a microplate in the same manner as in Example 2 (c). On the other hand, the POD-labeled antibody was prepared according to the method of Example 2 by using the monoclonal antibody against the pepsin-solubilized human type IV collagen peptide (clone No. 4H12) obtained in Example 1. (C) of Example 3
A standard curve was prepared by the method shown in Table 7 using the pepsin-solubilized human type IV collagen prepared by the method described in 1. as a standard sample.

[0049]

[Table 8]

The quantitative sensitivity of this sandwich method is 0.5 n.
g / well and the quantification range was 0.5-54 ng / well. The CV at that time is 0.3 to 6.8%.
Met. The immune reaction in this plate method was 60 minutes and the color reaction was 30 minutes, and human type IV collagen was measured with high sensitivity for a short time as in the measurement results shown in Example 2 (c). NO by this sandwich method
As a result of measuring the concentration of human type IV collagen in the sera of R, LC and HCC patients, the positive rates of LC and HCC patients were 71% and 100%, respectively, when the cutoff value was M + 2SD of NOR serum. 8 table).

[0051]

[Table 9]

[Brief description of drawings]

FIG. 1 is a diagram showing the use of polystyrene balls as a solid phase carrier 1.
It is a standard curve of pepsin-solubilized human type IV collagen by the step sandwich method (bead method).

FIG. 2 NOR, HCC, L measured by the bead method
The immunoreactive type IV collagen peptide concentration in the serum of each of C and CAH patients is shown. In the figure, the horizontal bar is M and the dotted line is N.
M + 2SD of OR, and the value in parentheses indicates the number of samples.

FIG. 3: HM, LM and M measured by the bead method
(-) Shows the concentration of immunoreactive type IV collagen in the serum of each patient. In the figure, the vertical bar indicates M ± SD, and the dotted line indicates NO.
The M + 2SD of R and the numerical value in parentheses indicate the number of samples.

FIG. 4 is a calibration curve of pepsin-solubilized human type IV collagen by a one-step sandwich method (plate method) using a polystyrene microplate as a solid phase carrier.

FIG. 5: NOR, HCC, L measured by the plate method
Immunoreactivity I in the sera of C, CAH and CIH patients
The V-type collagen peptide concentration is shown. The horizontal bar in the figure is M,
The dotted line indicates M + 2SD, and the value in parentheses indicates the number of samples.

FIG. 6 is an immunoblotting diagram of standard pepsin-solubilized human type IV collagen peptide. A: Fab '
(Clone No. 1D3) -Staining with POD B: Fa
b'clone No. 4H12) -POD staining.

FIG. 7 is a diagram showing a gel filtration pattern of an immune reaction product in human serum.

FIG. 8 shows a correlation diagram of recovered type IV collagen to added pepsin-solubilized human type IV collagen peptide to human serum by the ball method (A) and the plate method (B).

FIG. 9 is a diagram showing the inhibition rate of an antigen-antibody reaction against three types of monoclonal antibodies.

Claims (1)

[Claims] 1. A human IV comprising the following (1), (2), (3) and (4) as essential constituent components:
Reagent for sandwich enzyme immunoassay of type I collagen. (1) An enzyme-labeled antibody obtained by adding an enzyme label to an anti-human type IV collagen monoclonal antibody that cross-reacts with pepsin-solubilized human type IV collagen. (2) Buffer solution. (3) An antibody-bonded solid-phase carrier in which another anti-pepsin-solubilized human type IV collagen monoclonal antibody that recognizes a site different from the monoclonal antibody in (1) above is bonded to the solid-phase carrier. (4) Purified pepsin-solubilized human type IV collagen standard sample.