JPH021553A - Sandwich enzyme immunoassay of human iv type collagen - Google Patents

Abstract

PURPOSE:To determine human IV type collagen peptide with high accuracy with a small amt. of a sample by using the monoclonal antibody for the pepsin solubilization human IV type collagen and using the enzyme immunoassay based on a single-stage sandwich method. CONSTITUTION:The monoclonal antibody which makes cross reaction with the molecular center triplet spiral section of the pepsin solubilization human IV type collagen is labeled with enzyme to obtain the enzyme labeled antibody. The sample to be measured is diluted by using the buffer soln. of this labeled antibody to obtain a diluted sample liquid A. The monoclonal antibody which makes cross reaction only with the pepsin solubilization human IV type collagen is conjugated with a solid phase antibody to obtain an antibody conjugated solid phase carrier B. The antibody conjugated carrier B is intimately mixed with the above-mentioned diluted liquid A and the immune reaction is effected between the human IV type collagen in the above-mentioned measurement sample and the enzyme labeled antibody as well as the antibody in the solid phase antibody B. The solid phase antibody is thereafter fractionated and the enzyme activity thereof is measured, by which the molecular central triplet spiral section of the human IV type collagen is determined.

Description

Detailed Description of the Invention [Technical Field] The present invention provides human I.I.
This invention relates to an enzyme immunoassay method for V collagen peptide.

More specifically, the present invention provides a method for quantifying the central triple helical region of the human IV collagen molecule by an enzyme immunoassay based on the one-step Sand-Deutsch method using a monoclonal antibody against human IV collagen, and
Provides a method for quantifying collagen 7-S domain.

[Background technology]

Previously, it has been reported that radioimmunological measurements were performed on blood human ■ using polyclonal antibodies against procollagen N-terminal peptide, human ■ pear collagen N-terminal peptide 7-S domain, and C-terminal peptide MCI domain. (Rohdg et al., Eur, J.
Cl1n, Invest, +9. 451-4
5L 1979, Hagemann et al., C11n,
Chim-Acta.

144 1-10. 1984.5 chuppan et al.
J, Cl1n.

Invest, 78.244-248.1986).

However, regarding the central triple helical part of the molecule of human IV collagen in blood, as well as human IV collagen 7
Regarding the -S domain, there have been no reports yet regarding methods for quantifying them.

As a result of various studies regarding methods for specifically quantifying human IV collagen, the present inventors have found that, according to the present invention, using a monoclonal antibody against pepsin-solubilized human IV collagen or a monoclonal antibody against human IV [collagen 7-S domain], By performing an enzyme immunoassay based on a one-step sandwich method, we succeeded in providing a method for quantifying human IV collagen peptide that can be rapidly measured with good accuracy using a small amount of sample. The conventionally known sandwich method involves a reaction between the in-phase antibody and the antigen in the sample (first reaction), followed by a reaction between the immobilized antibody and the antigen in the sample.
Compared to the sandwich method, which requires a two-step immune reaction before the color reaction: the reaction between the antigen complex and the enzyme-labeled antibody (second reaction), and the color reaction with the enzyme substrate (third reaction), this method is more effective. Since the method of the invention uses a one-step sandwich method in which the first reaction and the second reaction are performed simultaneously, it is possible to easily measure a large number of specimens with higher accuracy and in a shorter time.

[Disclosure of the invention]

The present invention provides a method for quantifying human IV collagen by performing an enzyme immunoassay method based on the one-step Sand-Deutsch method described in (1) and (2) below.

(1) A method for quantifying the central triple helical part of the human IV collagen molecule by an enzyme immunoassay based on a sandwich method using a monoclonal antibody against pepsin-solubilized human IV collagen, comprising: (a) hepsin-solubilized human IV collagen; A sample dilution solution obtained by diluting a sample to be measured using a buffer solution of an enzyme-labeled antibody, which is an enzyme-labeled monoclonal antibody that cross-reacts with the central triple helical part of the collagen molecule, and (b) pepsin solubilization. Using an antibody-bound solid phase carrier in which a monoclonal antibody that cross-reacts only with human IV collagen is bound to the solid phase carrier, the above-mentioned antibody-bound solid phase carrier (b) is mixed in the above diluent (a), After performing an immunoreaction between the human IV collagen present in the sample to be measured, the enzyme-labeled antibody, and the antibody bound to the solid phase carrier, the solid phase carrier is separated, and the solid phase carrier A method for quantifying the central triple helical part of the human IV collagen molecule by measuring the enzymatic activity of.

(2) A method for quantifying human IV collagen 7-S domain by an enzyme immunoassay based on a sandwich method using a monoclonal antibody against human IV collagen 7-S domain, comprising: (a) pepsin-solubilized human IV collagen; A sample dilution solution obtained by diluting a measurement target sample using a buffer solution of an enzyme-labeled antibody obtained by attaching an enzyme label to a monoclonal antibody that cross-reacts with the 7-S domain; (b) Human IV collagen 7-S Using an antibody-bound solid-phase carrier in which a monoclonal antibody that cross-reacts only with the domain is bound to the solid-phase carrier, the above-mentioned antibody-bound solid-phase carrier (b) is mixed in the above-mentioned diluent (a), and the above-mentioned measurement is carried out. After performing an immunoreaction between the human IV collagen 7-S domain present in the target sample, the enzyme-labeled antibody described above, and the antibody bound to the solid phase carrier, the solid phase carrier is separated, and the solid phase carrier is separated. A method for quantifying human IV collagen 7-S domain by measuring the enzymatic activity of a phase carrier.

In the method of the present invention, the antibody to be given an enzyme label is obtained by fractionating the antibody-containing material with DEAE-Sephace
An IgG fraction purified by a protein A affinity column and a protein A affinity column is used.

In the method of the present invention, the monoclonal antibody used,
Polyclonal antibodies also include embodiments in which the specific binding portion F(ab') of these antibodies or Fab' itself is used.

As shown in the attached FIGS. 2 and 5, the measured values of the concentration of the central triple helical region of the human type IV collagen peptide molecule and the human IV collagen 7-S domain in the serum of patients with liver disease measured by the method of the present invention. was found to be significantly higher than that in the serum of healthy individuals, and by using the method of the present invention, the concentration of the central triple helical region of the human IV collagen peptide and the human IV collagen 7-S domain in the blood could be measured. It is possible to predict liver disease, especially liver fibrosis, liver cancer, and liver metastasis gastrointestinal cancer, without performing a biopsy that is burdensome for the patient.

ZTT (zinc sulfate turbidity reaction), GOT (glutamine pyruvate transaminase), GPT (glutamine pyruvate transaminase), ALP (alkaline 7-osphatase), LDH (lactate dehydrogenase), and γ-GTP (γ
- glutamyl transpeptidase), etc.
Fibrosis of liver tissue, liver cancer, and liver metastasis gastrointestinal cancer cannot be determined, and this has been confirmed by the present inventors. Therefore, the measurement of blood human prolyl hydroxylase concentration previously reported by the present inventors (Japanese Unexamined Patent Publication No. 61-20
By measuring the concentration of the central triple helical part of the human IV collagen peptide molecule and the human IV collagen 7-S domain in the blood using the method of the present invention and the method of the present invention, this type of disease can be detected at an early stage. expected to be discovered.

By the method of the present invention, it is possible to diagnose liver tissue fibrosis, liver cancer, and liver metastasis gastrointestinal cancer based on the measurement of the concentration of human IV collagen peptide, so the present invention is extremely useful. Hereinafter, the present invention will be specifically explained using an example. However, the present invention is not limited to these examples.

Example 1 Preparation of anti-human IV collagen peptide monoclonal antibody (a) Preparation of antigen-pepsin solubilized human IV collagen Using human placenta as material Artery, 7.262-2
80 (1980) according to the method of Mayne et al.
．． Homogenized with 5N acetic acid and digested with pepsin (1my/
After solubilizing collagen with m(1), sodium chloride was added to a final concentration of 2M to precipitate collagen. I and III were dissolved in 0.5N acetic acid and dialyzed against a 0.5N acetic acid solution containing 0.7M sodium chloride.
Type collagen was precipitated, and the supernatant was dialyzed against a 0.5N acetic acid solution containing 1.2M sodium chloride to precipitate type 2 and v type collagen. ■, 0.5M type v collagen fraction
Lis-hydrochloric acid buffer (5QmM) containing sodium chloride (p
H7,4) containing 2.2M sodium chloride 5
1Vfi with 0mM Tris-HCl buffer (pH 7,4)
Collagen was precipitated and separated from type V collagen. The purity of the obtained IVfi was determined by Biochem, Bioph.
ys, Res, Commun-, 72, 1472~
It was about 95% when examined by sodium itodecyl sulfate-polyacrylamide gel electrophoresis (5OS-PAGE) according to the method of 5ykes et al., described in 1480 (1976).

In addition, 174KD, 135KD, 108KD
, 92KD, 78KD, 68KD, 60KD,
56KD143KD, 34KD and 29.5KD
Eleven different bands were detected (in the presence of mercaptoethanol).

(b) Collagen by type, acid extraction ■, collagen, IVf
fi collagen? -S domain, IVW collagen NC
Preparation of I domain and laminin Pi fraction from human placenta and neonatal rat according to the method described in (a) above, in addition to pepsin solubilized IV.
, I [, ■ and ■ type collagen were fractionated and purified. In addition, pepsin-solubilized IV collagen was similarly prepared from newborn rats.

The human IV collagen N-terminal long 7-S domain is described above (
Using the pepsin-solubilized TVW collagen prepared in a) as a material, Eur, J., Biochem, +108+
239-250 (1980) prepared by the method of R15teli et al. I;

Acid-extracted IVW collagen is made from human placenta.
J. Biochem, 84.43-52 (197
8) and want.

255-263 (1979). In addition, the IVffi collagen C-terminal NCl domain is prepared using the above acid-extracted IVW collagen as a material.
3-211' (1981).

On the other hand, Ramiev 21 fraction is made from human placenta as a raw material.
em-J, 193.749-755 (1981
) was prepared according to the method of Rtsteli et al.

(C) Preparation of antibody-producing cells Pepsin-solubilized human IV collagen looμ9 was first administered intraperitoneally to two 8-week-old BALB/C female mice together with complete Freund's adjuvant.

From the second time onwards, 100% of the antigen was dissolved in 50mM Tris-HCl buffer (pH 7.4) containing 0.5M sodium chloride.
BALB/c female mice were boosted with μ9 2-4 times every 2-4 weeks. As a final immunization, spleen cells were prepared by intravenous administration 3 days before spleen extraction.

(d) m foam amount The following materials and methods are used.

RPMI 1640 medium: RPMI 1640 (
Difco Labora-Lories), sodium bicarbonate (12mM), sodium pyruvate (1m
M), L-glutamine (2mM), penicillin G potassium (50u/mα), streptomycin sulfate (50μ
g/rRQ), and amikacin sulfate (lOOug/l
112) and adjust the pH to 7.2 with dry ice.
Q.271m Filter to sterilize with a Toyo membrane filter.

N5-1 medium/ground: sterile-filtered calf fat serum (M, A, Bioproduc) was added to the above RPMI 1640 medium.
ts) to a concentration of 15% (v/v).

HAT medium: Hyboxanthin (100 μM), aminopterin (0.4 μM), and thymidine (16 μM) are further added to the above N5-1 medium.

IT medium HAT above except that niaminopterin was removed.
It has the same composition as the medium.

PEG 4,000 solution: Polyethylene glycol 4,000 (PEG 4.0) in RPMI 1640 medium
00. Merck & Go.

Prepare a 50% (w/v) serum-free solution.

8-azaguanine-resistant myeloma cells NS-1 (R3-
Fusion with NSI-1) is 5elected 1Jeth
od 1nCellular Immunology
(ed. B.B., Michelle and S.M.
Shiigi), W.H., Freeman and
Company (1980), 351-372, with some modifications. The nucleated splenic cells (viable cell rate 95%) and myeloma cells (viable cell rate 95%) prepared in (c) above are fused at a ratio of 5 to 6:1. The lung cells and myeloma cells were separately subjected to the above RPM.
Wash with 11640 medium. Next, suspend in the same medium,
Mix in the above ratio for fusion. Conical styrene resin test tube with a capacity of 50 mQ (Tvaki Glass
40 ma in RPMI 1640 medium using
Centrifuge for 0x9.10 minutes and aspirate the upper groove completely.

PEG 4.000 solution LmQ heated at 37° C. is added dropwise to the precipitated cells over 1 minute with gentle stirring, and the cells are further stirred for 1 minute to resuspend and disperse the cells. Next, 37℃ heating RP
Add 1 mQ of MI 1640 medium dropwise over 1 minute.

After repeating this operation once more, 7 mg of the same medium was added to
Add dropwise for 3 minutes with constant stirring to disperse the cells. This is centrifuged at 400Xy for 10 minutes, and the upper groove is completely removed by suction. Next, immediately add 10 mQ of N5-1 medium warmed at 37°C to the precipitated cells, and remove large clumps of cells by 10 mQ.
Disperse by carefully pipetting using a ++I2 pipette. Further dilute by adding 20ml (l) of the same medium,
Polystyrene 96-well microwell (Iwaki G
5.9 x 10S per well (10.1m)
(Inoculate the cells from Step 2. At this time, add Q and 2 mQ of NS-1 medium as a pretreatment for the I; 966 microwells, and keep warm in a carbon dioxide gas incubator (37'c) overnight.
Aspirate the medium before use. Microwells with completed cell fusion were placed in 7% carbon dioxide gas/93% air at a temperature of 37°C.
Incubate under 100% humidity.

(e) Selective proliferation of hybridomas using selective medium Add 2 drops (approximately 0.1 ml) of HAT medium to each day using a Pasteur pipette. 2.3.5.8.11 Day Replace half of the medium (0,1 m+2) 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 to 3 weeks. The solid phase-antibody binding test (EL) described in the next section (f) is performed on all hybridoma-growing wells.
Check for positive wells using the ISA method. Next, 1mα of HT medium containing 1Oy mouse thymocytes was added to a 24-well polystyrene cell well (Iwaki) as a feeder.
(manufactured by Glass) to transfer the entire contents of each positive hybridoma detected above. This was heated at 37°C in the presence of 7% carbon dioxide as in (d) above.
Incubate for about 1 week. During this time, replace 0.5 mQ of supernatant of each well with 0.5 mQ of fresh HT medium once or twice. ELISA when the hybridomas have grown sufficiently
Reconfirm the positive result using the method, and follow the next section (g) for each.
Cloning is performed by the limiting dilution method as described. In addition, the remaining liquid after being used for cloning was transferred to a 25 cm2 polystyrene tissue culture flask (manufactured by Iwaki Glass).
Prepare samples for cryopreservation.

(f) Search for hybridomas producing anti-human IV collagen peptide antibodies by ELISA method Ana1. Biochem, 104. 205
A method slightly modified from the method of Rennard et al., described in 214 (1980), is used. This method is suitable for detecting hybridoma antibodies. 96-well microtitration plate (Flow Laboratory
s, Inc,! A) is coated with 0.5 to 1.0 μ9 of pepsin-solubilized human IV collagen, and the other portions are further coated and blocked with 1% bovine serum albumin (BSA). A portion of the upper groove of the hybridoma growth well is added to this and incubated at room temperature for about 1 hour. 2 Horseradish-derived peroxidase (POD) as a blowing agent I!
Cappel goat anti-mouse immunoglobulin (Cappel La
b.

(manufactured by J.D.) and further incubated at room temperature for about 1 hour.

Next is hydrogen peroxide and the substrate. -Finyl diamine (
4 using a microplate reader (MPR-A4, manufactured by Toyo Soda Kogyo Co., Ltd.) to measure the degree of brown color produced.
Measure the absorbance at 92 nm.

(g) Cloning Since there is a possibility that two or more types of hybridomas are growing in each well, I recloning is performed in addition to the limiting dilution method to obtain monoclonal antibody-producing hybridomas. 10 as feeder per NS-1 medium I+IQ
Prepare a cloning medium containing ' mouse thymocytes and add 5, 1 and 0.5 hybridomas per well to 36, 36 and 24 wells of a 96-well microwell. 5th day, 122nd day 6 approx. 0.1
Add mQ NS-1 medium. After starting cloning 1
Sufficient hybridoma growth was observed within 4 to 15 days.
ELISA is performed on groups in which 50% or more of wells are negative for colony formation. If all the tested wells are not positive, check the number of colonies in the antibody positive wells, select 1 colony + 7) in all 4 to 6 wells and re-clon. Finally, 22 clones were obtained for pepsin-solubilized IV collagen.

(h) In vitro and in vivo proliferation of monoclonal antibodies Monoclonal antibodies are obtained from the culture supernatant by culturing each clone obtained in (g) above in an appropriate culture medium such as NS-1 medium (in vitro proliferation). (monoclonal antibody protein concentration is 10 to 100 μg7mQ). On the other hand, in order to obtain antibodies in large quantities, the tumorigenic agent pristane (2, 6, 10, 1
4-tetramethylpentadecane, Aldrich Ch
0.5 mff per mouse was intraperitoneally administered. After 1 to 3 weeks, the monoclonal antibody protein concentration was 4 to 7 mg/m in vivo by intraperitoneal administration of 1X10' hybridomas after 1 to 2 weeks.
(L of ascites can be obtained.

(i) Monoclonal antibody heavyweight, light chain isotype Each of the ascites obtained in (h) above was first placed in each row of wells of a microtitreion plate coated with pepsin-solubilized human IV collagen, and then subjected to the ELISA described above.
Bind the monoclonal antibodies in each ascites according to the method. After washing, isotype-specific rabbit anti-mouse! g Antibody (manufactured by Zymed Laboratories) is added.

After washing, POD-labeled goat anti-rabbit IgG (H+L) antibody was added, and heavy and light chain isotypes were determined using 2,2'-azinodi (3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide as substrates. Detected. The results are shown in Table 1. Of the obtained monoclonal antibodies against vezucin-solubilized human double-base collagen, 16 were directed against the immunoglobulin chain γl/, 2 against γ2b/, and 1 against a
/ and 3 had μ/.

C1 All D3 D6 EIO A7 D5 H1 A9 B1 H12 D10 F6 B5 C11 G5 C8 H2 B4 G12 A3 C7 Table 1 1gM 1gM G1 G1 G1 M G1 A G1 G1 G1 G1 G1 gGI gGI IgG2 b gGI gGI gGl [gGI I gG2 b gGl μ/ to μ/ to γl/ to γ1/ to γl/ to μ/ to γl/ to α/ to γl/ to γl/ to γ1/ to γl/ to γl/ to γl/ to γ2b/ to γl / to γl / to γl / to γl / to γ2b / to γl / (j) Purification of monoclonal antibodies Each ascites obtained in the above (h) was fractionated with ammonium sulfate (40% saturation)
After that, the IgG class was 40% containing 0.06M sodium chloride.
DEAE equilibrated with mM phosphate buffer (pH 8,0)
The non-adsorbed fraction of -5ephacel (manufactured by Pharmacia) was collected, and calf fat serum and mouse-derived protein in the medium were separated and removed. For purification of IgA and 1gM class
DEAE equilibrated with mM phosphate buffer (pH 8,0)
- Sodium chloride 0.1M to 1.0M in Sapha-cel column chromatography. Both fractions were eluted at the gradient end up to OM.

Furthermore, the IgG class was adsorbed on a Protein A Cellulofine (manufactured by Seikagaku Corporation) column equilibrated with O, 15M sodium chloride (50mM) lithium-hydrochloric acid buffer (pH 8.6), and the non-adsorbed fraction was removed. Purification was performed by elution with 50 mM acetate buffer (pH 4,0) containing 0.15 M sodium chloride. In addition, the eluate was immediately added to 1.
Neutralization was performed with 5M Tris-HCl buffer (pH 8, 9).

Example 2 Quantification of the central triple helical region of IV collagen in human serum (a) Enzyme-labeled monoclonal antibody (Fab'-POD
Preparation method of complex) (1) Preparation of Fab' fraction The IgG fraction obtained in Example 1 (j) was dissolved in 0.1 M acetate buffer (pH 4, 2) containing 0.1 M sodium chloride, The solution was digested with pepsin as described below. That is, 2% (v/
pepsin (v) was added and the mixture was digested at 37°C for 24 hours. Furthermore, the digestion reaction was stopped by adding 2M Tris solution to the digested product and adjusting the pH to 7.0, and the Kultrogel Ac equilibrated with O.1M phosphate buffer (pH 7.0) was added.
F(
ab') and both fractions were collected.

Next, this F(ab')' fraction was added to a 0.1 M phosphate buffer (p
After dialyzing in 0.1M phosphate buffer (pH 6.0) containing 5 mM EDTA and reducing at 37°C for 1.5 hours by adding aminoethanethiol (MEA) to a final concentration of 10mM. ) Ultrogel AcA equilibrated with
Gel filtration was performed using a 44 column, and a Fab' fraction was collected.

(2) Preparation of maleimide-labeled POD fraction Apart from the operation in (1) above, POD was labeled with maleimide as described below. That is, POD was dissolved in lOmy/m (0.1M phosphate buffer (1) H7,O in an amount of l),
To the POD, 25 times the molar amount of N-(ε-maleimidocaproyloxy)succinimide (EMC5) was added as a dimethylformamide solution, and
Allowed to react for minutes. This was mixed with 0.1M U acid buffer (pH
Gel filtration was performed using a Sephadex G-50 column equilibrated with 6,0), and a maleimide-labeled POD fraction was collected.

(3) Preparation of Fab'-POD complex fraction (1) above
The above (2)
), the two fractions were mixed so that the maleimide-labeled POD in the fraction obtained in step 2 was equimolar, and further added with 0.1M phosphate containing 5mM EDTA so that the final concentration of Fab' and maleimide-labeled POD was 100 μM. Acid buffer (pH
6.0). After reacting this mixture at 4°C for 20 hours, unreacted thiol groups were blocked with N-ethylmaleimide in an amount 10 times the molar amount of Fab'. Ultrogel A was equilibrated with 0.1M phosphate buffer (pH 6,5).
After gel filtration with a cA 44 column and separating the Fab'-POD complex fraction, 0.1% BSA and 0.005% thimerosal were added and stored at 4°C.

(b) Polystyrene pole (16,5 m
m, Precision Plastic Ba1l
Purified monoclonal antibody (clone N) against the pepsin-solubilized human IV collagen obtained in Example 1 (j)
o, 4H12) containing 0.1% sodium azide
M phosphate buffer (pH 7.5) to bring the concentration to 0.
Prepare TA to 1 mg/mQ. A polystyrene pole as a solid support is immersed in this antibody solution to coat the polystyrene pole with the antibody. Next, the antibody soaking solution was collected, and the polystyrene ball was washed with 10mM'J acid buffer (pH 7,0) containing 0.1% BSA, 0.1M sodium chloride, and 0.1% sodium azide, and incubated at 4°C. Save to.

When used, use antibody-bound polystyrene balls that have been washed with 10 mM phosphate-MaI buffer (pH 7.0) containing 0.1 M sodium chloride.

As a standard sample, purified pepsin-solubilized human IV collagen obtained in Example 1 (a) was added to 1% BSA.

0.1M sodium chloride, l150 (v/v) horse serum (
lJ, A.

Bioproducts) and 10mM phosphate buffer containing 0.05% thimerosal (pH 7,0) to prepare a solution of 40ag/20μa, serially dilute it, take 20μα of each, and bind the measurement sample I.
;.

On the other hand, as a serum sample, 20μ of serum from a healthy person (NOR) was used.
20a (l) of serum from patients with Q1 liver cancer (HCC), liver cirrhosis (LC), and chronic active hepatitis (CAI) was used. body, 1% BSA,
It was dissolved in 300 μa of 10 mM phosphate buffer (pH 7.0) containing 0.1 M sodium chloride, 1150 (v/v) horse serum and 0.05% thimerosal. Next, monoclonal antibody-conjugated polystyrene balls were added to each of these standard samples and serum samples, and after incubation at 37°C for 45 minutes (immune reaction), 10 mM phosphate buffer containing 0.1 M sodium chloride (pH 7,0 ).

Next, PO dissolved in 0.1M acetate buffer (pH 5,5)
Add 300μ+2 of D substrate, i.e. 0.0134% tetramethylbenzidine (TIJBZ), further j: 0.0
After adding 100μa of 1% hydrogen peroxide and incubating at 37°C for 30 minutes (color reaction), 1.33N sulfuric acid 60
The reaction is stopped by adding 0 μα. After stopping the reaction, the absorbance at a wavelength of 450 nm is measured using a Shimadzu microflow ultraviolet-visible spectrophotometer (UV-730) using water as a control, and the difference in absorbance between the blind test and the sample is determined. According to the calibration curve created from the standard sample, it corresponds to the absorbance of 20 μa of the sample! 1V
The amount of W collagen peptide was read and the value was multiplied by 50 to determine the amount of IV collagen per 1 mQ of sample (see Table 2).

FIG. 1 shows a calibration curve for pepsin-solubilized human IV collagen. As is clear from Figure 1, the fixed i sensitivity of this sandwich method is 0.31 ag/tube, and the quantitative range is 0.
．． It was 31-40ag/test tube. Further, the quantitative coefficient of variation (CV) was 2.6 to 9.5%. The reaction time for this bead method is 45 minutes for the immunological reaction and 30 minutes for the color development reaction, compared to the conventional two-step Sandersch method (e.g. JP-A-61-20
Measurement can now be performed in less than half the time (see Publication No. 2162).

NOR is measured by this sandwich method.

As a result of measuring IV collagen in serum of HCC, LC and CAH patients, M (mean value) of NOR serum + 2S
When D (standard deviation) was used as the cutoff value, the positive rates for each liver disease were 97%, 80%, and 80%, respectively (Figure 2).

In addition, a non-metastatic group (M(-)) in which gastric cancer patients do not have metastasis.
12 cases, 13 cases of hepatic metastases (HM) confirmed histologically or by imaging diagnosis, and 13 cases of lymphoid metastases (
LM) IV collagen in the serum of Example 1 was similarly measured by this sandwich method. As is clear from Figure 3, the IV collagen concentrations were 552±300.199±81 and 104±81 in HM, LM and M(-), respectively.
62n9/mQ□Jf SD).

When M±2SD of NOR serum is taken as the cutoff value,
The positivity rate for each disease is 100%, 80% and 2, respectively.
It was 5%.

(c) One-step sandwich method using polystyrene microblades (manufactured by Nunc) as a solid phase support Example 1 Purified monoclonal antibody (clone N) against pepsin-solubilized human IV collagen obtained in (j)
o, 4H12) containing 0.1% sodium azide
Dissolved in M phosphate buffer (pH 7,5) and adjusted its concentration to O
, 1 mg/mQ. To coat a polystyrene microplate as a solid support, 100 μa of this antibody solution is added per well and stored at 4°C. 1% BSA when used.

10mM phosphate buffer (pH 7.0) containing 0.1M sodium chloride and 0.1% sodium azide 300μα
After blocking the microplate with O, 1M sodium chloride and 0.1% (v/v) Tween 20
A microplate washed with 10+nM phosphate buffer (pH 7,0) is used.

As a standard sample, purified pepsin-solubilized human IV collagen obtained in Example 1(a) was added to 1% BSA.

Prepared at 50% g/50μ4 with 10mM phosphate buffer (pH 7.0) containing 0.1M sodium chloride, 1150 (v/v) horse serum, and 0.05% thimerosal, and serially diluted this to 1 liter each. 20 μa was used per well. -, normal subjects (NOR) or H as serum samples.
C.C.

20 μα of each serum from patients with LC, CAH, and chronic inactive hepatitis (CIH) was used per well. That is, 50μa of each of these samples was 0.8μ9/rn(
l Fab' (clone No. 1D3)-POD complex, 1% BSA, 0.1M sodium chloride, 1150
(v/v) horse serum and 0.05% thimerosal 1
The sample solution was dissolved in 200 μa of 0 mM phosphate buffer (pH 7,0) and added to a monoclonal antibody binding microplate at 100 μa per well.
After 1 hour incubation (immune reaction) at 0.1
M Sodium Chloride and 0.1% (v/v) Twee
l of 10mM phosphate buffer (pH 7,0) containing n20
Immune reactions were stopped by adding 100 μa per well, 0.1 M sodium chloride and 0.1% (v/
v) 10mM phosphate buffer containing Tween 20 (pH
Wash at 7.0). Next, OPD・2
Add 100 μα of a solution (pH 5, 0) prepared by dissolving hydrochloride to a concentration of 4 mg/mQ, incubate at room temperature for 15 minutes (color reaction), and add 100 μQ 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 using a microplate reader, and the difference in absorbance between the blind sample and the sample is determined.

The amount of IV collagen corresponding to the absorbance of 20 μQ of the sample was read from a calibration curve prepared from a standard sample, and the value was multiplied by 50 to determine the amount of IV collagen per LmQ of the sample (see Table 3).

Table 3 and Figure 4 show the calibration curve of pepsin-solubilized human IVW collagen. As clearly shown in the figure, the quantitative sensitivity of this sandwich method was 0.16% g/well, and the quantitative range was 0.16-20 ng/well. Moreover, the Cv value of this quantitative system was 0.5 to 6.0%. The reaction time for this plate method was 60 minutes for the immunological reaction and 15 minutes for the color development reaction, making it possible to measure a large number of specimens in less than half the time of the conventional two-step sandwich method. In addition, the simultaneous reproducibility and daily variation when measuring the same sample both have a Cv value of 5.
% (see Table 4) J Table 4 (A) Simultaneous reproducibility (B) Daily variation In addition, blood collagen peptides were quantified by using the method of the present invention. At the time, a significant increase in collagen peptides was observed in the serum of HCC%r-c 1CAH patients. The positive rate for each liver disease was HCC 92%, LC 87%, C
AH was 83% and CIH was 39% (Figure 5). This increase in collagen peptides in the serum of patients with liver disease is
The same tendency was observed as in the measurement by the one-step Sand-Deutsch method using polystyrene poles as the solid support.

Example 3 (a) Identification of antigen After subjecting the pepsin-solubilized human IV collagen purified in Example 1 (a) to 5DS-PAGE,
Using the Fab'-POD complex obtained in ), Western profiling was performed according to the method of Kyuto described in Cell Engineering 1 & 21061-1068 (1983) to obtain an enzyme-antibody staining pattern.

The pattern of proteins stained with Kumashi brilliant blue after 5DS-PAGE and the nitrocellulose membrane after Western blotting are shown in Example 2 (a).
Fab' (clone No.) obtained with ).

1D3)-POD complex immunostained pattern includes 190KD, 175KD, 125KD, 94
Five bands of KD and 86KD and two bands of aggregates (205KD and 220KD) above 200KD were observed (in the presence of mercaptoethanol). Same <
Fab' (Clone No., 4H12) -POD
The pattern of immunostaining with the complex includes 185KD,
170KD, 155KD, 120KD, and 9
5 bands at 0KD and 2 aggregates above 200KD (
A band of 200K [l and 220KD) was observed (Figure 6).

(b) Molecular weight of immunoreactant in human serum The molecular weight size of the immunoreactant in human serum was determined supplemented by the human IV collagen oxygen immunoassay described in Example 2(b) and (C). That is, serum from healthy subjects and patients with liver disease (HCC) (both containing 300 ng of human IV collagen) was mixed with 0.05% Tyeen 20.0.
20ml 1J phosphate buffer containing 15M sodium chloride (p
5ephacryl S-3 equilibrated with H7,0)
00 (1,6X 90cm, manufactured by Pharmacia), and the immunoreactant from each aliquot was filtered using Example 2 (
Quantification was performed by the one-step sandwich method using the polystyrene pole described in b). As shown in Figure 7, the immunoreactive products in the serum of healthy individuals (-・-) and patients with liver disease (-X-) had a single peak, and vezucin-solubilized human IV, which was used as a control, showed a single peak.
The molecular weight was slightly smaller than collagen peptide (300 ng, -〇-). The molecular weight of the immunoreactant in the serum was estimated to be 620 KD from a calibration curve using fibrinogen, immunoglobulin, bovine serum albumin, and peroxidase.

(c) Specificity Example 1 Human r, ■, ■ prepared in (b) (acid extraction)
, V, type VI collagen, 7-S domain, NCI domain, laminin P1 and pepsin solubilized run 1-
Type IV collagen was quantified by the one-step sandwich method using polystyrene poles as described in Example 2(b), and compared with the quantitative value of pepsin-solubilized human IV collagen. Fifth
As shown in the table, there is some cross-reactivity (approximately 7
No cross-reactivity was observed with any other collagen or Ramiev 21 fraction, except that %) was observed.
.

(d) Recovery rate IV collagen 93. On9/mff and 234.0
31-50 for two types of human serum with n9/mQa degree, respectively.
Pepsin-solubilized human collagen was added in the range of 0 ng/m (l), and the recovery rate was examined by a one-step sandwich method using the polystyrene pole and polystyrene plate described in Examples 2(b) and 2(c). As shown in Figure 8, the correlation coefficient and recovery rate of the recovered IV collagen to the added pepsin-solubilized IV collagen peptide in the Pall method and the plate method were γ-0,9997 and γ-0,9997, respectively.
99.4±10.3% (M f 5D) 8 J: Biy
= 0.9993, lol, 6f 7.1%.

(e) Antigenic determinants As shown in the immunoblotting diagram in FIG.
The antigen recognition site by the solid-phase antibodies (clone No. 4H12 and PODi recognition antibody (clone No. 1D3) used in the one-step sandwich method using poles or plates described in (b) and 2(c) above is slightly different. In order to clarify the antigen recognition site of this solid-phase antibody and POD-labeled antibody, the antigen-antibody inhibition rate was investigated.That is, 10 ng of pepsin-solubilized human IV collagen and 0 to 10μ9 monoclonal antibody (clone) were used per test tube. No.
, + ID3.3A9 and 4H12) in 1% BSA
Monoclonal antibody from clone No. 4H12, preincubated in 10 mM phosphate buffer (pH 7,0) containing 0.1 M sodium chloride at 37° C. for 160 minutes and then prepared according to the method of Example 2(b). Add coated polystyrene pole and heat to 37°C160
Let it react for a minute. Next, add 40 ng Fab' (clone No. 3A9)-POD complex per test tube and hold at 37°C.
! , After reacting for 60 minutes, TMBZj-,' and hydrogen peroxide solution were added, and after standing at 30°C for 160 minutes, Ats.

was measured. As shown in Figure 9, when standard pepsin-solubilized human IV collagen was treated with the monoclonal antibody from clone No. 1D3 (-X-), no inhibition of the antigen-antibody reaction was observed; Inhibition was observed when treated with monoclonal antibodies from clone No. 3A9 (-@) and clone No. 4H12 (-0-) used for antibodies and complexes. From this result, the solid-phase antibody (clone No. 4H) used in the one-step sandwich method disclosed herein
It is clear that POD-labeled antibody (Clone No. 12) reacts specifically with different antigenic determinants of pepsin-solubilized human IV collagen.

From the above results (a) to (e), it is clear that the one-step sandwich enzyme immunoassay method using the pole and microplate disclosed herein is a method that specifically recognizes the central triple helical region of the IV collagen molecule. It can be said that it is a system.

Example 4 (a) Preparation of anti-human IV collagen 7-S domain monoclonal antibody Human type IV collagen 7 according to the method described in Example 1
-17 monoclonal antibodies against the S domain were prepared. Four of these clones were immunoglobulin chain γl
/ni, and the other 13 had μ/ni (Table 6).

Clone no.

1-IG4 31-2H12 1-3D2 31-4H9 1-5H8 31-6'H4 1-7E4 1-8G9 31-9H8 31-10F5 31-11DII 31-12G12 31-13H3 31-15B4 31-17H6 31-18H5 31-19F4 Table 6 Isotype gGI 1gM gGI 1gM 1gM 1gM 1gM gGI 1gM 1gM [gM 1gM gGI 1gM 1gM 1gM 1gM Chain γl/to μ/to γ1/to μ/to μ/to μ/to μ/to γl/ to μ / to μ / to μ / to μ / to γ1 / to μ / to μ / to μ / to (b) Polystyrene microbrakes h (
Purified monoclonal antibody against the human IV collagen 7-S domain obtained in (a) above (clone No. 3)
18G9) was coated on a microplate in the same manner as in Example 2(c). On the other hand, the POD-labeled antibody in Example 1
Monoclonal antibody against pepsin-solubilized human IV collagen peptide (clone No. 4H12) obtained in
) according to the method of Example 2.

Human type IV collagen 7- prepared in Example 1 (b)
A calibration curve was prepared using the S domain as a standard sample by the method shown in Table 7 (FIG. 10).

As is clear from Figure 1O, the quantitative sensitivity of this sandwich method is 0.5 ng/well, and the quantitative range is 0.5 to 5 ng/well.
The amount was 4 ng/well. Also, the CV at that time was 0.3
It was ~6.8%. The immune reaction with this plate method is 6
The color reaction time was 30 minutes, making it possible to measure the human IV collagen 7-S domain in a short time and with high sensitivity, similar to the measurement of the central triple helix portion of human IV collagen shown in Example 2(c). Ta.

As a result of measuring the human IV7-S domain concentration in the serum of NOR1 LC and HCC patients using this sandwich method, when M + 23D of NOR serum was used as the cutoff value, the positive rate for LC and HCC diseases was 71%, respectively. It was 100% (Table 8).

(C) Recovery rate Type IV collagen 7-S domain 5B/mα concentration of human serum is added with human IV collagen 7-S domain in the range of 40 to 1280 ng/mQ, and the microplate described in (b) above is used. Their recoveries were investigated using the step-sandwich method. As shown in Figure 11, the correlation coefficient and recovery rate of recovered 7-S domain to added 7-S domain are γ = 0.9991 and 94.2 ± 1, respectively.
It was 4.7% (M:I:SD). From this result,
The one-step sandwich method using a microplate described in (b) above can be said to be a specific method for measuring the human collagen 7-S domain.

[Brief explanation of the drawing]

FIG. 1 is a test line of pepsin-solubilized human IV collagen by a one-step sandwich method (bead method) using polystyrene poles as a solid phase carrier. Figure 2 shows the NOI measured by the bead method. , 1(C.C.
1 Immune reactivity in the serum of each patient with LC and CAH■? Collagen peptide concentration is shown. In the figure, the horizontal bar indicates M, the dotted line indicates M + 25D of NOR, and the numbers within 0 indicate the number of specimens. Figure 3 shows HM, LM and M measured by the bead method.
(-) indicates the immunoreactive IV collagen concentration in the serum of each patient. In the figure, the vertical bar represents M±SD, and the dotted line represents NOR.
The value within 0 indicates the number of specimens. FIG. 4 is a calibration curve of pepsin-solubilized human IV collagen obtained by a one-step sandwich method (plate method) using a polystyrene microplate as a solid phase carrier. FIG. 5 shows NOR measured by the plate method. Immunoreactive IV collagen peptide concentrations in serum of HCClLC, CAH and CIH patients are shown. In the figure, the horizontal bar indicates M, the dotted line indicates M+2SD, and the numbers within 0 indicate the number of specimens. FIG. 6 is an immunoblotting diagram of standard pepsin-solubilized human IV collagen peptide. A: Fab' (clone No. 1D3) -POD
Staining B: Fab' (clone No. 4H12)
- Staining with POD. FIG. 7 is a diagram showing a gel pattern of an immune reaction product in human serum. Figure 8 shows pepsin-solubilized human IV added to human serum by the Paul method (A) and plate method (B).
A correlation diagram of recovered IV collagen with respect to W collagen peptide is shown. FIG. 9 is a diagram showing the inhibition rate of the antigen-antibody reaction against three types of monoclonal antibodies. FIG. 1O shows a calibration curve for human IV collagen 7-S domain. Figure ti shows human IV added to human serum.
Recovered collagen 7- to collagen 7-S domain
A correlation diagram of the S domain is shown. Patent applicant: Fuji Pharmaceutical Co., Ltd. Figure Figure Recovery IV Colladen (n?/m1) Figure 0.00064 0.0032 0.016 0.08 0.4 Monoclonal antibody (μ2/test tube) Procedural amendment form Heisei July 21, 1 year

Claims (1)

[Scope of Claims] 1) A method for quantifying the central triple helical region of the human type IV collagen molecule by an enzyme immunoassay based on a sandwich method using a monoclonal antibody against pepsin-solubilized human IV collagen, comprising: (a ) A sample dilution solution obtained by diluting the sample to be measured using a buffer solution of an enzyme-labeled antibody, which is an enzyme-labeled monoclonal antibody that cross-reacts with the triple helical region in the center of the molecule of pepsin-solubilized human type IV collagen. (b) using an antibody-bound solid phase carrier in which a monoclonal antibody that cross-reacts only with pepsin-solubilized human type IV collagen is bound to the solid phase carrier; Carrier (b)
After performing an immunoreaction between the human type IV collagen present in the sample to be measured, the enzyme-labeled antibody, and the antibody bound to the solid phase carrier,
A method for quantifying the central triple helical region of human type IV collagen molecule by fractionating a solid phase carrier and measuring the enzyme activity of the solid phase carrier. 2) A method for quantifying human type IV collagen 7-S domain by an enzyme immunoassay based on a sandwich method using a monoclonal antibody against human type IV collagen 7-S domain, comprising: (a) pepsin-solubilized human IV; A sample dilution solution obtained by diluting a sample to be measured using a buffer solution of an enzyme-labeled antibody obtained by attaching an enzyme label to a monoclonal antibody that cross-reacts with type IV collagen 7-S domain; (b) human type IV collagen; Using an antibody-bound solid-phase carrier in which a monoclonal antibody that cross-reacts only with the 7-S domain is bound to the solid-phase carrier, the above-mentioned antibody-bound solid-phase carrier (b) is added to the diluent (a).
After performing an immunoreaction between the human type IV collagen 7-S domain present in the sample to be measured, the enzyme-labeled antibody, and the antibody bound to the solid phase carrier, the solid phase Human type IV collagen 7-S was detected by fractionating the carrier and measuring the enzyme activity of the solid phase carrier.
How to quantify domains.