JP3537331B2 - Type IV collagen immunoassay and reagents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying type IV collagen by latex agglutination and a reagent therefor.

[0002]

BACKGROUND OF THE INVENTION The determination of human type IV collagen in blood
Since it is useful for diagnosis of liver diseases, particularly hepatic fibrosis, liver cancer, gastrointestinal hepatic metastasis, etc., various methods have been conventionally proposed. Rhode et al., European Journal of Clinical Investigation (Eur. J. Clin. Invest.) Vol. 9,
451-459, 1979; Hogman
emann) et al., Clinical Chemical Acta (C
lin. Chem. Acta) 144 volumes, 1-10 pages,
1984; Shuppan et al., Journal of Clinical Investigation (J. Clin. Invest.) 78, 244-2.
48, 1986, radioimmunology using polyclonal antibodies of blood human type III procollagen N-terminal peptide, human type IV collagen N-terminal peptide 7S domain, and human type IV collagen C-terminal peptide NC1 domain. There have been reports that have made objective measurements.

[0003] However, all of these quantification methods are based on polyclonal antibodies to the type 7 collagen 7S domain from human placenta or human IV from human placenta.
It uses a polyclonal antibody for the type 1 collagen NC1 domain, and its quantification accuracy is extremely inaccurate. Therefore, the quantification results obtained by these quantification methods are sufficient even when used for diagnosis of liver disease and the like. It was not something.

[0004] In order to solve such problems, the inventors of the present invention used a monoclonal antibody against pepsin-solubilized human type IV collagen and carried out an enzyme immunological quantification method based on a one-step sandwich method. Proposed a method for quantifying human type IV collagen peptide which can be rapidly measured with good accuracy [Obata et al., Clin. Chem. Acta 181].
Vol., 293-304, 1989].

[0005] However, such an enzyme immunoassay is superior in clinical accuracy to the above-mentioned radioimmunoassay, but a simpler and faster method is required. Was.

Therefore, it has been considered to use latex conventionally used for immunochemical agglutination or agglutination inhibition as an antibody reagent. Usually, an enzyme immunoassay (EIA method) or a radioimmunoassay (RI
A), detection methods using an immunoreaction such as latex agglutination method may differ in sensitivity etc. depending on the detection means if the same antigen or the same antibody is used, but the same performance is obtained for the specificity. Be expected.

[0007] However, there has been no technique using a latex reagent as a detecting means for measuring collagen, and no literature report has been found, so that there is a great technical obstacle to developing a reagent using latex. Is inevitable.

In recent years, in the field of immunological diagnostics, monoclonal antibodies have been widely used as materials for sensitively and selectively detecting a target substance. Unlike antibodies, they are expected to have high sensitivity and specificity.However, since monoclonal antibodies have a single reactive site, many technical studies are required when binding to a solid support such as latex particles. It has been.

[0009]

SUMMARY OF THE INVENTION In view of the above, the present invention uses a latex reagent which can rapidly, simply and accurately quantify type IV collagen and is applicable to a fully automatic analyzer. An object of the present invention is to provide an immunoassay for type IV collagen and a measurement reagent for performing the immunoassay.

[0010]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that, when quantifying type IV collagen in a human sample, a latex on which a monoclonal antibody that specifically reacts with type IV collagen is immobilized. When the particles were reacted with type IV collagen in the specimen, the latex particles were found to specifically aggregate due to the antigen-antibody reaction,
The present invention has been completed.

That is, the present invention relates to a type IV collagen immunoassay for quantifying the amount of type IV collagen in a specimen by utilizing the agglutination reaction of a latex reagent comprising latex particles having a monoclonal antibody immobilized thereon. is there. Hereinafter, the present invention will be described in detail.

The specimen used in the present invention is not particularly limited as long as it is necessary to determine the amount of type IV collagen, and examples thereof include human blood, serum, plasma, tissue fluid, urine, saliva, sweat and the like. Can be The type IV collagen preferably has a 7S domain and a molecular triple helix domain so that it can react with a latex reagent described later. The sample may be used after dilution, if necessary.

The monoclonal antibody used in the present invention uses, as antigens, type IV collagen obtained from human tissues, recombinant antigens obtained by genetic recombination techniques, and peptides or modified peptides obtained by peptide synthesis. In the field of cell fusion technology, for example, methods known per se, such as the method described in Tatsuo Iwasaki et al., “Monoclonal Antibody Hybridoma and ELISA” (published by Kodansha), are appropriately selected. By combining the techniques, a monoclonal antibody-producing fusion cell line can be formed, and then the monoclonal antibody can be produced and obtained using the cell line.

In the present invention, the monoclonal antibody may further be obtained by fractionating the monoclonal antibody-containing substance obtained as described above with ammonium sulfate, and then subjecting it to DEAE-Sephac.
El and an IgG fraction purified by a protein A affinity column are used. In the present specification, the above-mentioned monoclonal antibodies also include embodiments in which the specific binding portion F (ab ') 2 in these antibodies or Fab' itself is used.

In general, when the above monoclonal antibody is bound to a carrier, the structure and arrangement of the reaction site, which is the antigen recognition site, are greatly changed, and the active site may be located at a position unsuitable for aggregation between latexes. In addition, since there is only one reaction site, specificity to the antigen may be lost. For this reason, in the present invention, it is preferable that the monoclonal antibody does not lose its binding ability to type IV collagen even after being immobilized on latex particles described later, provided that it has such properties. The subclass and the like of the monoclonal antibody are not particularly limited. The binding ability of the monoclonal antibody to type IV collagen can be appropriately measured by a neutralization method using type IV collagen, an ELISA method, or the like.

After immobilization on the latex particles, I
Monoclonal antibodies that do not lose the ability to bind to type V collagen can be obtained by selecting by screening or the like. The screening method is not particularly limited, and for example, a method used in a normal EIA method or an RIA method such as an SRID method or an ELISA method can be used.

In the present invention, it is preferable to use two kinds of anti-human type IV collagen antibodies as the above-mentioned monoclonal antibody, and in particular, an antibody reacting with any of pepsin-solubilized type IV collagen and type IV collagen 7S domain. It is preferable to use a combination of monoclonal antibodies that react with pepsin-solubilized type IV collagen and do not react with the type IV collagen 7S domain. Among them, clone numbers 4H12 (hereinafter referred to as “clone 4H12”) and clone numbers 1D6 (hereinafter referred to as “clone 1”) obtained in Example 1 described later.
D6 ”).

In the present invention, the above monoclonal antibody is used after being immobilized on latex particles. The latex particles are not particularly limited. For example, fine particle carriers generally used in immunochemical agglutination reactions and agglutination inhibition reactions can be used.

As the carrier for the fine particles, organic fine particles that can be industrially mass-produced are preferable, but are not limited thereto. The organic fine particles that can be mass-produced industrially are not particularly limited, and include, for example, a homopolymer or copolymer of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylates, and methacrylates. Coalesced; fine particles such as butadiene-based copolymers such as styrene-butadiene copolymer and methyl methacrylate-butadiene copolymer; carboxyl group, primary amino group, carboamino group (-
CONH ₂ ), a hydroxyl group, an aldehyde group, etc., and
Reactive organic fine particles in which the substrate is composed of organic fine particles are exemplified. Among them, polystyrene-based latex particles are preferred because they have excellent antibody-adsorbing properties and can stably maintain biological activity for a long period of time.

The average particle size of the latex particles is not particularly limited, and aggregates resulting from an agglutination reaction caused by an antigen-antibody reaction between the immobilized monoclonal antibody and an antigen substance to be measured are visually or optically aggregated. What is necessary is just to show the magnitude | size which is sufficient for the detection. More preferably, it is 0.01 to 1.0 μm, further preferably,
It is 0.05 to 0.500 μm.

The method for immobilizing the monoclonal antibody on the surface of the latex particles is not particularly limited, and a known method can be used. For example, a method of physically adsorbing the monoclonal antibody to the surface of the latex particles, a method of sensitizing the surface of the latex particles having a functional group by a known method, such as a chemical bonding method or a covalent bonding method, may be used.

When several kinds of mouse anti-human type IV collagen antibodies are immobilized, for example, one kind of each antibody is separately immobilized on latex particles to prepare several kinds of monoclonal antibody-immobilized latex. After that, the mixture may be mixed, or several kinds of monoclonal antibodies may be mixed in advance and immobilized on latex particles at once.

When mixing the latex particles on which the above-mentioned several types of monoclonal antibodies are immobilized, the mixing ratio is not particularly limited, and the particle size of the latex particles used, the amount of the monoclonal antibody, and the required It is appropriately selected according to the sensitivity and the like.

The amount of the monoclonal antibody immobilized on the surface of the latex particles is not particularly limited. In the present invention, an antigen-antibody reaction is performed, and the amount is sufficient to induce aggregation of latex sufficient to be detected, and, due to excess monoclonal antibody, non-specific agglutination reaction via the antibody itself or in blood It is sufficient that the non-specific agglutination reaction with the coexisting substance does not occur.

When the above-mentioned several kinds of mouse anti-human type IV collagen antibodies are immobilized, the amount of immobilization of each monoclonal antibody may be the same, and the activity and non-specific reaction of each monoclonal antibody may be demonstrated. What is necessary is just to select suitably for every production lot according to a degree. Preferably, the amount is 5 to 50 mg per 1 g of latex particles, but is not necessarily limited to this range.

In the present invention, the latex particles having the monoclonal antibody immobilized thereon are suspended in a buffer and used as a latex reagent. The buffer is not particularly limited, and includes, for example, phosphate-based and glycine-based buffers; and buffers generally used in biochemical experiments, such as Good's buffer. Preferred is a Good's buffer comprising tris (hydroxy) aminomethane and a buffer having a sulfonic acid group. Examples of the buffer having tris (hydroxy) aminomethane and a sulfonic acid group include 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS).
O), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) and N-tris (hydroxymethyl) methyl-2-aminopropanesulfonic acid (TES) are preferred.

The pH of the buffer is preferably 5.0 to 9.0. If the ratio is less than 5.0 or exceeds 9.0, the reaction between the monoclonal antibody and type IV collagen does not sufficiently occur. More preferably, it is 7.0 to 8.5.

In the present invention, bovine serum albumin (hereinafter referred to as Bovine serum albumin) is used for stabilizing and improving sensitivity.
Additives that do not show an activity specific to an antigen-antibody reaction, such as “BSA”), casein, casamino acid, etc., may be added.

Further, to the latex reagent, globulin obtained from the same animal species or another animal species immobilized on the latex particles, products thereof, and the like may be added. The globulin or the like is used after being immobilized on latex particles. The method for immobilizing the globulin or the like on the latex particles may be the same as the method for immobilizing the monoclonal antibody on the latex particles, or may be a different method.

In the present invention, the amount of type IV collagen in the sample is determined by mixing the sample containing type IV collagen and a latex reagent containing latex particles having the monoclonal antibody immobilized thereon. I do. The reaction between the latex particles on which the monoclonal antibody is immobilized and the type IV collagen is an antigen-antibody reaction and an accompanying agglutination reaction.

In the antigen-antibody reaction, the liquid component to be added, for example, a buffer for diluting a sample, etc. may be a buffer generally used for biochemical applications, for example, a phosphate type, a glycine type, or the like. A buffer such as a tris-based buffer can be used.

The pH of the reaction solution in the above antigen-antibody reaction
Is preferably 7.0 to 8.5, and in particular, 7.5 to 8.5.
5 is preferred. In the above reaction solution, BS was used as a stabilizer.
A, sucrose; water-soluble polysaccharides such as polyethylene glycol and dextran to enhance sensitivity; sodium azide as a preservative; sodium chloride and the like for salt concentration adjustment.

The reaction conditions between the sample and the latex particles on which the monoclonal antibody is immobilized are not particularly limited as long as the above-mentioned antigen-antibody reaction can occur, but the reaction is preferably carried out at a constant temperature, and particularly preferably at 25 ° C. It is preferably carried out at -37 ° C. The reaction time is not particularly limited, either.
Seconds to 15 minutes are preferred.

The method for measuring the degree of aggregation of the latex particles is not particularly limited. For example, when the aggregation is measured qualitatively or semi-quantitatively, the degree of aggregation of the conjugate can be visually determined from comparison with the degree of turbidity of a known sample. When the aggregation is quantitatively measured, for example, it is preferable to measure the aggregation optically from the viewpoint of simplicity and accuracy.

The optical measuring method is not particularly limited, and includes, for example, a turbidity method in which the formation of aggregates is regarded as an increase in turbidity; a particle size distribution in which the formation of aggregates is regarded as a change in particle size distribution or average particle size. Measurement method: a change in forward scattered light due to the formation of aggregates is measured using an integrating sphere, and an integrating sphere turbidity method for comparing the ratio with transmitted light intensity is exemplified.

Each of the above measuring methods can utilize a speed test. The kinetic test, also called a rate assay, is a test in which at least two measurements are obtained at different time points, and the increment of the measurement value between these time points, that is, the degree of aggregation is determined based on the rate of increase. In the present invention, it is preferable to perform a speed test using a turbidimetric method from the viewpoint of simplicity and quickness of measurement.

The latex reagent comprising the latex particles having the monoclonal antibody immobilized thereon can be applied to a measuring device such as a fully automatic analyzer, so that the results do not differ depending on the practitioner. A more accurate measurement result can be obtained and can be provided as a reagent for measuring type IV collagen. If necessary, a buffer for dilution of a sample or a standard type IV collagen may be attached to the measurement reagent. A reagent for measuring type IV collagen comprising the latex reagent is also one of the present invention.

In the immunoassay for type IV collagen of the present invention, a latex reagent comprising latex particles having a type IV collagen monoclonal antibody immobilized thereon is used. Quick, simple, and accurate quantification of type IV collagen can be performed.

In the present invention, beads as a solid phase in the EIA method include constituent substances, size, charge state of the solid phase surface,
Although latex particles having a large difference in roughness, hydrophobicity, etc. are used, as the above-mentioned monoclonal antibody, those that specifically react with type IV collagen even when immobilized on latex particles are selected and used. Therefore, it has the same specificity as the EIA method, and also has excellent performance.

In the present invention, when the clones 4H12 and 1D6, which are anti-human type IV collagen antibodies, are used
Since the specificity to type collagen is excellent, more accurate quantification of type IV collagen can be performed. Clone 4H12 [mouse-derived monoclonal anti-human type IV collagen antibody-producing hybridoma (IV-4H
12)] is clone 1D6 (hybridoma IV-1D6) as accession number FERM BP-2847.
Is the accession number FERM P-17012 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture (Postal code 305-
8566) at the National Institute of Advanced Industrial Science and Technology (NIBH) of the Ministry of International Trade and Industry.

Furthermore, according to the type IV collagen immunoassay of the present invention, type IV collagen can be quantified in about 10 to 20 minutes. The quantification can be performed in a very short time compared to the required 3 to 4 hours, and a large number of samples can be easily measured with high accuracy.

[0042]

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

Embodiment 1Anti-human type IV collagen pepti
Of monoclonal antibodies (A) Preparation of antigen-pepsin solubilized human type IV collagen
Made Method of Mayne et al. Using human placenta as material
[Artery, Vol. 7, 262-280
P. 1980] with 0.5 N acetic acid.
And collagen with pepsin digestive enzyme (1mg / ml)
After solubilizing the sodium chloride, the sodium chloride was adjusted to a final concentration of 2M.
Was added to precipitate collagen. 0.5N vinegar
Dissolved in acid, 0.5N acetic acid containing 0.7M sodium chloride
By dialysis with a solution, type I collagen, type III
Collagen is precipitated, and the supernatant is washed with 1.2 M sodium chloride.
Dialysed against 0.5N acetic acid solution containing
, V-type collagen was precipitated.

The type IV collagen and type V collagen fractions were dissolved in 50 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, and 50 mM Tris-HCl buffer (pH 7.4) containing 2.2 M sodium chloride. ) In IV
Collagen was precipitated and separated from type V collagen.
The purity of Form IV obtained was determined by the method of Sykes et al. [Biochemical and Biophysical Research Communications (Biochem. Bio).
phys. Res. Commu. ) 72 volumes, 1472-
1480, 1976], sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-P
AGE) was about 90%. In this case, 174 kD, 135 kD, 108 kD, 92 kD
D, 78 kD, 68 kD, 60 kD, 56 kD, 43 k
Eleven different bands at D, 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 together with complete Freund's adjuvant was used for an 8-week-old BALB / c.
Two female mice were first intraperitoneally administered. From the second time on,
100 μg of antigen dissolved in 50 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride was used for 2 to 4
Each week, BALB / c female mice were boosted 2-4 times. As final immunization, spleen cells were prepared by intravenous administration 3 days before removal of the spleen.

(C) Cell fusion The following materials and methods are used. -RPMI 1640 medium: RPMI 1640 (Di
fco Laboratories), sodium bicarbonate (12 mM) and sodium pyruvate (1 m
M), L-glutamic acid (2 mM), potassium penicillin G (50 u / ml), streptomycin sulfate (50
μg / ml) and amikacin sulfate (100 μg / m
l), adjust the pH to 7.2 with dry ice, and add 0.2
The cells were sterilized and filtered through a μm Toyo membrane filter. -NS-1 medium: Fetal calf serum (manufactured by MA Bioproducts), which had been sterilized and filtered, was added to RPMI 1640 medium to a concentration of 15% (v / v).

HAT medium: NS-1 medium was further supplemented with hypoxanthine (100 μM) and aminopterin (0.4 μM).
M) and thymidine (16 μM) were added. HT medium: except that aminopterin was removed
It was prepared in the same manner as the AT medium. -PEG 4000 solution: polyethylene glycol 4000 (PEG 4000, Mer in RPMI 1640 medium)
ck & Co. Inc.) (50% (w / w) serum-free medium).

8-Azaguanine-resistant myeloma cells NS
-1 (P3-NS1-1) is fused with Oi (Oi).
[Selected Method in Cellular Immunology (Edited by BB Michel and S.M.S.)]
cted Method in Cellular I
Munology (ed.BB Mischel)
and S.M. M. SHiigi), WH
Freeman & Company (WH Freem)
an and Company), 1980, 351
352] was slightly modified.

The nucleated splenocytes (viable cell rate 95%) and myeloma cells (live cell rate 95%) prepared in the above section (b) were fused at a ratio of 5 to 6: 1. Splenocytes and myeloma cells were separately washed with RPMI 1640 medium.
Next, the cells were suspended in the same medium and mixed at the above ratio for fusion. Using a 50 ml conical styrene resin test tube (manufactured by Iwaki Glass), the mixture was centrifuged in 40 ml of RPMI 1640 medium at 400 × g for 10 minutes, and the supernatant was completely sucked out. 37 ° C heated PE for precipitated cells
1 ml of the G4000 solution was added dropwise for 1 minute with gentle stirring, and the mixture was further stirred for 1 minute to resuspend and disperse the cells.

Next, 1 ml of an RPMI 1640 medium heated at 37 ° C. was added dropwise over 1 minute. After repeating this operation once more, 7 ml of the same medium was added dropwise with constant stirring for 2 to 3 minutes to disperse the cells. This was centrifuged at 400 × g for 10 minutes, and the supernatant was completely removed by suction. Next, 10 ml of NS-1 medium warmed at 37 ° C. was quickly added to the precipitated cells, and large clumps of cells were carefully dispersed by pipetting using a 10 ml pipette. Furthermore, the same medium 2
0 ml was added for dilution, and 5.9 × 10 ⁵ cells / 0.1 ml of cells were seeded per well into a polystyrene 96-well microwell (manufactured by Iwaki Glass). In addition, 96-well microwells were prepared as 0.2 ml as a pretreatment.
NS-1 medium was added, and the medium was kept in a carbon dioxide incubator (37 ° C.) overnight, and the medium was removed by suction when used. The microwell in which the cell fusion was completed was incubated at 37 ° C. and 100% humidity in 7% carbon dioxide / 93% air.

(D) Selective Growth of Hybridoma in Selection Medium On the first day of culture, 2 drops (about 0.1 ml) of HAT medium were added using a Pasteur pipette. On days 2, 3, 5, 8, and 11, half (0.1 ml) of the medium was replaced with fresh HAT medium. On day 14, the medium was switched to the HT medium, and the same operation was repeated every 3 to 4 days thereafter. Usually, sufficient hybridoma growth is observed in 2 to 3 weeks.

For all hybridoma-growing wells,
Positive wells were checked by the solid phase-antibody binding test (ELISA) described in (e). Next, 1 ml of HT medium containing 10 ⁷ mouse thymocytes was used as a feeder.
To a polystyrene 24-well cell well (Iwaki Gl)
(manufactured by Ass Inc.), and the entire contents of each positive hybridoma detected above were transferred. In the presence of 7% carbon dioxide gas,
Incubated at 37 ° C. for about one week. Meanwhile, 1-2
Each time, 0.5 ml of the supernatant of each well was replaced with 0.5 ml of fresh HT medium.
Replaced with ml. When the hybridomas had grown sufficiently, the positivity was confirmed again by ELISA, and each was cloned by the limiting dilution method described in (f). The residual solution used for cloning was used as a 25 cm ³ polystyrene tissue culture flask (Iwaki G).
(manufactured by Lass Inc.) to prepare a sample for cryopreservation.

(E) Search for hybridomas producing anti-human type IV collagen peptide antibody by ELISA method Renard et al. [Analytical Biochem.
104, 205-214, 1980]. This method is suitable for detecting a hybridoma antibody. 96-well microtitration plate (Flow Laboratories, Inc.
0.5-1.0 μg of pepsin-solubilized human IV
Was coated with type I collagen, and the others were coated with 1% BSA and blocked. A portion of the supernatant of the hybridoma growth well was added thereto and incubated at room temperature for about 1 hour. Horseradish peroxidase (POD) -labeled goat anti-mouse immunoglobulin (Cap
pel Lab. Was added, and the mixture was further incubated at room temperature for about 1 hour. Next, hydrogen peroxide and the substrate o-
Phenylenediamine (OPD) was added, and the degree of brown generated was measured for absorbance at 492 nm using a microplate reader (MPR-A4, manufactured by Toyo Soda Kogyo KK).

(F) Cloning Since there is a possibility that two or more types of hybridomas may grow in each well, cloning was performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. 10 feeders per ml of NS-1 medium
A cloning medium containing ^seven mouse thymocytes was prepared, and 5, 1, and 0.5 hybridomas were added per well to 36, 36, and 24 wells of a 96-well microwell. On days 5 and 12, 0.1 ml of each NS-1 medium was added. After 14 to 15 days from the start of cloning, sufficient hybridoma growth was observed, and ELISA was performed on a group in which the number of colony formation negative wells was 50% or more. If all the tested wells were not positive, the number of colonies in the antibody-positive wells was confirmed, and 4 to 6 wells of one colony were selected and re-cloned. Finally, 22 clones were obtained for pepsin-solubilized type IV collagen.

(G) In vitro growth and in vivo growth of monoclonal antibody Each clone obtained in the above section (f) was cultured (in vitro growth) in an appropriate culture medium such as NS-1 medium, and each of the culture supernatants was used. A monoclonal antibody was obtained (the concentration of the monoclonal antibody protein was 10 to 100 μg /
ml). On the other hand, in order to obtain a large amount of antibodies, animals that are syngeneic to the animal from which the splenocytes and myeloma cells are derived (BALB / c,
In mice), the tumor formation promoter pristane (2, 6, 10,
14-tetramethylpentadecane, Aldrich C
chemical) (0.5 ml per mouse). Hybridoma 1 × 10 after 1-3 weeks
By intraperitoneal administration of ^{7 of} them, 1
~ 2 weeks later, monoclonal antibody protein concentration 4-7
mg / ml ascites could be obtained.

(H) Isotypes of heavy chain and light chain of monoclonal antibody Each ascites obtained in the above (g) was firstly added to each row of wells of a microtitration plate coated with pepsin-solubilized human type IV collagen. Then, each monoclonal antibody in each ascites fluid was bound according to the ELISA method. After washing, isotype-specific rabbit anti-mouse I
g (H + L) antibody was added, and heavy chain and light chain isotypes were detected using 2,2'-azino-di (3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide as substrates. The results are shown in Table 1. Looking at each of the obtained monoclonal antibodies, among these monoclonal antibodies, 16 had immunoglobulin γ1 / κ and two had γ.
2b / κ, one with α / κ, and three with μ / κ.

[0057]

[Table 1]

(I) Purification of monoclonal antibody Each ascites fluid obtained in (g) above was subjected to ammonium sulfate fractionation (40% saturation) and then equilibrated with 40 mM phosphate buffer (pH 8.0) containing 0.06 M sodium chloride. DEAE-Seph
A non-adsorbed fraction of acel (manufactured by Pharmacia) was collected, and fetal calf serum and mouse-derived protein in the medium were separated and removed. Further, the protein A was adsorbed to a protein A cellulofine (manufactured by Seikagaku Corporation) column equilibrated with a 0.15 M sodium chloride-containing tris-hydrochloric acid buffer (pH 8.6), and a non-adsorbed fraction was removed. Purification was achieved by elution with a 50 mM acetate buffer (pH 4.0) containing sodium chloride. The eluate was immediately used for 1.
Neutralized with 5M Tris-HCl buffer (pH 8.9).

(J) Reactivity of monoclonal antibody The reactivity of the purified monoclonal antibody obtained in the above (i) was examined by tissue staining and Western blot. For tissue staining, liver cells frozen in dry ice-acetone were cut into 4 μm-thick frozen sections, immersed in acetone for 5 minutes, and air-dried. The obtained tissue section and the purified monoclonal antibody were incubated at room temperature for 30 minutes. Fluoroisothiocyanate (FI) as a secondary antibody
TC) labeled goat anti-mouse immunoglobulin (Cappel
Lab. Was added, and the mixture was further incubated at room temperature for 30 minutes and observed.

In the Western blot, the pepsin-solubilized type IV collagen and pepsin-solubilized type IV collagen obtained in the above (a) were treated with bacterial collagenase, and the triple helix portion other than the 7S domain of type IV collagen was removed. After decomposition, 0.05% Tween
Sephacryl equilibrated with en20, 10 mM phosphate buffer (pH 7.0) containing 0.1 M sodium chloride.
The type IV collagen 7S domain obtained by gel filtration with S-400 (Pharmacia) was subjected to SDS-
After PAGE, the protein was transferred to a nitrocellulose membrane.

The membrane was incubated with the purified monoclonal antibody for 3 hours at room temperature. POD-labeled goat anti-mouse immunoglobulin (Cappel Lab.
Was further incubated at room temperature for 3 hours, and the reactivity with the pepsin-solubilized type IV collagen having the central triple helix and the 7S domain and the pepsin-solubilized type IV collagen 7S domain without the central triple helix was determined. Examined. The results are shown in Table 2. Eleven of the obtained monoclonal antibodies reacted with liver tissue sections.
In addition, 13 of the obtained monoclonal antibodies were pepsin-solubilized type IV collagen and pepsin-solubilized IV.
Reacted with the type 7 collagen 7S domain, the remaining 7
It reacted with pepsin-solubilized type IV collagen and did not react with pepsin-solubilized type IV collagen 7S domain.

[0062]

[Table 2]

Embodiment 2Type IV collagen measurement
Method of preparing reagent (1) Material Latex: polystyrene latex (average particle size 0.3
μm, manufactured by Sekisui Chemical Co., Ltd.). Buffer: • Monoclonal antibody dilution buffer: 50 mM sodium phosphate
Thorium buffer (NaPB) (pH 7.5) 50 mM NaH_Two PO_Four ・ 12H_Two O and 50 mM N
a_Two HPO_Four ・ 2H_Two Mixed with O to bring the pH to 7.5
The adjusted one was used. -Blocking buffer: 1% BSA-NaPB (pH
7.5) 100 mM NaH_Two PO_Four ・ 12H_Two O and 100 mM
  Na_Two HPO_Four ・ 2H _Two O and pH 7.5.
NaN_Three 0.1% (w / v), B
SA (Fraction V, Reagent gra
de) was dissolved to 1.0% (w / v).
Was used.

Latex suspension buffer: 1% BSA
10 mM N-2-hydroxyethylpiperazine-N '
-2-ethanesulfonic acid (HEPES) buffer (pH
7.5) NaN was added to 10 mM HEPES (pH 7.5)._Three To
0.1% (w / v) and BSA to 1.0% (w / v).
Used in this manner. -Sample dilution buffer: 0.35% polyvinylpyrrolidone
(PVP) 100 mM NaPB (pH 7.5) 100 mM NaH_Two PO_Four ・ 12H_Two O and 100 mM
  Na_Two HPO_Four ・ 2H _Two O and pH 7.5.
NaN_Three 0.1% (w / v), B
SA (Fraction V, Reagent gra
de) is 1.0% (w / v), PVP (PK-90, B
ASF) (0.35% (w / v))
What was made to use was used.

Standard: Normal human pool serum (THS, C
DP Co., Ltd.) was freeze-dried, reconstituted with purified water, and the concentration of type IV collagen measured by the method described in JP-A-2-1553 was used as a human serum standard.

(2) Preparation of latex reagent Take 60 μl of latex solution (solid content: 10%) and dilute it into a monoclonal antibody dilution buffer [50 mM NaPB (pH
7.5)] and mixed well with 540 μl. Monoclonal antibody [clone number 1D6 (clone 1D6), 1
[4.7 mg / ml] was diluted to 5 mg / ml with a monoclonal antibody dilution buffer, 15 μl of the solution was added, and 1235 μl of a monoclonal antibody dilution buffer was added and mixed well. After incubating each at room temperature for 30 minutes, the monoclonal antibody diluent was added while the latex solution was well stirred on a magnetic stirrer.
After stirring for 60 minutes as it was, 3 ml of blocking buffer was added, and the mixture was further stirred for 90 minutes. afterwards,
Centrifugal washing (15000 rpm, 4
(15 ° C., 15 minutes), the supernatant was gently discarded by decantation, and 5 ml of a latex suspension buffer was added to the obtained latex pellet to disperse well. This operation was repeated three times. After the third washing, the obtained latex pellet was well dispersed in 3.6 ml of a latex suspension buffer, and the mixture was mixed with an ultrasonic cell disrupter (Astrason,
W-850). The obtained latex suspension (solid content: 0.17%) was used as a latex immobilized with 1D6 antibody.

The 4H12 antibody-immobilized latex is
As a monoclonal antibody, clone number 4H12
(Clone 4H12, 32.1 mg / ml), except that it was obtained as described above. Furthermore, clone numbers 1D3, 8G12, and 9C7 (hereinafter, “clone 1D3” and “clone 8G1
2 "and" clone 9C7 ") to prepare latex immobilized with 1D3 antibody, latex immobilized with 8G12 antibody, and latex immobilized with 9C7 antibody, respectively.

(3) Evaluation of the binding ability between the antibody-bound latex and type IV collagen The type IV collagen recognition site of the antibody on the various antibody-immobilized latexes prepared in the above section (2) was bound to the latex to form the antigen-antibody. The following method was used to determine whether the reaction was inhibited. The antibody-immobilized latex and pepsin-solubilized type IV collagen were incubated at 37 ° C. for 10 minutes to react the type IV collagen with the antibody-immobilized latex. This is centrifuged and unbound I
Type V collagen was measured by the method described in JP-A-2-1553.

The binding ability between the antibody-bound latex and type IV collagen was calculated as follows. Binding capacity (%) = ([Amount of added antigen]-[Amount of antigen in supernatant])
/ [Amount of added antigen] × 100 The results are shown in Table 3. As is evident from the results, clone 1D3 whose antigen was detectable by the method described in JP-A-2-1553 (EIA method) lost its binding ability by immobilizing it on latex. . Similarly, clone 9C7 and clone 8G12, which were confirmed to react with the antigen by ELISA in Example 1 (e), also lost their binding ability by immobilization on latex. On the other hand, clones 1D6 and 4H12 retained their binding ability even when immobilized on latex. From these results, it was found that an antibody suitable as a material for the latex reagent was present, and that an antibody suitable for the latex reagent could be selected by evaluating its binding ability. Such an antibody is considered to have a configuration suitable for binding to the antigen on the latex.

[0070]

[Table 3]

Embodiment 3Measurement of type IV collagen standard Combination of the following reagents
As a drug, the sensitivity was measured using a standard product. ・ Latex reagent (1D6 antibody fixed at 0.17% solid content)
Latex and 4H12 antibody-immobilized latex reagent
1: 1 mixed in equal volume) ・ Sample dilution buffer ・ Standard product (Type IV collagen concentration 700, 350, 17)
5, 83, 0 ng / ml)

The type IV collagen was measured using a latex reagent using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.). The measurement conditions were as follows. Sample volume: 20 μl Latex reagent: 90 μl Sample dilution buffer: 180 μl Measurement wavelength: 700 nm Measurement temperature: 37 ° C.

After the sample and the sample diluent buffer were well mixed in advance, the increase in absorbance (ΔOD700) was measured about 80 seconds and about 320 seconds after the addition of the latex reagent, and this was measured at 10,000. The doubled value was defined as the change in absorbance. The above operation was performed using a standard sample as a sample to prepare a calibration curve.

COMPARATIVE EXAMPLE 1 As a latex reagent, 1D6 antibody-immobilized latex (solid content: 0.17%) was used instead of a 1: 1 mixture of 1D6 antibody-immobilized latex and 4H12 antibody-immobilized latex in an equal amount of 1: 1. Except for this, the measurement was performed in the same manner as in Example 3.

Comparative Example 2 Instead of latex reagents in which 1D6 antibody-immobilized latex and 4H12 antibody-immobilized latex were mixed in equal amounts of 1: 1, 1D6 antibody-immobilized latex (solid content 0.17%) and 8G12 antibody were used. The measurement was carried out in the same manner as in Example 3 except that a mixture of immobilized latex (solid content: 0.17%) in an equal amount of 1: 1 was used.

Comparative Example 3 Instead of latex reagents in which 1D6 antibody-immobilized latex and 4H12 antibody-immobilized latex were mixed in equal amounts of 1: 1, 1D6 antibody-immobilized latex (solid content 0.17%) and 9C7 antibody were used. The measurement was carried out in the same manner as in Example 3 except that a mixture of immobilized latex (solid content: 0.17%) in an equal amount of 1: 1 was used.

Comparative Example 4 As a latex reagent, 4H12 antibody-immobilized latex (solid content: 0.17%) was used instead of a mixture of 1D6 antibody-immobilized latex and 4H12 antibody-immobilized latex in an equal amount of 1: 1. Except for this, the measurement was performed in the same manner as in Example 3.

Comparative Example 5 As latex reagents, 1H6 antibody-immobilized latex and 4H12 antibody-immobilized latex were mixed in an equal amount of 1: 1 instead of 4H12 antibody-immobilized latex (solid content 0.17%) and 1D3 antibody. The measurement was carried out in the same manner as in Example 3 except that a mixture of immobilized latex (solid content: 0.17%) in an equal amount of 1: 1 was used.

Comparative Example 6 As latex reagents, 1D3 antibody-immobilized latex (0.17% solids) and 8G12 antibody were used instead of 1D6 antibody-immobilized latex and 4H12 antibody-immobilized latex in the same 1: 1 mixture. The measurement was carried out in the same manner as in Example 3 except that a mixture of immobilized latex (solid content: 0.17%) in an equal amount of 1: 1 was used.

Comparative Example 7 As latex reagents, 1D3 antibody-immobilized latex and 4H12 antibody-immobilized latex were mixed in equal amounts of 1: 1 instead of 1D3 antibody-immobilized latex (solid content 0.17%) and 9C7 antibody. The measurement was carried out in the same manner as in Example 3 except that a mixture of immobilized latex (solid content: 0.17%) in an equal amount of 1: 1 was used.

Table 4 shows the amount of change in absorbance of Example 3 and Comparative Examples 1 to 7. As apparent from Table 4, clone 1D
Latex reagents in which only one of monoclonal antibodies 6 and 4H12 was immobilized, and latex reagents in which one of clones 1D6 and 4H12 was mixed with latex in which other antibodies were immobilized, were used as 1D6 antibody. Unlike the case where a latex reagent was prepared by mixing the immobilized latex and the 4H12 antibody-immobilized latex, the change in absorbance of the standard product did not increase, and only the combination of clone 1D6 and clone 4H12 showed IV in human serum. It was found to be a reagent capable of specifically detecting type collagen.

[0082]

[Table 4]

Embodiment 4Type IV collagen in human serum
Measurement: Correlation with EIA method Healthy humans, human blood including liver cancer (HCC) and cirrhosis (LC)
For 30 samples, 1D6 antibody immobilized latex and 4
Equally mixed 1: 1 with H12 antibody-immobilized latex reagent
Type IV collagen in the sample using the latex reagent
Was measured. Using the calibration curve obtained in Example 3
And 30 types of human serum whose type IV collagen concentration is unknown
From the amount of change in absorbance of
I asked. Table 5 shows the results.

Comparative Example 8 The concentration of type IV collagen in 30 human serum samples used in Example 4 was measured by the EIA method (the method described in JP-A-2-1553). Table 5 shows the results.

[0085]

[Table 5]

Based on the above results, the correlation between the measured value of Example 4 and the measured value by the EIA method is shown in FIG. As is clear from these results, it was found that the latex reagent used in Example 4 showed the same correlation as that of the EIA method (the method described in JP-A-2-1553).

Embodiment 5Type IV collagen in human serum
Measurement: Simultaneous reproducibility EIA method (method according to JP-A-2-1553)
Type IV collagen-positive human pooled sera (1
00, 310, 580 ng / ml) in Example 3
The measurement was repeated 10 times each using the above reagents. Flatness of measured value
Average, standard deviation, variation (CV) (%) value of measured value
I asked. The results are shown in Table 6.

Comparative Example 9 The type IV collagen-positive pooled human serum used in Example 5 was repeatedly measured 10 times using the EIA method described in JP-A-2-1553. Average, standard deviation, C.I. V. (%) Value was determined. The results are shown in Table 6.

[0089]

[Table 6]

From the above results, it was found that the latex reagent used in Example 5 exhibited simultaneous reproducibility (measurement accuracy) superior to the method described in JP-A-2-1553.

[0091]

As described above, the immunoassay for type IV collagen of the present invention is as described above, so that type IV collagen can be quantified quickly, easily and accurately, and is useful for diagnosis of liver disease and the like. Can be used. Further, since the latex reagent used in the present invention can be applied to a fully automatic analyzer, it is also suitable as a reagent for measuring type IV collagen.

[Brief description of the drawings]

FIG. 1 is a graph showing a correlation between a measured value of Example 4 and a measured value by an EIA method (Comparative Example 8). The vertical axis is the measured value of Example 4, and the horizontal axis is the measured value by the EIA method.

────────────────────────────────────────────────── ─── Continued on the front page (72) Kenichi Obata, Inventor Kenichi, Takaoka, 530 Fuji Pharmaceutical Company Limited (56) References JP-A-2-1553 (JP, A) JP-A-7-72148 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/543 G01N 33/53

Claims (4)

(57) [Claims] Claims: 1. The amount of type IV collagen in a sample was determined by using clone 4 as an anti-human type IV collagen monoclonal antibody.
Latex particles on which H12 and clone 1D6 are immobilized, and / or latex particles on which clone 4H12 is immobilized.
Latex with immobilized tex particles and clone 1D6
An immunoassay for type IV collagen, characterized in that it is quantified by utilizing an agglutination reaction of latex reagents containing dextran particles . 2. The immunoassay for type IV collagen according to claim 1, wherein the type IV collagen is human type IV collagen having a 7S domain and a central triple helix domain. 3. The agglutination reaction of a latex reagent comprising latex particles on which a monoclonal antibody is immobilized,
The immunoassay for type IV collagen according to claim 1 or 2 , wherein the method is performed by mixing a sample and the latex reagent. 4. A reagent for measurement of type IV collagen which is used for performing an immunoassay of type IV collagen according to claim 1, wherein, as the monoclonal antibody
Latex particles with immobilized clone 4H12 and clone 1D6 and / or immobilized clone 4H12
Immobilized latex particles and clone 1D6
A type IV collagen measurement reagent, comprising a latex reagent containing the obtained latex particles .