JPH08119998A - Human protein 1 specific monoclonal antibody - Google Patents

Abstract

PURPOSE: To obtain a new monoclonal antibody recognizing human protein 1 (P1). CONSTITUTION: The characteristic of this monoclonal antibody has the following reactivity. (1) The monoclonal antibody is reacted with a human P1 dimer. (2) This monoclonal antibody is reacted with a human P1 monomer obtained by reducing the human P1 dimer and cleaving the SS bond. (3) The reactivity to the human P1 monomer is equal to or more than that to the human P1 dimer. A hybridoma to produce the monoclonal antibody and the use of the monoclonal antibody are provided. Tissue comprising P1 as an index can be subjected to immune dyeing by using a monoclonal antibody and a clear dyed image can be obtained by a monoclonal antibody having strong affinity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a monoclonal antibody and its use. Specifically, human pro
tein 1 (urinary low molecular weight protein 1 or UP1
(Hereinafter, also referred to as P1), a monoclonal antibody that recognizes an antigenic determinant, a production hybridoma, and uses thereof.

P1 is a protein identified in urine having a molecular weight of 14 kDa and an isoelectric point of 4.7 (J. Chromatogr. 452:
359-367, 1988), it exists as a homodimer composed of two structurally identical subunits in the living body, and its presence has been confirmed at least in the seminiferous gland by tissue staining.
(Journal of Clinical Laboratory Analysis 7: 394-40
0,1993). The two subunits constituting P1 form a homodimer by two SS bonds, and when the SS bond is cleaved with a reducing agent, two subunit monomers are produced. P1 is a human cl secreted by lung epithelium
It is known to be the same protein as the ara cell 10kDa protein (also called HCC10 or CCP)
(J. Chromatogr. 577: 25-35, 1992). P1 does not show a difference between male and female in the serum of a healthy person (adult),
In the urine, about 12.5 μg / day is excreted in males and about 1.4 μg / day in females. In men, P1 is secreted in the lower urethra. The amount of P1 excreted in male urine is higher than that in female because it contains a large amount of P1 derived not from the kidney but from the prostatic fluid in the early stage of urination. Therefore, it is possible to use it as a marker without being aware of the difference between men and women, if the mind is to collect the intermediate urine when collecting urine. While markers such as PSA, PA, and γSM, which are currently known as indicators of prostate cancer, cannot be differentiated from prostatic hypertrophy, P1 is an index for excellent differentiation because it is specifically elevated in prostate cancer. Could be. Moreover, since P1 can be detected in urine, it is easier to procure a sample than a marker that appears in blood. P1 in blood decreased in smokers and P1 in alveolar lavage fluid (hereinafter abbreviated as BALF) in asthmatics (Lan
cet Vol.339; June, 27, 1992), etc., and is useful for diagnosis of lung health and inflammatory symptoms. Furthermore, it is known to be detected in sarcoidosis, lung cancer, serum of patients with renal failure and the like, and may be an index for these diseases. Furthermore, since P1 is a low-molecular-weight protein, it can be used as a marker for renal tubular disorder like α1-microglobulin.

[0003]

[Problems of the prior art] In advancing research on in-vivo substances,
Immunological techniques are essential. In particular, a monoclonal antibody that specifically recognizes a substance to be studied is one of the powerful tools for separating / purifying, detecting, and identifying the substance. Attempts have also been made to produce a monoclonal antibody for P1 (J. Chromatogr. 577; 25-35, 1992). The present inventors have obtained a monoclonal antibody which is a useful tool for various immunoassays and have already applied for a patent (Japanese Patent Application No. 6-7).
2625).

However, although the monoclonal antibodies obtained so far have no problem in terms of specificity for P1,
It was not always sufficient in terms of affinity. For example, the monoclonal antibodies TY-1 to 4 obtained by the present inventors certainly recognize P1 specifically, but do not show sufficient reactivity with tissues containing P1 and the like. Therefore, although it can be used for immunoassay of free P1, it is insufficient as a tool for immunologically analyzing tissues.

If a new monoclonal antibody showing sufficient reactivity not only with free P1 but also with P1 in tissues can be obtained, immunostaining, which has hitherto been resorted to a polyclonal antibody, can be carried out with a highly specific monoclonal. So useful.

Further, not only TY-1 but also conventional monoclonal antibodies have little or no affinity for the P1 monomer produced by reduction of P1, or even if they do have weak reactivity as compared with the affinity for P1. Was only shown. In such a monoclonal antibody, P1
It cannot be used as a tool for monomer analysis or purification.

[0007]

The first object of the present invention is to provide a novel monoclonal antibody having a high affinity for P1. The second object of the present invention is to provide a hybridoma producing this monoclonal antibody. Further, the present invention provides, as a third object, the use of the novel monoclonal antibody provided by the present invention.

[0008]

The object of the present invention is solved by a monoclonal antibody which recognizes human P1 characterized by the following reactivity. (1) React with human P1 dimer (2) React with human P1 monomer obtained by reducing human P1 dimer to cleave the SS bond (3) Reactivity with human P1 monomer to human P1 dimer Is equal to or more than

The monoclonal antibody of the present invention is simply P1.
Not only does it react specifically with, it also has a strong affinity for P1 that has never been seen. Due to this strong affinity, both P1 monomer cleaved SS bond by reduction treatment and P1
It shows reactivity equal to or higher than that for dimer. The P1 monomer can be easily obtained by treating the homodimer with a reducing agent such as 2-mercaptoethanol or dithiothreitol. The monoclonal antibodies known so far show reactivity with P1 in the homodimer state, but have no practical reactivity with P1 monomer. Therefore, it is presumed that the monoclonal antibody according to the present invention acquires a strong affinity by using an epitope in a region that is less likely to be affected by the P1 conformational change.

The strong reactivity of the monoclonal antibody according to the invention is demonstrated by the experimental data given in the examples. That is, conventional monoclonal antibodies show almost no detectable binding to immobilized P1 at an antibody concentration of 0.02 μg / ml or less. In contrast, the monoclonal antibody of the present invention exhibits a sufficiently detectable binding activity even at an antibody concentration of 0.004 μg / ml or less.

As described above, the monoclonal antibody of the present invention which recognizes a specific epitope reacts with a sufficient affinity to P1 of a tissue which cannot be sufficiently reacted by the conventional monoclonal antibody, and therefore, can detect a P1-containing tissue. It is useful. P1-containing tissues include HC, which is a structurally identical protein in addition to spermatic gland tissue, uterine cancer tissue, prostate cancer tissue, and the like.
Although lung epithelial cell tissues and the like expressing C10 are known, it becomes possible to search for other P1-containing tissues by the monoclonal antibody of the present invention.

Monoclonal antibody TY-5 can be mentioned as a monoclonal antibody satisfying such conditions. Monoclonal TY-5 in the present invention
Is P1 (J. Chromatogr. 57 purified by a known method.
7; 25-35, 1992) was used as an immunogen to immunize BALB / c mice, and their splenocytes were converted to mouse myeloma cells (P3-NS).
It was obtained by cell fusion with 1). The obtained hybridomas were screened by the reactivity of the culture supernatant with respect to P1 to obtain hybridomas producing the monoclonal antibody according to the present invention. The hybridoma thus cloned is deposited at the Patent Depositary Center under the deposit number FERM P-14564. Although the operation itself for obtaining the hybridoma of the present invention is not novel, the obtained monoclonal antibody is P
It is a novel monoclonal antibody with a strong affinity for 1 that has never been seen before. The monoclonal antibody produced by the hybridoma TY-5 according to the present invention is IgG ₁ κ.
Belong to. Monoclonal antibody TY of the present invention
-5 is P1 after immobilization similar to the polyclonal antibody
Reacts strongly with contained tissues. In contrast, known monoclonal antibodies are almost unable to bind to fixed tissue. The affinity of the monoclonal antibody of the present invention is very strong, for example, TY- which is a conventional monoclonal antibody.
1: When contacted with the P1 conjugate, the monoclonal antibody of the present invention displaces TY-1.

The following are examples of uses of the monoclonal antibody which is the third object of the present invention. Use A: Immunological detection of P1 Since the monoclonal antibody according to the present invention has a strong affinity for P1 and also reacts with P1 in tissues, the immunological detection of P1 is used as an index. It is useful for immunostaining. Immunostaining for P1 is useful for identifying P1 production sites such as lung cancer tissue and endometrial tissue. For immunostaining, the monoclonal antibody is directly labeled with an enzyme such as peroxidase or β-galactosidase, or a fluorescent dye such as FITC, or indirectly used by reacting with avidin-labeled as a biotinylated antibody. Various staining methods are known, such as a method of labeling with an antibody or indirectly with a second antibody.

Direct binding between the labeling agent and the monoclonal antibody can be carried out by the periodic acid method, glutaraldehyde method, p-benzoquinone method, N, N'-phenylenedimaleimide (OPDM), bis-succinic acid-N-hydroxyl. Succinimide (BSNHS), carbodiimide, tolylene-2,4-diisocyanate, 4,4'-difluoro-
A method using a bifunctional linker compound such as 3,3′-dinitrophenylsulfonic acid (FNPS) is known. A monoclonal antibody used as a labeled antibody may be advantageous when digested with pepsin, plasmin or the like and using only its variable region because a nonspecific reaction is less likely to occur.
The reaction conditions between the monoclonal antibody and P1 may be set according to a general immunological reaction. Specifically, pH 5-10, preferably 6-8, temperature 5-50
C., preferably 20 to 40.degree. C., time is 10 minutes or longer, preferably 30 minutes or longer. In addition, it is possible to add a surfactant or to adjust the concentration of the buffer component or the salt, if necessary, in order to prevent the non-specific reaction or to accelerate the reaction.

Further, the strong affinity of the monoclonal antibody of the present invention can be applied to detection by P1 particle agglutination reaction. The particle agglutination reaction is carried out by following the formation of P1-mediated aggregates of antibody-sensitized particles, but an antibody having a strong affinity is required to realize sufficient sensitivity and a measurement range. Therefore, the monoclonal antibody of the present invention is suitable for a particle agglutination reaction. When the particle agglutination reaction system is constituted by the monoclonal antibody according to the present invention,
Higher sensitivity can be expected by combining a monoclonal antibody that recognizes an epitope different from that of the monoclonal antibody of the present invention. When combining monoclonal antibodies, do not prevent binding to each other's epitopes,
It is important to choose the ratio of monoclonal antibodies used to obtain good sensitivity. The monoclonal antibody required for immunological detection of the present invention is convenient because it can be used for various detection operations immediately when provided as a reagent.

Furthermore, the monoclonal antibody necessary for the immunological detection of P1 according to the present invention, the substrate for detecting the label, the standard sample and the like can be supplied as a pre-assembled kit. In the case of a kit, inactive proteins, sugars, water-soluble polymer compounds and the like can be added in advance to stabilize enzymes such as monoclonal antibodies and labeling agents such as peroxidase. Specific examples of substances useful as stabilizers include bovine serum albumin (abbreviated as BSA) and normal animal serum as inactive proteins, sucrose and dextrose as sugars, and water-soluble polymer compounds as insoluble proteins. Examples thereof include polyethylene glycol. A preservative may be used in combination with the stabilizer. A preservative such as sodium azide is used as a preservative. Further, a buffering agent that gives a pH suitable for an immunological reaction and an additive for suppressing a nonspecific reaction may be used together. The Fc fragment of IgG or P1 is used as the additive.
Mouse IgG fractions having no reactivity with, non-specific mouse monoclonal antibodies, etc. are known. Each of these constituent components may be supplied in a solution state or in a dried form by freeze-drying or the like.

Use B: Purification of P1 Since the monoclonal antibody of the present invention has a strong affinity for P1, it is also useful for a purification operation by immunoadsorption. In the so-called immunoaffinity chromatography, it is desirable to use an antibody having a strong affinity in order to obtain a high yield. Furthermore, since the monoclonal antibody of the present invention shows reactivity not only with P1 homodimers but also with P1 monomers, it enables purification of P1 monomers. Although the monoclonal antibody of the present invention has reactivity with both the P1 dimer and the monomer, it can be used for purification of only one of them. Before contacting both with the monoclonal antibody of the present invention, it may be fractionated based on the difference in molecular weight. Gel filtration is effective for fractionation based on the difference in molecular weight. The immunoaffinity chromatograph comprises the steps of applying a P1-containing sample to a column on which the monoclonal antibody of the present invention is immobilized, washing the column, and then applying conditions for dissociating the immunological bond to recover P1 adsorbed on the column. If the affinity of the antibody used at this time is insufficient, P1 will be washed away by washing, and a high recovery rate cannot be expected. Since the monoclonal antibody of the present invention has high affinity, high yield can be expected. In the method for purifying P1 according to the present invention, a column such as an agarose column is used as a column, various buffers are used for washing, and extremely pH is used for dissociation of immunological bond.
A buffer solution having a different pH (for example, pH 2.5), high-concentration urea, guanidine, or the like can be used. For the agarose column, Sepharose 4B (manufactured by Pharmacia, trade name) activated with bromocyan is known. P1 recovered
The fraction is dialyzed against an appropriate buffer to obtain purified P1.

[0018]

FUNCTION The monoclonal antibody TY-5 of the present invention
Has new features of strong affinity for P1 dimers and strong reactivity with P1 monomers. Further, the monoclonal antibody of the present invention also realizes a strong reaction with P1 contained in tissues, which could not be realized by conventional monoclonal antibodies. Based on such characteristic reactivity, it is possible to utilize the specificity of the monoclonal antibody in various applications which were difficult with conventional monoclonal antibodies. A particularly advantageous application is P
Immunological detection of tissues containing 1 and application to a method for purifying P1 can be demonstrated.

[0019]

According to the present invention, P having a novel reactivity
A monoclonal antibody against 1 is provided. The novel reactivity of the monoclonal antibody of the present invention enables immunological detection of P1 contained in tissues. Conventional monoclonal antibodies cannot be used for this type of application because of insufficient reactivity, and the present situation is that they rely solely on polyclonal antibodies. The present invention enables detection using a monoclonal antibody, and can be expected to have high specificity surpassing that of a polyclonal antibody. In particular, the monoclonal antibody of the present invention gives not only a simple reaction but also a clear staining result when used for immunostaining because it has a strong affinity. There is a concern that the bound antibody may be washed away by washing with an antibody having insufficient affinity, but since the monoclonal antibody of the present invention has strong affinity, such a problem can be solved. When P1 producing cells become cancerous, P1
Is likely to be produced as a monomer instead of a normal dimer. In such a case, it cannot be detected by a conventional monoclonal antibody having a weak reactivity with P1 monomer, but according to the present invention, it can be detected in the same manner as P1 dimer.
It is possible to detect one monomer.

Further, the strong affinity of the monoclonal antibody of the present invention can exert a great effect in the purification method utilizing the P1 immunoreaction. Since it is a monoclonal antibody, it can specifically bind to P1, and since it has a strong affinity, it is less likely to be missed and the washout due to the washing operation can be minimized. Due to such characteristics, the monoclonal antibody of the present invention greatly improves the efficiency of the P1 purification operation.

[0021]

【Example】

1. Purification of P1 Purification of P1 used as immunogen and standard sample P1 was purified according to a known method (J. Chromatogr. 577; 25-35, 1992). The specific operation is as follows.
About 3.5 liters of pooled urine from patients with chronic renal failure was used as the starting material, and 6
The supernatant from the 0% ammonium sulfate fraction was collected, and the 90% ammonium sulfate precipitate was collected from this supernatant fraction. The precipitate is 10 mM Tris hydrochloric acid buffer (pH
7.4, hereinafter simply referred to as Tris buffer), and dialyzed against the same Tris buffer. 150 ml of the obtained concentrated fraction was applied to a Sephadex A-25 column (40 cm x
2.5 cm I. D. , Pharmacia-LKB). P1 is adsorbed on this column. Tri
After washing with s buffer, the adsorption fraction was adjusted to 0
Elution with a linear gradient of Tris buffer changed to -0.3 M gave P1 a NaCl concentration of 0.18.
It is eluted near M. An immunoaffinity column (6 cm × 2.5 cm I.D.) on which anti-P1 antibody was immobilized was combined with the obtained elution fractions (about 80 ml) having a NaCl concentration of about 0.18 M.
D. ) Was applied. After washing with 50 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl, P1 was eluted with 0.2 M glycine HCl buffer (pH 2.5) containing 0.5 M NaCl, and the elution fraction was 100 mMT.
It was dialyzed against ris-HCl buffer (pH 7.4). Further, 12 ml of the dialyzed eluate was equilibrated with a 0.05% trifluoroacetic acid solution for reverse phase chromatography (μBondasphere 5 μm C4-300 angstrom, 150 mm × 3.9 mm ID, Water.
s). Then, elution was performed with a linear gradient in which the concentration of acetonitrile was changed from 0 to 100% in 0.05% trifluoroacetic acid. Fractions with a concentration of acetonitrile of about 55% were pooled, lyophilized, and dissolved in 1 ml of purified water. To obtain purified P1. This purified P1
Was analyzed by SDS-PAGE, it was confirmed that P1 was separated as an almost pure protein. Also EL
When the amount of protein was measured by the ISA method (J. Chromatogr. 452: 359-367, 1988), the result was 544.8 μg.

2. Preparation of Monoclonal Antibody 50 μg of the purified P1 obtained in 1 was suspended in an equal amount of FCA to immunize BALB / c mice. 50μ after 2 weeks
Booster immunization with g of P1 (suspended in an equal volume of FCA), then 2
After 30 weeks, 30 μg of P1 dissolved in physiological saline was intraperitoneally injected, and 3 days later, the spleen was extracted. Splenocytes prepared from the excised spleen were used as mouse myeloma cells P3-
Cells are fused with NS1 and seeded in a well of a 96-well microplate at 3 to 5 × 10 ⁵ cells / well, and HA
Selection with T medium was applied. The immunological reactivity to P1 was checked for the culture supernatant of the wells in which cell proliferation was observed, and the wells that were antibody-positive were further cloned by the limiting dilution method. The antibody activity was checked by an ELISA method using a microplate in which purified P1 was physically adsorbed in a well and a commercially available POD-labeled anti-mouse immunoglobulin rabbit antibody (manufactured by Dako). By such an operation, clone TY-5 was finally established. TY-5 has been deposited at the Patent Microorganisms Depositary Center as FERM P-14564. TY-5 (1 × 10 ⁷ cells) was inoculated into the abdominal cavity of BALB / c mice, and ascites was collected from 4 to 5 days later. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the supernatant was subjected to 50% ammonium sulfate fractionation, dialysis with a phosphate buffer (pH 7.2, hereinafter abbreviated as PBS), and protein A affinity chromatography (Protein A SepharoseCL). -4B)
And purified by dialysis to obtain a monoclonal antibody. This clone is a monoclonal antibody TY- that recognizes P1.
Produces 5. The monoclonal antibody TY-5 is IgG ₁
It belonged to κ.

3. Reactivity of TY-5 The reaction of the monoclonal antibody TY-5 of the present invention by concentrated urine containing a high concentration of P1 and Western blotting using the P1 monomer obtained by reducing the purified P1 obtained in 1 as an antigen. I confirmed the sex. 60-90% in 1
The concentrated urine obtained by dialysis of the ammonium sulfate fraction was converted into SDS,
Electrophoresis on 10% polyacrylamide gel (Anal. Bioche
m.166,368-379,1987), PVDF membrane Immobilon Transf
er Membranes (Millipore) (Proc.Natl.A
cad.Sci.USA.76,4350-, 1979). After transfer, each PVDF membrane blocked with 1% BSA was reacted with each monoclonal antibody, and PBS containing 0.05% Tween 20 (pH 7.
2, hereinafter simply PBS. It is washed with T) and further 1μ
g / ml streptavidin-horseradish peroxidase was reacted. After washing, Konica Immunostain (produced by Konica, trade name) was colored according to the package insert, and the result is shown in FIG. It was confirmed that both the monoclonal antibody according to the present invention and the conventional monoclonal antibody specifically reacted with the band corresponding to P1. The monoclonal antibody TY-5 of the present invention and a conventional monoclonal antibody against P1 (J. Chromatogr. 577; 25-35, 19) were used as controls.
92) was prepared. Each monoclonal antibody (about 1 mg / ml) 1
Biotin (N-hydroxysuccinimidobiotin; = NHS-Bi
250 μg of otin and PIERCE) in DMSO (Dimethy)
l sulfoxide, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was reacted at room temperature for 4 hours to be biotinylated. Biotinylated monoclonal antibody was dialyzed against PBS and centrifuged to give 1%
PBS with BSA. The antibody concentration was adjusted to 5 μg / ml with T.

Next, the reactivity with the P1 monomer obtained by the reduction treatment was compared. The operation is as follows. SDS-formation: Purified P1 was purified with 4% SDS, 12% glycerol, 50 mM Tris-HCl, 0.01% Serva.
Reduction treatment was performed at 90 ° C. for 5 minutes in the presence of Blue G and 2% 2-mercaptoethanol (pH 6.8). By this treatment, the two SS bonds of P1 are almost completely cleaved to form P1 monomer. S, not reduction process
When converting to DS, the composition was such that only 2-ME was removed.

Western blotting method: Both SDS-modified P1 monomer (20 ng / lane) and P1-containing concentrated urine (10 ng / lane) were applied to 10% polyacrylamide gel in an amount of 10 μl each. 2 for anode buffer
00 mM Tris-HCl (pH 8.9) was used as the cathode buffer, and 100 mM Tris.100 mM Tricine.
0.1% SDS (pH 8.5) was used (Annal. Biochem.
166,368-379,1987). After running the gel, use Pro.Natl.Acad.Sc
PVDF Membrane Immobilon according to i.USA, 76,4350,1979
It was transferred to Transfer Membranes (manufactured by Millipore). The thickness of the gel was 1 mm, the distance between the electrodes of the transfer device was 3 cm, the transfer buffer was 25 mM Tris.20% methanol, 192 mM glycine, and transfer was performed at 21 V for 1 hour. On the other hand, as the monoclonal antibody, the monoclonal antibody TY- of the present invention is used.
5, and TY-1 ~ as conventional monoclonal antibodies
4 (as described in Japanese Patent Application No. 6-72625), and TY
-1 and the like were reacted with TY-6 to 8 and the reactivity was confirmed by the same operation.
1) and FIG. 3 (P1 monomer). The compared monoclonal antibodies all have similar specificity and affinity for purified P1. However, the reactivity of the conventional monoclonal antibody with respect to the P1 monomer obtained by the reduction treatment is remarkably reduced.
It was confirmed that only Y-5 maintained a strong affinity.

4. Affinity of TY-5 With respect to the monoclonal antibody TY-5 of the present invention, the affinity for P1 was compared with that of a conventional monoclonal antibody.
The purified P1 obtained in 1 was adjusted to 0.5 μg / ml with 50 mM carbonate buffer (pH 9.6), and 100 μl thereof was dispensed into the wells of a polystyrene microplate (Nunc) (50 ng per well). After left overnight at 4 ° C. for physical adsorption, the solution was removed by suction and PBS. Wash 3 times with T.
After washing, 200 μl of the same buffer containing 1% BSA was dispensed and blocked at 37 ° C. for 40 minutes, the solution was removed by suction, and the plate was washed with the same buffer to prepare a P1 solid-phased plate. The monoclonal antibody TY-5 of the present invention was prepared as a monoclonal antibody, and the conventional monoclonal antibody against P1 (J. Chromatogr. 577; 25-35, 1992) was prepared as a control. To 1 ml of monoclonal antibody (about 1 mg / ml), biotin (N-hydroxysuccinimidobiotin; = NHS-Biotin, P
250 μg of IERCE DMSO (Dimethyl sulfox)
ide, manufactured by Wako Pure Chemical Industries, Ltd.) and added, and reacted at room temperature for 4 hours for biotinylation. The biotinylated monoclonal antibody was dialyzed against PBS, and PBS. T
The antibody concentration was adjusted to 10-0.00016 μg / ml with.

100 μl of the monoclonal antibody solution was dispensed into the wells of the microplate and reacted at 37 ° C. for 90 minutes to remove the reaction solution by suction. PBS. 100 μl of 1 μg / ml streptavidin-horseradish peroxidase (VECTOR) was added to wells washed 6 times with T.
LABORATORIES) and dispense at 30
Let react for minutes. After the reaction, the reaction solution is aspirated and PBS. T
Wash 8 times with 100 μl enzyme substrate solution (1 mg / ml o
Citrate buffer pH 5. containing phenylenediamine
0, 0.03% H ₂ O ₂ content) was dispensed and reacted at room temperature. Five minutes after the start of the enzymatic reaction, 150 μl of 1M sulfuric acid was added to stop the reaction, and the reaction was stopped with a microplate reader.
The absorbance at 90 nm was measured. The results are shown in Table 1. The numerical values in the table are the measured values of the absorptiometer. It was confirmed that the monoclonal antibody TY-5 of the present invention shows quantitative reactivity even under 0.0008 μg / ml under these conditions. On the other hand, with conventional monoclonal antibodies (indicated by M.Ab. in the table), 0.004 μg / ml
The loss of quantification was confirmed, and the affinity strength of the monoclonal antibody of the present invention was confirmed.

[0028]

[Table 1]

5. Immunostaining An attempt was made to immunostain P1-containing tissues with the monoclonal antibody TY-5 of the present invention. As the tissue, an adenocarcinoma of Clara cells was used, and a conventional monoclonal antibody (J. Chromatogr.
7; 25-35, 1992) and a commercially available anti-P1 polyclonal antibody (manufactured by Dako). An adenocarcinoma tissue of human Clara cells was fixed with formalin by a conventional method and embedded in paraffin to prepare a section. The monoclonal antibody 2 of the present invention was added to this section.
μg / ml (the antibody concentration was adjusted with PBS.T containing 1% BSA) was reacted, and a commercially available POD-labeled anti-mouse IgG rabbit antibody was stained as a secondary antibody. The staining result with the monoclonal antibody of the present invention was almost the same as that of the commercially available polyclonal antibody, while showing much stronger reactivity than the staining result with the conventional monoclonal antibody. Since the monoclonal antibody TY-5 of the present invention has a strong reactivity to paraffin sections, it has a novel reactivity to recognize an antigenic determinant that is stable against fixation treatment with formalin or dehydration. It was speculated that

[Brief description of drawings]

FIG. 1 is a monoclonal antibody TY according to the present invention.
It is the figure which showed typically the result which confirmed the specificity of -5 with respect to the P1-containing concentrated urine by Western blotting. In the figure, lane 1: molecular weight marker, lane 2: conventional monoclonal antibody, lane 5: TY-5 are shown.

FIG. 2 is a monoclonal antibody TY according to the present invention.
It is the figure which showed typically the result which confirmed the reactivity with respect to P1 (10 ng / lane) of -5 by the western blotting method. In the figure, lane 1: molecular weight marker, lane 3: TY-1, lane 4: TY-2, lane 5: TY-.
3, Lane 6: TY-4, Lane 7: TY-5, Lane 8: TY-6, Lane 9: TY-7, Lane 10: TY.
-8 is shown.

FIG. 3 is a monoclonal antibody TY according to the present invention.
It is the figure which showed typically the result which confirmed the reactivity with respect to the P1 monomer (20 ng / lane) of -5 by the western blotting method. In the figure, lane 1: molecular weight marker, lane 3: TY-1, lane 4: TY-2, lane 5: TY-3, lane 6: TY-4, lane 7: TY-.
5, lane 8: TY-6, lane 9: TY-7, lane 10: TY-8.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/53 D 33/535 33/577 B // C12N 15/02 (C12P 21/08 C12R 1 : 91)

Claims (10)

[Reference number] P-000308 [Claims] 1. A human prot characterized by the following reactivity:
A monoclonal antibody that recognizes ein 1. (1) React with human protein 1 dimer (2) Reduce human S protein 1 dimer to S
React with human protein 1 monomer obtained by cleaving S bond (3) Reactivity with human protein 1 monomer is equivalent to that for human protein 1 dimer,
Or more 2. The monoclonal antibody according to claim 1, which is the monoclonal antibody TY-5. 3. The anti-human protein according to claim 1.
Hybridoma TY- that produces 1 monoclonal antibody
5. 4. Hybridoma TY-5 deposited under accession number FERM P-14564. 5. A method for detecting human protein 1, which is characterized by the following reactivity:
1. A detection method, which comprises using a monoclonal antibody that recognizes 1. (1) React with human protein 1 dimer (2) Reduce human S protein 1 dimer to S
React with human protein 1 monomer obtained by cleaving S bond (3) Reactivity with human protein 1 monomer is equivalent to that for human protein 1 dimer,
Or more 6. The detection method according to claim 5, wherein human protein 1 is contained in a biological tissue. 7. A reagent for detecting human protein 1 containing a monoclonal antibody characterized by the following reactivity as an antibody. (1) React with human protein 1 dimer (2) Reduce human S protein 1 dimer to S
React with human protein 1 monomer obtained by cleaving S bond (3) Reactivity with human protein 1 monomer is equivalent to that for human protein 1 dimer,
Or more 8. The reagent for detection according to claim 7, wherein the monoclonal antibody is labeled. 9. The label is selected from enzymes, coenzymes, fluorescent substances, luminescent substances, particles, and radioactive substances.
Reagent for detection of. 10. Human protein 1 by immunoadsorption
A method for purifying dimers and / or monomers, the method comprising:
The following purification method using a monoclonal antibody characterized by reactivity as a human protein 1 ligand. (1) React with human protein 1 dimer (2) Reduce human S protein 1 dimer to S
React with human protein 1 monomer obtained by cleaving S bond (3) Reactivity with human protein 1 monomer is equivalent to that for human protein 1 dimer,
Or more