JP2000002703A - Detecting method of human complement control factor and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To judge the diagnosis and critical degree of renal disease by bringing a second antibody having a labeled molecule connected thereto into contact with a biological sample and a subject having a first antibody brought into contact with the surface of a solid granule, and detecting the labeled molecule connected to a complement control factor. SOLUTION: With respect to urine and serum of a normal person and a renal disease patient, MCP quantity (membrane cofactor protein) is measured. A magnetic bead having a monoclonal antibody 177, a subject and a POD labeled monoclonal antibody M160 are reacted to focus and fix the bead by the magnet, and the supernatant is disposed. After washing, a substrate solution is added to further react them, and the bead is focused and fixed by the magnet, and the supernatant is collected to measure the absorbance. The serum MCP value is measured by use of the monoclonal antibody M160 and also by use of the POD labeled monoclonal antibody M160 as the detecting antibody. As a result of the measurement, the MCP values in urine and serum are distributed in high level in the renal disease patient, compared with the normal person, and also high in a more critical patient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a membrane cofactor protein (hereinafter referred to as MCP) contained in a biological sample such as urine or blood.
A method for detecting complement control factors such as
The present invention also relates to a method for evaluating renal function, which comprises measuring the amount of a complement control factor excreted in urine. Furthermore, the present invention relates to a reagent for diagnosing renal function comprising an anti-complement regulatory factor monoclonal antibody.

[0002]

2. Description of the Related Art Kidney transplantation is the only treatment for end-stage renal failure in which no renal hemofiltration or detoxification functions. However, in Japan, the supply system for transplanted kidneys is well established. However, it is difficult to say that transplantation therapy itself has not gained public recognition, and the current situation is that dialysis therapy must be used as a renal replacement therapy.

At present, the estimated number of dialysis patients in Japan is about 170,000, but the treatment cost per person requires about 6 million yen per year, which is one of the major causes of pressure on the medical insurance system. Further, since the patient is detained for dialysis treatment for 2 to 3 days a week for 4 to 6 hours a day, the social activity of the patient himself is greatly hindered.

[0004] Renal failure is a condition that ultimately leads to kidney disease patients, but the cause and history are not uniform, and drug intoxication,
There are many cases in which renal damage occurs due to lesions other than the kidney such as infectious diseases, malignant tumors, diabetes, and systemic lupus erythematosus (SLE), leading to renal failure.

[0005] In such renal disorders, since remarkable subjective symptoms do not appear until the end stage, that is, near renal failure, the occurrence is easily overlooked, and the kidney is already irreparably damaged at the time of onset. Often. Therefore, it is important to detect renal impairment as early as possible before observing the onset of subjective symptoms, in order to prevent the transition to renal failure and to avoid pressure on insurance finances due to dialysis treatment. .

Conventionally, as a clue for diagnosing renal disorder,
Examination of urine protein and urine sediment by so-called urinalysis is widely performed. However, urinary protein is transiently increased even in healthy subjects due to extreme exercise, mental stress, heavy eating, and premenstrual periods. There are also urinary proteins not derived from renal diseases, such as orthostatic proteinuria, which is common in young people (about 0.5% of healthy people). Urine protein is also found in urinary tract diseases, bladder diseases, female genital diseases and the like. Therefore, it is impossible to make a definite diagnosis of renal disorder by urine protein test.

[0007] Urine sediment is obtained by centrifuging urine and observing the sediment with a microscope. Erythrocyte sediment is also observed in healthy people and may be derived from organs related to the urinary system other than renal disorders. Again, this is not sufficient for a definitive diagnosis of renal impairment. In addition, serum creatinine (Cr) and blood urea nitrogen (BUN) are also measured for the purpose of examining blood retention of urine components, but these tests are also easily affected by diet. As described above, even if abnormal values appear in the examination of urine protein, serum Cr, and BUN, the abnormal values do not always result from renal impairment, and abnormal values often appear in healthy persons and other diseases.

In addition, urinary β2-microglobulin, N-
Diagnosis of renal impairment by measurement of various substances such as acetyl-β-D-glucosamidase, IgG, transferrin, and interleukin-6 has been attempted, but often does not match the severity of renal impairment, and neither is effective. . Other than these, no testing method having sufficient sensitivity and specificity for diagnosing renal damage and determining the severity has been known yet. Therefore, histological diagnosis by renal biopsy is indispensable for the diagnosis of kidney damage and the final determination of the severity.

[0009] However, renal biopsy is an invasive test, and there is always the risk of complications such as bleeding and infection. In addition, in order to perform the examination, it is necessary to be hospitalized in a well-equipped facility with a specialist, and the physical and social burden on the patient cannot be ignored. As described above, the urinalysis test is a simple and excellent test method capable of processing a large number of samples, but is not satisfactory from the viewpoint of a definitive diagnosis of renal disorder. On the other hand, the use of renal biopsy, although reliable in diagnosing renal damage and judging the severity, must be extremely limited. Therefore, there has been a demand for a method for diagnosing renal disorders that combines the simplicity of urine analysis and the accuracy of renal biopsy.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems and to diagnose a kidney disease and determine the severity of the disease. First, the present inventors thought that in order to confirm the diagnosis and determine the severity of renal disorder, it was necessary to measure a specific protein closely related to the disease state. Therefore, first, IgA nephropathy, which is a typical pathological condition of renal disorder, and accounts for 40% of nephritis, is a type of antibody, IgA nephropathy.
Attention was paid to the fact that an immune complex of A and the antigen was deposited on the glomeruli of the kidney. In addition, glomerulonephritis is a type III allergic reaction in which an immune complex in which an antigen and an antibody are bound is deposited on the tissue and the renal tissue is damaged by the activated complement system.
Focused on being a seed.

[0011] The complement system refers to an antibody that is recruited to infection, inflammatory response, immune response, etc., exhibits various biological activities such as lysis, hemolysis, etc., and reacts non-specifically to an immune complex by heat denaturation. It is a biological defense system composed of auxiliary protein components. In addition, the present inventors have focused on complement regulators present in the complement system. That is, the complement control factor is a protein that suppresses the disorderly activation of the complement system, and it is known that when the complement system is enhanced, the expression of the complement control factor also increases. And, as described above, the completion of nephritis involves the immune complex and the complement system. Thus, the present inventors have found that the complement system is enhanced in glomeruli and tubules,
If the expression of the complement control factor increases, the complement control factor leaks into the urine, and the amount of the leakage may increase as the disease progresses. It was thought that the degree of inflammation in the local kidney associated with the complement system could be estimated by measuring. Thus, an attempt was made to measure complement control factors using a normal immunoassay such as ELISA, but the sensitivity was low and was not always practical.

[0012]

Means for Solving the Problems On the other hand, the present applicant has developed a monoclonal antibody which specifically reacts with human sperm after acrosome reaction as a reagent for evaluating the fertility of human sperm. Were combined to develop a sperm fertility test granule (Japanese Patent No. 2651249). According to the present invention, a monoclonal antibody that specifically reacts with human sperm after the acrosome reaction is bound to solid granules to form test granules,
By binding sperm to this, by counting the bound sperm, it was possible to evaluate the fertility of human sperm, radioactivity, without the need for complicated measurement means such as fluorescence,
Easy and reliable fertility testing in a short time. Further, since the solid granules have magnetism, the solid granules can be easily focused by a magnet, and the measurement of a small amount of sample has become possible.

The present inventors have thought that by applying this technique to the detection of complement control factors, it will be possible to simply and accurately detect complement control factors, and have reached the present invention. That is, the present invention provides (1) a granule for complement control factor detection contained in a biological sample, which is obtained by binding an anti-complement control factor antibody to the surface of a solid granule, and (2) a biological sample. A method for detecting a complement control factor contained therein, comprising the following steps: (a) complementing a biological sample with a first anti-complement control factor antibody (a) on the surface of a solid granule; (B) a second sample in which a labeled molecule is bound to a sample that has been brought into contact with the granule for detecting a complement control factor,
(C) detecting the labeled molecule bound to the complement control factor via the second anti-complement control factor antibody (b). A method for detecting a complement control factor contained in a biological sample, comprising the steps of: (3) (a) the granule for detecting a complement control factor according to (1), and (b) A kit for detecting a complement control factor, comprising a magnet, (4) a method for evaluating renal function comprising comparing a measured value of a complement control factor in a sample urine with a complement control factor value in normal urine, and (5) An object of the present invention is to provide a reagent for renal function diagnosis containing an anti-complement regulatory factor monoclonal antibody.

The complement inhibitory factors to be measured in the present invention include C1 inactivating factor, C4 binding protein, factor I,
In addition to factor H, there are membrane cofactor protein (MCP), a decay accelerating factor (DAF), CD59, and type 1 complement receptor (CR1) which have a function of protecting against a self-complement reaction. Although not limited, particularly preferred is MCP.

MCP is an acidic glycoprotein having a molecular weight of 45,000 to 70,000 present in whole blood cells other than red blood cells, and was identified in 1985 as one of the cellular complement regulators (Proceedings of the National Co., Ltd.). Academy of Sciences of the United States of America (Proc. Natl.
Acad. Sci. USA), Vol. 82, 859-863
1985).

The measurement method used in the present invention is not limited, and examples thereof include a light scattering method, a radioimmunoassay method, a fluorescence and luminescence immunoassay method, and an enzyme immunoassay method. Can be. There are no restrictions on the radioactive substance, fluorescent / luminescent substance, and enzyme used for these substances.
5, I131, C14, H3, etc., and the fluorescent and luminescent materials are FITC, umbelliferone, luminol,
There are acridinium derivative, luciferase and the like, and as the enzyme, β-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase and the like can be used. Polyclonal antibodies and monoclonal antibodies can be used without limitation, but monoclonal antibodies are particularly preferred from the viewpoint of specificity.

For example, as a monoclonal antibody specific to MCP, hybridoma strain M75 (available from
Monoclonal antibody M75 produced from ERM BP-2894), monoclonal antibody M160 produced from hybridoma strain M160 (FERM BP-2895 deposited by Microtechnical Research Institute), and hybridoma strain M177 (FERM BP-2896 deposited from Microtechnical Research Institute). Monoclonal antibody M177, hybridoma strain MH-61
Monoclonal antibody MH-61 and monoclonal antibody CD46 (manufactured by Cosmo Bio), which are produced from (FERM BP-2257 deposited by Micro Engineering Laboratory). Further, these monoclonal antibodies can be bound to a label by a conventional method.

Other immunoassays can be performed according to known methods for all of the substrates, detection methods, and the like. There is also no particular limitation on the type of renal disorder that is the target disease, for example, IgA nephropathy, acute progressive glomerulonephritis, acute glomerulonephritis, acute pyelonephritis, chronic pyelonephritis, focal glomerulosclerosis, microscopic sclerosis Altered nephrotic syndrome,
Membranous glomerulonephritis, membranous proliferative glomerulonephritis, diabetic nephritis, lupus nephritis, renal amyloidosis, chronic renal failure, etc. can be widely measured by the method of the present invention.

Among the above antibodies, antiserum (polyclonal antibody) which binds to human MCP is available from European Journal of Immunology (Eur. J. Immunol.),
18, pp. 1289-1294, 1988. Moreover, the anti-human MCP monoclonal antibody and the antibody-producing hybridoma cells can be produced according to the description in JP-A-4-45796.

Hereinafter, the present invention will be described in detail. (A) Production of antibodies (1) Immunization of animals and preparation of antibody-producing cells Immunization of animals is performed, for example, as follows. Known methods, for example, Journal of Experimental Medicine (J. Exp. Med.), Vol. 163, 837-85.
Human MCP purified as described on page 5, 1986 is used to immunize mammals such as rats and mice. As the mammal, it is preferable to use an animal of the same strain as the partner of the cell in which the cells are fused. The age of the animal is preferably, for example, 6 to 8 weeks in mice. Sex may be either male or female. In the immunization method, purified human MCP is mixed with an appropriate adjuvant (for example, Freund's complete adjuvant or aluminum hydroxide gel-pertussis vaccine, etc.) to form an emulsion, and then administered subcutaneously, intraperitoneally, intravenously, etc. to the animal. . Thereafter, this immunization operation was
Perform 2-5 times at 2 week intervals. The final immunization is 0.5-2μ
g of human MCP is administered intraperitoneally to the animals. A polyclonal antibody is obtained from the body fluid of the immunized animal. Three to seven days after each immunization, blood was collected from the fundus venous plexus, and the antibody titer of the serum was determined by the protein A rosette assay shown below (European Journal of Immunology (Eur. J. Immunol.)).
4, 500-507, 1974), and when the antibody titer has sufficiently increased, the antibody or antibody-producing cells are collected.

Protein A rosette assay: A human erythroid cell line K562 (Japanese Cancer Research, Inc.) was placed on a 72-well Terasaki plate (Falcon).
Resources Bank (JCRS)), and Dulbecco's PBS (2.90 g disodium phosphate, 0.20 g monopotassium phosphate, 8 g sodium chloride, 0.1 g potassium chloride).
(2 g, 1 L of distilled water), and the mixture is left in a CO ₂ incubator at 37 ° C. for 30 minutes. After washing with PBS, sheep erythrocytes coated with protein A (manufactured by Amersham Pharmacia Biotech) are added, and the formation of a rosette is observed with a microscope. Human MCP as described above
The antibody-producing cells are collected from the animal immunized with. Antibody-producing cells can be obtained from spleen, lymph nodes, peripheral blood, etc., with spleen being particularly preferred. For example, 3 to final immunity
Four days later, the spleen was aseptically removed, shredded in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), disentangled with forceps, and 1200 rpm × 5.
After centrifugation under the conditions of
The cells are treated with a hydrochloric acid buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, and further washed three times with a MEM medium to obtain spleen cells for cell fusion.

(2) Preparation of Permanently Proliferating Cells As the perpetually proliferating cells to be fused, any cells having permanently proliferating properties can be used, and myeloma cells are particularly popular. Permanently proliferating cells are preferably derived from animals of the same species as the antibody-producing cells. For example, in the case of a mouse, a bone tumor cell line P3-X63Ag8-U1 (P3-U) derived from an 8-azaguanine-resistant mouse (BALB / c)
1) (Current Topics in Microbiology and Immunology (Current Topics in
Microbiol. and Immunol. ), Vol. 81, 1-7
P. 1978), P3 / NS1 / 1-Ag4-1 (N
S-1) (European Journal of Immunology (Eur. J. Immunol.), Vol. 6, 511-519)
P. 1976), SP2 / 0-Ag14 (SP-2).
(Nature, Vol. 276, 269-270
P. 1978), P3-X63-Ag8653 (65
3) (Journal of Immunology (J. Immuno
l.), 123, pp. 1548-1550, 1979.
Year), and P3-X63-Ag8 (X63) (Nature, 256, 495-497, 19).
75 years). These permanently proliferating cell lines were prepared in 8-azaguanine medium (glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 −5 M) in RPMI-1640 medium, gentamicin (10 μg / m 2).
L) and fetal calf serum (FCS, CLS) (10%)
8-azaguanine (15 μl)
g / mL) and subcultured for 3 hours for cell fusion.
The cells are passaged to a normal medium 4 days before, and the number of cells is 2 × 10 ⁷ or more on the day of fusion.

(3) Cell fusion Cell fusion is performed, for example, as follows. The antibody-producing cells obtained in (1) and the permanently proliferating cells obtained in (2) were combined with Minim
al Essential Medium (MEM) medium or PBS
And mix them so that the cell number is in a ratio of 5 to 10: 1. After centrifugation at 1200 rpm for 5 minutes, the supernatant was removed, and the precipitated cell group was thoroughly dissociated.
(PEG-1000) 1-4 g, MEM medium 1-4 mL
And a mixture of 0.5 to 1.0 mL of dimethyl sulfoxide and 0.1 to 1.0 mL / 10 ⁸ cells are added to cause cell fusion. Thereafter, 3 mL of MEM medium is added several times every 10 minutes, and the MEM medium is added and diluted so that the total volume becomes 50 mL, thereby stopping cell fusion. Next, centrifugation (1
The supernatant was removed at 500 rpm × 5 minutes, and the cells were gently dissociated.
% FCS), and the cells are gently suspended by suction and blowing with a female pipette. This suspension was dispensed at 100 μL / well into a 96-well culture plate, and the suspension was added at 37 ° C. in a 5% CO ₂ incubator at 37 ° C.
Incubate for ~ 5 days. 100 μL / well of HAT medium (hypoxanthine (10-4
M), thymidine (1.5 × 10 −5 M) and aminopterin (4 × 10 −7 M) are added), and the cells are further cultured for 3 days. Thereafter, half the volume of the culture supernatant was removed every three days, and the same amount of HAT medium was newly added, and 5% CO ₂
Incubate at 37 ° C for about 2 weeks in an incubator.

(4) Screening and cloning of hybridomas From the wells in which the fused cells were observed to grow in colonies, half of the supernatant was removed and the HT medium (HT
A medium without aminopterin) and add the same amount
Incubate for 4 days. A part of the culture supernatant is collected, and the antibody titer against MCP is measured by the protein A rosette assay described above. The cloning was repeated 4 times by the limiting dilution method for the wells that reacted with MCP, and stable MC
Those exhibiting the antibody titer of P are selected as hybridoma strains producing anti-MCP monoclonal antibody. As a specific example of the hybridoma obtained by the above operation, hybridoma strain M75 (FERM BP-289 deposited by Micro Engineering Laboratory)
4), hybridoma strain M160 (FERM deposited with MIC)
BP-2895), hybridoma strain M177 (FERM BP-2896 deposited by Micro Engineering Research Institute) and hybridoma strain MH-61 (FERM BP-225 deposited by Micro Engineering Research Institute)
7).

(5) Preparation of monoclonal antibody The hybridoma obtained as described above is cultured in vitro and in vivo to produce a monoclonal antibody. When culturing in vivo, the hybridoma is transplanted into any animal, and it is preferable to use an animal of the same species as the animal from which the spleen cells used for cell fusion have been collected. For example, 8 to 10-week-old B treated with pristane (0.5 mL of 2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneally administered and bred for 2 weeks).
In the ALB / c female mouse, 2 to 4 × 10 ^{6 of} the anti-MCP monoclonal antibody-producing hybridoma cells obtained in (4)
Administered intraperitoneally per animal. In a few weeks, ascites containing a high concentration of the monoclonal antibody accumulates in the abdominal cavity of the mouse, and the abdomen becomes enlarged. Ascites was collected from the mouse, and centrifuged (3000 rpm x 5 minutes) to remove solids.
Salt out using 50% ammonium sulfate and 1-
Dialyze for 2 weeks. The dialyzed fraction is passed through a protein A sepharose column, and the IgG fraction is collected to obtain a purified monoclonal antibody.

The antibody isotype can be determined by the octaloni (double immunodiffusion) method (introduction to immunology experiments, biochemistry experiments 15,
74, 1981). The protein amount was determined by the Folin method and 280 nm.
At 1.4 (OD ₂₈₀ ) ≒ 1 mg / mL immunoglobulin. The inhibitory activity of the obtained monoclonal antibody against MCP-dependent factor I cofactor activity was determined by a known method (Journal of
Experimental Medicine (J. Exp. Me)
d. 163, 837-855, 1986).
And quantification by a liquid phase measurement method. That is, fluorescently labeled C3b and Factor I are mixed with a certain amount of MCP previously treated with an antibody, and incubated for a certain period of time. After that, the conversion of C3b to C3bi is analyzed by SDS-polyacrylamide gel electrophoresis, and a fluorometer is used. Measure using

(6) Characteristics of Monoclonal Antibody The characteristics of the monoclonal antibody obtained as described above include, for example, (1) HSB in which the cell surface is labeled with iodine.
-2, Immunoprecipitation reaction using human lymphocyte-derived cell lines such as K562 (Journal of Immunology (J. Immunol.), 138, 2850-3855)
1987), (2) Inhibition test on C3b degradation reaction by factor I in which MCP acts as a cofactor (Biochemical Journal (Biochem. J.),
264, 581-588, 1989) and (3) Enzyme-linked immunosorbent assay (ELISA) (Journal
Of Immunology (J. Immunol.), Vol. 142,
2743-2750, 1989).

(7) Preparation of Labeled Monoclonal Antibody The purified monoclonal antibody obtained is obtained by the glutaraldehyde method (Immunochem.
Vol. 43, 1969), periodate method (J. Histochem. Cytochem.), Vol. 22, 108.
4, 1974), maleimide method (Journal of Biochemistry, J. Biochem.), Vol. 79,
233, 1976) and the pyridyl disulfide method (Biochemical Journal (Biochem. J.), 173, 723, 1978).
Enzyme labeling is possible.

For example, when the periodic acid method is used, 50 μL of periodic acid (38.5 mg / mL) is added to the peroxidase solution (4 mg / mL) while stirring, and the mixture is reacted at room temperature for 20 minutes, and then reacted with 1 mM Acetate buffer (pH 4.
The buffer is exchanged using Sephadex (registered trademark) G-25 packed column PD-10 (manufactured by Amersham Pharmacia Biotech) substituted in 5). Next, 40 μL of 0.2 M sodium hydroxide is added. 10m for this one
10 mg of the monoclonal antibody dialyzed against M carbonate buffer (pH 9.5) is added and reacted at room temperature for 2 hours. After completion of the reaction, the mixture is cooled on ice, and 100 μL of a sodium borohydride solution (4 mg / mL) is added, followed by a reaction for 2 hours. Reaction solution is PD
After replacing with PBS using -10, 3000 rpm x 3
After centrifugation for 0 minutes, the supernatant was separated from Sephacryl S200HR2.
6 x 30 (manufactured by Amersham Pharmacia Biotech)
The solution is subjected to gel filtration, and the absorbance at 403 and 280 nm is measured to collect a fraction of the labeled monoclonal antibody.
Bovine serum albumin (10 mg / mL) is added to the obtained fraction.
And store at −20 ° C., and use PBS-Tween immediately before use.
Dilute with (registered trademark) 20.

(B) Preparation of Test Granules The test granules used in the present invention are prepared by physically or chemically binding the antibodies used in the present invention to suitable granules, for example, a gel for chromatography. Preferred is a method of binding the antibody used in the present invention to the activated granules, and particularly preferred is binding of the antibody used in the present invention to the granules activated tosylation by p-toluenesulfonyl chloride. Is the way. As the granules, beads made of glass, agarose, sepharose, agarose-filled porous diatomaceous earth, hydrophilic copolymerized acrylic gel, polystyrene, or the like are used. Preferably, a magnetic material (eg, Fe ₂ O ₃ ) is used, for example, in the core. To have superparamagnetism. If magnetic beads are used, the beads can be focused using a magnet or the like, so that a small amount of sample can be measured. Also, when beads having a large specific gravity are used, convergence of the beads becomes easy, and the same effect as when using beads having magnetism can be obtained. Furthermore, in this case, since the conditions for centrifugation for concentrating the beads can be slowed down, it is convenient when using an antibody that is easily detached from the binding.

The shape of the granules is arbitrary, such as spherical and irregularly crushed, but spherical is preferred. The particle size is not particularly limited, and may be, for example, several to several tens to several hundreds μm. In order to chemically bind a secondary antibody, protein A or protein G to the above-mentioned granules, it is preferable to activate the granules and then bind them. For the activation of the granules, any activation method for binding proteins to such granules can be selected. Such activation methods include the tosyl chloride method, the Bromcian method, the bromoacetyl method, the glutaraldehyde method, and the like. Some activated granules are commercially available. Such activation and binding of the activated granules to a protein such as a secondary antibody, protein A or protein G can be performed by a conventional method.

Further, granules already bound with a secondary antibody, protein A or protein G, etc. are also commercially available. Such granules include Dynabeads (registered trademark) M-45, which is imported from Nippon Dynal Co., Ltd.
0, M-280 sheep anti-mouse IgG coated type,
Goat anti-mouse IgG coat type, sheep anti-rat Ig
G coat type, sheep anti-rabbit IgG coat type,
Goat anti-mouse IgG (H & L) carboxylate beads, goat anti-rabbit IgG (H & L) carboxylate beads, protein A carboxylate beads, goat anti-rabbit IgG (H) manufactured by Polysciences Incorporated.
& L) Micro magnet particles, goat anti-rabbit Ig
G (H & L) micro magnet particles, protein A micro magnet particles, sheep anti-mouse I
gG (H & L) micro magnet particles. In order to bind the antibody used in the present invention to the above-mentioned granules, the granules suspended in a suitable medium are treated with a protein solution to prevent non-specific adsorption, and then the ascites containing the antibody or the purified antibody. Mix the solution.

(C) Test Method To carry out the test method of the present invention, blood or urine is collected from a subject, and the supernatant after centrifugation is used as a specimen. The diluted specimen and the labeled conjugated antibody obtained in (7) are added to the test granules produced as described above, and incubated at room temperature for 2 hours. After completion of the reaction, the mixture was washed, centrifuged to remove granules, and the supernatant was transferred to a microplate.
Measure the absorbance at 90 nm. Similarly, a sample of a normal person is measured and the values are compared.

(D) Kit In order to carry out the above experiment, it is convenient to prepare materials necessary for the execution into a kit. Such a kit may include the test granules and magnet described above. It may further include an antibody to which a label molecule is bound. In addition, the kit can include test tubes, centrifuge tubes, other similar containers, pipettes or similar suction devices, or microscopes.
Instead of the test granules, a combination of solid granules as a raw material for the production and an antibody can also be used.

[0035]

EXAMPLES The present invention will be described in more detail with reference to Examples and Examples. Reference Example (Purification of Monoclonal Antibody) 8 to 10-week-old BAL treated with pristane (manufactured by Sigma)
Hybridoma strain M75 (FERM BP-2894 deposited by MRI) and hybridoma strain M16 were added to B / c female mice.
0 (FERM BP-2895 deposited by the Japan Institute of Technology), and hybridoma strain M177 (FERM BP-289 deposited by the Institute of Fine Technology)
6) and hybridoma strain MH-61 (deposited by
ERM BP-2257) was injected intraperitoneally at 1 × 10 ⁷ cells / animal. After 2-3 weeks, the hybridomas grew as ascites cancer cells. Ascites was collected and centrifuged (3000 rpm x 5 minutes) to remove solids. 5
After salting out with 0% ammonium sulfate and dialyzing with PBS, the pet fraction was passed through a protein A sepharose column (manufactured by Pharmacia) with a volume of 20 mL to collect the IgG fraction. From the hybridoma strains M75, M160, M177 and MH-61, Purified monoclonal antibodies M75, M160, M177 and MH-61 were obtained, respectively.

Example 1 (Preparation of POD-Labeled Monoclonal Antibody) Horseradish peroxidase solution (4 mg / mL)
After adding 0 μL of periodic acid (38.5 mg / mL) with stirring and reacting at room temperature for 20 minutes, a column packed with Sephadex (registered trademark) G-25 replaced with 1 mM acetate buffer (pH 4.5). Buffer exchange is performed using PD-10 (Amersham Pharmacia Biotech).
Next, 40 μL of 0.2 M sodium hydroxide is added. To this, 10 mg of monoclonal antibody M160 dialyzed against 10 mM carbonate buffer (pH 9.5) was added.
React for hours. After completion of the reaction, the mixture is cooled on ice, and 100 μL of a sodium borohydride solution (4 mg / mL) is added, followed by a reaction for 2 hours. After exchanging the reaction solution with PBS using PD-10, the mixture was centrifuged at 3000 rpm for 30 minutes, and the supernatant was subjected to gel filtration using Sephacryl S200HR26 × 30 (manufactured by Amersham Pharmacia Biotech) to obtain 403
Then, the absorbance at 280 nm is measured, and the fraction of the labeled monoclonal antibody is collected. Bovine serum albumin (10 mg / mL) is added to the obtained fraction, stored at −20 ° C., and diluted with PBS-Tween® 20 immediately before use.

Example 2 (Production of Test Granules) A 1 mM hydrochloric acid suspension of Dynabeads (registered trademark) M-450 Tosyl Activated (tosyl activated uniform paramagnetic polystyrene beads: manufactured by Nippon Dynal) was used. Wash once with sterile distilled water. If necessary, stir briefly to obtain a uniform suspension. Next, purified IgG antibody was added at 0.2.
Dissolve at a concentration of 150 μg / mL in M borate buffer (pH 9.5). An equal volume of activated bead suspension is added to the I
Add to gG solution (antibody / beads = 75 μg / 15 m
g). Incubate for 24 hours at 22 ° C. with gentle agitation. The antibody-bound beads were collected using a magnet, and the supernatant was discarded with the beads still attached to the magnet.
(2) 5 mL of 1 M ethanolamine containing 0.1% Tween® 20 in 1 M PBS for 10 minutes
(3) 0.1 M sodium chloride, 0.1% bovine serum albumin (BSA), 0.01 hour with hydrochloric acid (pH 9.5)
12% with 5 mL of 0.05 M Tris (pH 7.5) containing 0.1% methylthiolate and 0.1% Tween 20
And (4) 5 times with the buffer of (3) without Tween 20.
Wash in order of mL and 2 hours. After washing, the beads are collected by a magnet, the supernatant is discarded, and the suspension is suspended in PBS / BSA to a concentration of about 4 × 10 ⁸ beads / mL (30 mg / mL). The resulting IgG-bound beads are stable at 4 ° C for at least 6 months. For the storage buffer, use 0.05M Tris containing 0.1M sodium chloride and 0.1% BSA. Take 50 μL of beads and add the same amount of monoclonal antibody M177. After binding the antibody to the beads while shaking at 37 ° C. for 1 hour, 1 mL of PBS was added, and 1000 rp was added at 4 ° C.
Centrifuge mx 5 minutes. The precipitate is washed twice with PBS to obtain monoclonal antibody M177-bound beads, which are used as test granules.

Example 3 (Serum MCP Measurement Protocol) Serum MCP was measured according to the following procedure. (1) Primary antibody (monoclonal antibody M177) PBS (p) was added to each well of a 96-well plate (manufactured by Dynatech).
H7.4) Add 100 μL of diluted solution (5 μg / mL). (2) Leave at 4 ° C. overnight. (3) Wash 5 times with 300 μL of ion exchange water. (4) To each well, 350 μL of 25% Block Ace (registered trademark: Cosmo Bio) is added, and left at room temperature for 1 hour to perform blocking. (4) Prepare a dilution series of the sample, and add 100 μL of each dilution series. However, a standard solution is prepared each time.
Mix with 0% BSA / PBS solution). (5) Let stand at room temperature for 2 hours. (6) 300 μL of 0.005% Tween® 2
Wash 5 times with 0 / saline. (7) Wash once with 300 μL of ion exchanged water. (8) Add 100 μL of horseradish peroxidase-labeled monoclonal antibody M160 diluted to 0.1 μg / mL with PBS (pH 7.4) (the antibody was adjusted to the target double concentration, and an equal amount of 10% BSA / Mix with PBS solution). (9) Let stand at room temperature for 2 hours. (10) Wash 5 times with 300 μL of 0.005% Tween 20 / saline. (11) Wash once with 300 μL of ion exchange water. (12) Add 100 μL of a substrate solution having the following composition. Substrate solution (in 200 mL) o-phenylenediamine 20 mg Citric acid-phosphate buffer 200 mL (51.4 mL of 0.1 M citric acid (21.0 g / L)) (0.2 M sodium hydrogen phosphate (71.628 g / L) )
51.4 mL) (100 mL of distilled water) 10 μL of 30% hydrogen peroxide solution (13) Leave at room temperature for 30 minutes. (14) Add 100 μL of stop solution (1N sulfuric acid). (15) Measure the absorbance at 490 nm.

Example 4 (Test Method) The urine of 12 normal subjects and 21 patients with renal disease and the sera of 10 normal subjects and 20 patients with renal disease were subjected to MC.
The amount of P was measured. The breakdown of patients with kidney disease was Ig in urine samples
There are 14 cases of A nephropathy (IgAN) and 7 cases of rapidly progressive glomerulonephritis syndrome (RPGN). In addition, in serum samples, IgA
There were 14 cases of nephropathy and 6 cases of rapidly progressive glomerulonephritis syndrome. Urine and serum were collected at any time, frozen and stored at -80 ° C, thawed at the time of use, and centrifuged at 3000 rpm for 5 minutes. The POD-labeled monoclonal antibody M160 obtained in Example 1 and the magnetic beads M177 bound to the monoclonal antibody M177 obtained in Example 2 were used. Urine was a stock solution, and serum was diluted 2 to 10 times using PBS as a specimen.

Magnetic beads (Dynabeads (registered trademark) M-450) 50 to which monoclonal antibody M177 is bound
10,000, 500 μL of sample, and 1000 using PBS
500 μL of a 1: 2 dilution of the POD-labeled monoclonal antibody M160 was added to a pre-blocked 1.5 mL plastic tube, and reacted at room temperature for 2 hours while rotating. After focusing and fixing the beads using a magnet, the supernatant is discarded. After washing 5 times with PBS containing 0.02% Tween (registered trademark) 20 and 10% block ace, a substrate solution (o-phenylenediamine 4 mg, hydrogen peroxide 2 μL, citrate-phosphate buffer (pH 5) 10 m)
L) Add 150 μL and rotate for another 15-30 minutes.
After reacting for 2 minutes, 2N sulfuric acid was added to stop the reaction. After focusing and fixing the beads using a magnet, 200 μL of the supernatant was taken, transferred to a microplate, and the absorbance at 490 nm was measured using an absorptiometer (manufactured by Molecular Devices). Serum MCP value was measured using monoclonal antibody M1.
Using POD-labeled monoclonal antibody M160 as a detection antibody, the measurement was carried out by a sandwich ELISA method.

FIG. 1 shows the results of measurement of urinary MCP values (ratio of creatinine to urine) of normal subjects and patients with renal disease, and FIG. 2 shows the results of measurement of serum MCP values. As a result, urinary M
In the case of CP value, p <p between normal and IgA nephropathy patients
0.05, showed a significant difference of p <0.01 between normal subjects and patients with rapidly progressive glomerulonephritis syndrome. Also, a significant difference of p <0.05 was shown between IgA nephropathy patients and patients with rapidly progressive glomerulonephritis syndrome. In addition, the serum MCP value was p <0.001 between normal subjects and IgA nephropathy patients, and p <0.001 between normal subjects and patients with rapidly progressive glomerulonephritis syndrome.
001 was significant. Moreover, p <0.0 was found between IgA nephropathy patients and patients with rapidly progressive glomerulonephritis syndrome.
A significant difference of 01 was shown.

As is evident from these results, urinary and serum MCP values are more highly distributed in renal disease patients than in normal subjects. In addition, in patients with IgA nephropathy and patients with rapidly progressive glomerulonephritis syndrome, the values were higher in patients with more severe rapidly progressive glomerulonephritis syndrome. Therefore, according to the method of the present invention, it was revealed that not only the determination of renal impairment but also the determination of the pathological condition of renal disease was possible.

[Brief description of the drawings]

FIG. 1 shows the results of measuring urinary MCP values (ratio to urinary creatinine) of normal subjects and patients with renal disease.

FIG. 2 shows the results of measurement of serum MCP values (ratio to urinary creatinine) of normal subjects and renal disease subjects.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // A61K 9/16 A61K 39/395 D 4H045 39/395 U C07K 16/18 C07K 16/18 C12N 5 / 00 B F-term (reference) 2G045 AA13 AA16 AA25 CA25 CB03 DA36 DA77 FA36 FB03 FB07 FB15 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA91X AB05 AC14 CA25 CA46 4C076 AA31 CC50 DD22 DD23 DD26 DD50 EE04 AE16 A14 EB04 A11 BB CC05 CC17 CC22 CC23 DD22 DD33 DD35 DD38 DD63 DD73 DD74 DD75 DD88 EE03 FF02 FF19 FF20 GG02 GG04 GG06 4H045 AA30 CA42 DA76 EA50 FA72 GA15 GA22 GA26

Claims (21)

[Claims] 1. A granule for detecting a complement control factor contained in a biological sample, which is obtained by binding an anti-complement control factor antibody to the surface of a solid granule. 2. The complement control factor is a membrane cofactor protein (MCP), a decay accelerating factor (DAF),
The detection granule according to claim 1, which is selected from CD59 and type 1 complement receptor (CR1). 3. The detection granule according to claim 1, wherein the anti-complement regulatory factor antibody is a monoclonal antibody. 4. The detection granule according to claim 1, wherein the solid granule has magnetism. 5. The solid granule according to claim 1, wherein the specific gravity of the solid granules is 1 or more.
The granules for detection according to the above. 6. A method for detecting a complement control factor contained in a biological sample, comprising the following steps: (a) applying a biological sample and a first anti-complement control on the surface of a solid granule; Contacting the granule for complement control factor detection with the factor antibody (a) bound thereto,
(B) After contacting a second anti-complement control factor antibody (b) bound with a labeling molecule to the sample contacted with the granule for complement control factor detection, (c) the second anti-complement Detecting the labeled molecule bound to the complement control factor via the body control factor antibody (b), comprising the step of: detecting the complement control factor contained in the biological sample. . 7. The complement control factor is MCP, DAF, CD5.
7. The detection method according to claim 6, wherein the detection method is selected from No. 9 and CR1. 8. A first anti-complement control factor antibody (a) and a second
7. The detection method according to claim 6, wherein the anti-complement control factor antibody (b) is a monoclonal antibody. 9. The first anti-complement regulatory factor antibody (a) and the second
The detection method according to claim 6, wherein the anti-complement control factor antibody (b) is an anti-complement control factor antibody having the same recognition site. 10. The detection according to claim 6, wherein the first anti-complement control factor antibody (a) and the second anti-complement control factor antibody (b) are anti-complement control factor antibodies having different recognition sites. Method. 11. The detection method according to claim 6, wherein the biological sample is urine. 12. The detection method according to claim 6, wherein the biological sample is blood. 13. A kit for detecting a complement control factor, comprising (a) the solid granule for detecting a complement control factor according to claim 1 and (b) a magnet. 14. A kit for detecting a complement control factor, comprising: (a) an anti-complement control factor antibody; (b) solid granules capable of binding the anti-complement control factor antibody to the surface; and (c) a magnet. 15. The kit for detecting a complement regulatory factor according to claim 13, further comprising an anti-complement regulatory factor antibody bound to a labeling molecule. 16. The complement control factor is MCP, DAF, CD
16. The detection kit according to claim 13, which is selected from 59 and CR1. 17. A method for evaluating renal function, comprising measuring the amount of a complement control factor in a sample. 18. A method for measuring the amount of a complement control factor in urine of a sample,
A method for evaluating renal function, comprising comparing with a measured value of a complement control factor amount in normal urine. 19. The complement control factor is MCP, DAF, CD
19. The method for evaluating renal function according to claim 17, which is selected from 59 and CR1. 20. A reagent for diagnosing renal function comprising an anti-complement regulatory factor monoclonal antibody. 21. The complement control factor is MCP, DAF, CD
The reagent for renal function diagnosis according to claim 19, which is selected from 59 and CR1.