JP2000065829A - Antibody against stanniocalcin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a means for providing a monoclonal antibody or a polyclonal antibody against human stanniocalcin. SOLUTION: This antibody has an immunoantigen of a modified peptide formed by modifying an amino acid composing a peptide including the whole or a part of a specific part of an amino acid sequence of a human stanniocalcin protein, in the range where identity as a whole is not lost. The immunoantigen of this antibody is an immunoantigen combined with a hapten, and the hapten is KLH(keyhole lympet hemocyanin).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibody in the technical field related to an antibody and a detection method using the same, and more specifically, a glycoprotein which may be involved in the control of ions such as human calcium. The present invention relates to an antibody against stanniocalcin, a method for detecting stanniocalcin using the antibody, and a detection kit containing the antibody.

[0002]

BACKGROUND OF THE INVENTION Staniocalcin is a glycoprotein that acts on fish gills and has a function of controlling calcium in fish blood. At first, mammals including humans do not have gills. Was considered not to be. However, in recent years, the presence of stanniocalcin also in these mammals, due to cloning of stanniocalcin gene, etc.
(For the cloning of the mouse stanniocalcin gene, Andy C.-M.Chang et al., Mo.
lecular and Cellular Endocrinology, 124, pp185-187 (1
996), and for further cloning of the human stanniocalcin gene, see Andy C.-M.
al., Molecular and Cellular Endocrinology, 112, pp241
-247 (1995): see Genebank HSU25997]. Thus, the role of stanniocalcin, also found in mammals, has not been elucidated so far, but it has at least worked to suppress calcium reabsorption in the kidney and intestine and promote phosphorus reabsorption. Has been reported (eg, Graham F. Wagner et al., Journal of Bone
and Mineral Research, vol. 12, No. 2, pp. 165-171).

[0003]

As described above, to identify stanniocalcin in humans, which is considered to play an important role in ion control in vivo, to elucidate the function of stanniocalcin, It is very important in providing a means for diagnosing a disease associated with the disease. As a typical means for easily and accurately specifying this stanniocalcin, an antibody against human-derived stanniocalcin is produced, and a specific means based on an antigen-antibody reaction between this antibody and stanniocalcin based on the principle of specifying stanniocalcin. It is conceivable to provide.

However, it has been found that the amino acid sequences of stanniocalcin of other mammals such as the above-mentioned mouse and that of human-derived stanniocalcin are almost identical. Is difficult to manufacture. That is, even when a human-derived stanniocalcin is directly used as an immunizing antigen and immunized against a mammal such as a mouse, in this mammal, human-derived stanniocalcin cannot be recognized as non-self, and human-derived stanniocalcin cannot be recognized. It is very difficult to produce antibodies against

[0005] Therefore, the problem to be solved by the present invention is to overcome such difficulties, provide a means for producing an antibody against human-derived stanniocalcin, and further provide a human-derived antibody using the resulting antibody against stanniocalcin. It is to provide a means for identifying stanniocalcin from the source.

[0006]

Means for Solving the Problems The inventor of the present invention has made intensive studies to solve this problem. As a result, a mammal is immunized with a synthetic peptide obtained by modifying one amino acid of the amino acid sequence in a specific region of the amino acid sequence deduced from the nucleotide sequence of the known stanniocalcin gene to another amino acid as an immunizing antigen. Accordingly, the mammal recognized the synthetic peptide as non-self, and as a result, found that an antibody against the synthetic peptide could be produced in the mammal. The inventors have found that the antibody thus obtained can exhibit excellent cross-reactivity with human-derived stanniocalcin, and completed the present invention.

That is, in the present application, the present inventors have modified the amino acids constituting the peptide containing all or a part of the amino acid sequence represented by SEQ ID NO: 1 within a range not to lose the identity as a whole. Provided is an antibody (monoclonal antibody or polyclonal antibody) against stanniocalcin (hereinafter, stanniocalcin may be referred to as STC) using a peptide as an immunogen. In addition, the above-mentioned "range which does not lose the identity as a whole" means a range in which the modified peptide can be recognized as a partial sequence of stanniocalcin on immunology, and specifically, 1 to several amino acids constituting a peptide containing all or part of the amino acid sequence represented by SEQ ID NO: 1.

[0008] The present invention also provides a method for specifying human-derived stanniocalcin using the antigen-antibody reaction between an antibody to stanniocalcin and human-derived stanniocalcin as a specific principle.

Further, the present invention provides a specific kit for human-derived stanniocalcin, comprising an antibody against this stanniocalcin. In the present invention, the word “specific” is used as a word meaning both “detection” and “quantitation”. In addition, unless otherwise specified, hereinafter, when stanniocalcin (STC) is described, this is used as the meaning of human-derived stanniocalcin (STC).

[0010]

Embodiments of the present invention will be described below. A. Antibody to stanniocalcin according to the present invention (hereinafter also referred to as anti-STC antibody of the present invention): The present invention S
The TC antibody is produced using a modified peptide obtained by modifying an amino acid constituting a peptide containing all or a part of the amino acid sequence represented by SEQ ID NO: 1 as an immunogen as long as the identity of the peptide is not lost as a whole. Antibody.

The amino acid sequence represented by SEQ ID NO: 1 is represented by Tyr Asn Arg Leu Val Arg SerLeu Leu Glu Cys Asp
Gln Asp Thr Val Ser Thr Ile, which is a peptide fragment corresponding to the sequence from position 161 to position 179 of the amino acid sequence of the STC protein deduced from the cDNA of human stanniocalcin.

As described above, the amino acid sequences of STC proteins are almost the same between mammals such as humans and mice. Therefore, even if an attempt is made to produce an antibody using the peptide fragment as an immunizing antigen as it is, it is difficult for an immunized animal to recognize the peptide fragment as non-self, and it is very difficult to obtain an antibody against the desired STC. Difficult.

In the present invention, the modified peptide fragment is used in an immunized animal by substituting any of the amino acid residues constituting the above-described peptide fragment with another amino acid residue and using the amino acid residue as an immunizing antigen. , Can be recognized as non-self.

The number of amino acid residues in the above-mentioned peptide fragment represented by SEQ ID NO: 1 which is substituted with another amino acid is determined by the fact that an antibody prepared using the modified peptide fragment as an immunizing antigen can cross-react with stanniocalcin as much as possible. Therefore, it is preferable that the number is as small as possible.

In the present invention, in the above-mentioned region (positions 161 to 179 of the STC amino acid sequence) selected from the amino acid sequence of natural stanniocalcin, amino acid residues within a range not losing the overall identity are determined. By modification, an antibody having cross-reactivity with the desired stanniocalcin can be obtained, but for the reasons described above, the number of amino acids to be modified is preferably at least one.

The above region (161 of the STC amino acid sequence)
To 179), the specific amino acid residue to be modified is not particularly limited, and can be arbitrarily selected from the above regions. However, as described below, when a hapten is bound to the modified peptide, it is preferable to eliminate as much as possible the influence of the cross-linking agent used for the binding.

In this sense, it is necessary to modify the amino acid residue having a sulfide bond, which is generally highly reactive with a crosslinking agent, from the N-terminal cysteine residue to another amino acid residue. , At least as a modification program. Specifically, the type of the amino acid residue to be substituted with this cysteine residue is not particularly limited, but is alanine whose structure is relatively similar to the cysteine residue, from the viewpoint of preventing steric hindrance or the like by the substitution. It is preferred to select residues.

The above-mentioned modified peptide serving as an immunizing antigen when producing the anti-STC antibody of the present invention can be easily synthesized by a generally known peptide synthesis method, for example, a solid phase method or a liquid phase method. . Of course, it is also possible to synthesize using a peptide synthesizer.

A hapten can be bound to the modified peptide thus obtained in order to improve its immunoreactivity. As the hapten, any substance that can be usually selected as a hapten can be arbitrarily selected. For example, umbrella hemocyanin (KLH),
Chicken egg albumin (OVA), bovine serum albumin (BSA) and the like can be selected.

Further, a cross-linking agent can be used to bind the modified peptide to the hapten. Such a crosslinking agent can be appropriately selected according to the type of the selected hapten. For example, as in an embodiment described later,
If you choose umbrella hemocyanin as a hapten,
m-Maleinimide benzyl-N-hydroxysuccinimide ester (MBS) or the like can be used. The necessary treatment can be applied to the above-mentioned modified peptide depending on the properties of the selected crosslinking agent. For example, M
When BS is used as a cross-linking agent, it is necessary to add a cysteine residue having a sulfide bond necessary for the binding reaction to either of the two ends of the modified peptide. Further, as described above, the cysteine residue in the above-mentioned region (positions 161 to 179 in the STC amino acid sequence) can be protected by substituting it with another amino acid residue or attaching a protecting group. preferable. Thus, an immunizing antigen of the anti-STC antibody of the present invention in a form in which the hapten is bound is provided.

[0021] The anti-STC antibody of the present invention can be produced using the above-mentioned immunizing antigen. The anti-STC antibody of the present invention may take a form as a polyclonal antibody or a form as a monoclonal antibody. In both cases, the anti-STC antibody of the present invention can be generally used using a known method.

First, when the anti-STC antibody of the present invention is a polyclonal antibody, it can be obtained by immunizing an immunized animal with the above-mentioned immunizing antigen and then purifying it from the serum of the immunized animal. Immunization can be performed by a general method, for example, by administering the above-mentioned immunizing antigen to an animal to be immunized by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like. More specifically, immunization can be carried out by administering the above-mentioned immunizing antigen to an animal to be immunized several times every 2 to 14 days by the above-mentioned means, optionally in combination with a usual adjuvant. The animal to be immunized is not particularly limited, and mice, rats, and the like can be widely used.

When the anti-STC antibody of the present invention is a monoclonal antibody, it is necessary to obtain immunocytes for producing a monoclonal antibody from an animal immunized as described above, for example, spleen cells after immunization. .

When a monoclonal antibody is produced, the myeloma cell as the other parent cell to be fused with the immune cell is already known, for example, SP2 / 0-Ag.
14, P3-NS1-1-Ag4-1, MPC11-4
5,6. TG1.7 (all derived from mouse); 210. R
CY. Ag 1.2.3 (from rat); SKO-00
7, GM15006TG-A12 (all derived from humans) and the like can be used. Cell fusion between the above-mentioned immune cells and the myeloma cells is performed by a known method, for example, the method of Kohler, G. and Milstein, C., Natu.
re , 256 , 495 (1975)).

More specifically, the cell fusion can be carried out by a known fusion promoter such as polyethylene glycol (PE).
G) or in the presence of Sendai virus (HVJ) or the like, a hybridoma is prepared in a usual culture medium to which an auxiliary agent such as dimethyl sulfoxide is added as necessary to improve the fusion efficiency.

The desired hybridoma can be isolated by culturing it in a usual selection medium, for example, a HAT (hypoxanthine, aminopterin and thymidine) medium. That is, hybridomas can be separated by culturing them in this selection medium for a time sufficient to kill cells other than the target hybridomas. The hybridoma thus obtained can be subjected to a search for a target monoclonal antibody and a single cloning by a usual limiting dilution method.

The target monoclonal antibody producing strain can be searched by, for example, ELISA, plaque, spot,
It can be performed according to a general search method such as an agglutination reaction method, an octalony method, and an RIA method.

The hybridoma producing the desired monoclonal antibody recognizing stanniocalcin thus obtained can be subcultured in a usual medium, and can be stored in liquid nitrogen for a long time.

The monoclonal antibody can be collected from the hybridoma by culturing the hybridoma according to a conventional method, obtaining a culture supernatant thereof, or administering the hybridoma to an animal that is compatible with the hybridoma and growing the hybridoma. A method for obtaining the ascites can be used.

The polyclonal antibody and the monoclonal antibody obtained as described above can be prepared by a conventional method such as affinity chromatography using a carrier to which protein A or an antigen peptide is bound, salting out, gel filtration and the like. It can be purified by means.

The anti-STC of the present invention obtained as described above
Antibodies (both polyclonal and monoclonal)
Is an antibody having cross-reactivity specifically with stanniocalcin.

The anti-STC antibody of the present invention can be prepared according to the type of means for detecting stanniocalcin using the anti-STC antibody.
If necessary, it can be labeled with a labeling substance and used in the selected means for detecting stanniocalcin.

Such a labeling substance can be used alone or by reacting the labeling substance with another substance.
It is a labeling substance that provides a detectable signal, and specifically, for example, horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase , Fluorescent substances such as penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothiocyanate, ¹²⁵ I, ¹⁴ C, ³
Radioisotopes such as H; chemical substances such as biotin, avidin and digokesigenin; and chemiluminescent substances.

The anti-STC of the present invention using these labeling substances
As a method for labeling the antibody, a known labeling method can be appropriately used depending on the type of the labeling substance to be selected. Further, the anti-STC antibody of the present invention (including a labeled antibody) can be used in an STC protein detection method as an immobilized antibody, which is immobilized on an insoluble carrier.

As such an insoluble carrier, various insoluble carriers already used as insoluble carriers for antibodies can be used. Specifically, for example, a plate represented by a microplate, a test tube, a tube, a bead,
For affinity chromatography of balls, filters, membranes, or cellulosic carriers, agarose carriers, polyacrylamide carriers, dextran carriers, polystyrene carriers, polyvinyl alcohol carriers, polyamino acid carriers, or porous silica carriers. Examples include the insoluble carrier used.

The anti-S of the present invention in these insoluble carriers
As a method for immobilizing a TC antibody, a method for immobilizing an antibody that has already been established on various insoluble carriers can be appropriately selected depending on the type of the insoluble carrier to be selected. Thus, the anti-STC antibody of the present invention, which can be used for detection of STC protein, can be produced.

B. Regarding the method for specifying stanniocalcin according to the present invention (hereinafter also referred to as the method for specifying the present invention): A typical embodiment of the method for specifying the present invention is the anti-STC of the present invention described above
This is a method for detecting stanniocalcin in a specimen (mainly, a blood specimen) using an antigen-antibody reaction between an antibody and a stanniocalcin protein.

The identification method of the present invention is a method for detecting stanniocalcin in a specimen by utilizing a specific antigen-antibody reaction of the anti-STC antibody of the present invention with a stanniocalcin protein. Examples of the specific method of the present invention utilizing the antigen-antibody reaction include, for example, enzyme immunoassay, radioimmunoassay, analysis by flow cytometry, and Western blotting.

In all of the methods of the present invention, it is often preferable to use the anti-STC antibody of the present invention as a monoclonal antibody. The enzyme immunoassay is also referred to as enzyme immunoassay, and is a detection method using an enzyme as a labeling substance among labeled immunoassay methods (a detection method using a radioisotope is a radioimmunoassay method). Enzyme immunoassays include:
"Heterogeneous enzym requiring so-called B / F separation"
e immunoassay "and" hom, which does not require B / F separation
ogeneous enzyme immunoassay ".

In the specifying method of the present invention, among these, the former "heterog", which is generally said to have high measurement sensitivity, is used.
It is more preferable to select “eneous enzyme immunoassay”, and “enzyme-linked immunity using immunosorbent”
It is more preferred to select the "sorbent assay (ELISA)".

As a more specific specific embodiment, an enzyme immunoassay method by a so-called sandwich method (hereinafter, also referred to as a sandwich method) can be exemplified. Such a sandwich method is one of the particularly preferred specific embodiments, particularly in view of simplicity of operation and economical convenience, especially versatility as a clinical test.

When performing the sandwich method, the anti-STC antibody of the present invention as a monoclonal antibody
It is possible to use an immobilized monoclonal antibody immobilized on a microplate having a large number of wells, such as a well microplate, and a polyclonal antibody labeled with an enzyme such as horseradish peroxidase or biotin as described above. preferable.

The sandwich method comprises at least the following (a)
And an enzyme immunoassay method comprising the steps of (b). (a) a step of immobilizing the anti-STC antibody of the present invention as a monoclonal antibody or a polyclonal antibody on an insoluble carrier such as a microplate, and reacting the immobilized antibody with a sample represented by a blood sample such as human plasma; In this step (a), usually, after the reaction, the used microplate or the like is washed, and the unreacted sample is removed from the immobilized monoclonal antibody or polyclonal antibody.

(B) an antigen-antibody complex formed by the binding of the immobilized monoclonal antibody or the polyclonal antibody to human stanniocalcin in the sample, a monoclonal antibody or a monoclonal antibody labeled with horseradish peroxidase, biotin, or the like; A step of reacting the anti-STC antibody of the present invention as a polyclonal antibody. In this step (b), usually, after the reaction, the used microplate or the like is washed, and the unreacted labeled anti-STC antibody of the present invention as a monoclonal antibody or polyclonal antibody is removed from the microplate or the like. .

In this step (b), it is necessary to make the label signal visible using a means for expressing the label signal according to the type of the label in the reacted second antibody. For example, when biotin is used as the labeling substance, the labeling signal can be made visible using avidin or the like. Also, for example, when horseradish peroxidase is selected as a labeling substance,
The substrate can be added, if necessary, together with a chromogenic substance to make the label signal visible.

In this way, by detecting the color signal that has become apparent by using a signal specifying means corresponding to the type of the color signal, stanniocalcin in a desired sample can be specified. .

Next, in the analysis by flow cytometry, the stanniocalcin in the sample is labeled with the anti-STC antibody of the present invention as a fluorescently labeled monoclonal antibody, and the fluorescence intensity in the sample is specified. Is a method for identifying stanniocalcin.

Further, in the Western blotting method, a preparation of a sample separated by electrophoresis is transferred to a nitrocellulose membrane or the like, and the stanniocalcin protein in the preparation is identified using the anti-STC antibody of the present invention. Is the way.

In the present invention, the anti-STC antibody of the present invention is used for use in the method of the present invention.
A kit for specifying stanniocalcin containing the TC antibody is also provided (hereinafter, referred to as the specific kit of the present invention).

In one embodiment of the kit of the present invention, an element for providing the anti-STC antibody of the present invention for specifying stanniocalcin in a sample, for example, the anti-STC antibody of the present invention as the labeled monoclonal antibody or And the immobilized anti-STC antibody of the present invention.

[0051]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited by these examples.

Example 1 Preparation of Antigen 16 mg of mussel hemocyanin (KLH) was dissolved in 2 mL of 0.01 M phosphate buffer (pH 7.2), and 2.6 mg of m-maleinimide benzyl-N-hydroxysuccinimide was added thereto. A solution of the ester (MBS) dissolved in 1 mL of dimethylformamide was added and reacted at room temperature. After the reaction, excess MBS was removed through a Sephadex G-25 column, and 1.8 mL of 3 mg of the peptide represented by SEQ ID NO: 2 (peptide synthesizer model 430A: synthesized by Applied Biosystems) chemically synthesized in the reaction solution was used. Of 0.1 M disodium hydrogen phosphate was added and reacted at room temperature for 6 hours.
H-peptide conjugate was prepared.

Example 2 Preparation of Hybridoma 50 μg of KLH-peptide complex prepared in Example 1 above
And complete Freund's adjuvant were mixed in equal amounts to form an emulsion, and the mice were immunized subcutaneously and intraperitoneally. Two weeks later, an emulsion in which 50 μg of the KLH-peptide complex and incomplete Freund's adjuvant were mixed in equal amounts was boosted subcutaneously and intraperitoneally. After confirming the increase in specific antibodies in the immunized mice by solid phase immunoassay, booster immunization was performed in the same procedure as described above.

Three days after the final immunization, 1 × 10 ⁸ spleen cells and 3 × 10 ⁷ myeloma cells of the immunized BALB / c mouse were used.
Mixed in RPMI 1640 medium. Next, the mixture was centrifuged, and the mixed precipitate of cells obtained was washed with RPMI1640 medium to completely remove the supernatant, and the remaining cell precipitate was kept at 37 ° C. A 50% polyethylene glycol (average molecular weight: 1500) -PBS solution was added thereto with stirring over 45 seconds to suspend the remaining cell precipitate. While uniformly suspending the cells, 30 mL of RPMI1640 medium was added, followed by centrifugation to precipitate the cells again.

The cells thus obtained were suspended in a HAT medium, and seeded on a 96-well plate at a concentration of 1 × 10 ⁵ cells / 50 μL per well, and then incubated at 37 ° C. and 5% CO ₂ using an incubator. And cultured. After the start of the culture, 5
On days 7, 7 and 9, 50 μL of HAT medium was added per well for supplementation and cells were observed. After the start of culture
On the 10th to 14th days, cell growth is observed in most of the wells. A certain amount of each culture supernatant is collected, and a complex of bovine serum albumin and peptide (KLH and (Prepared in the same manner as for the peptide conjugate) at room temperature for 1 hour. After washing with a PBS washing solution containing 0.05% Tween20, it was allowed to react with rabbit anti-mouse IgG labeled with horseradish peroxidase at room temperature for 30 minutes. After washing the plate again, 0.015% aqueous hydrogen peroxide was added to 0.5 mg / mL of OPD and reacted at room temperature for 10 minutes. After stopping the reaction with 2N sulfuric acid, the absorbance at 492 nm was measured.

Example 3 Preparation of Monoclonal Antibody and
Purification From the wells that had reacted with the complex of bovine serum albumin and peptide in Example 2 above, single clones were selected by the limiting dilution method. That is, the number of cells in a well was measured, and a medium in which cells were suspended so that the number of cells became 0.2 per well was dispensed into a 96-well plate and cultured. This operation was performed three times. The culture supernatant of the selected single clone was again isolated on a plate, and the reactivity of the peptide with the bovine serum albumin complex was examined. The 9E12D clone having the highest reactivity was obtained. From this clone, a monoclonal antibody against the desired STC protein was produced by a conventional method, purified, and used in the following Examples.

Example 4 Preparation of Polyclonal Antibody and
Emulsions were prepared by mixing 400 μg of the purified KLH-peptide complex and complete Freund's adjuvant in equal volumes, and immunized subcutaneously and intraperitoneally in rabbits. Two weeks later, an emulsion obtained by mixing an equal volume of 400 µg of the KLH-peptide complex and incomplete Freund's adjuvant was boosted subcutaneously and intraperitoneally.
After confirming the rise of the specific antibody in the rabbit by solid phase immunoassay, booster immunization was performed four times in the same procedure as described above. Two weeks after the final immunization, whole blood was collected from the carotid artery of the rabbit, coagulated at room temperature for 60 minutes, and centrifuged to obtain serum. The antiserum thus prepared was purified by affinity chromatography using a carrier having protein A and an antigen peptide bound thereto, and used in the following Examples.

Example 5 Human Kidney Immunostaining Human kidney tissue fixed with 4% valaformaldehyde was embedded in paraffin, cut into a thickness of 2 μm, mounted on a slide glass, and sectioned. This section was treated with 0.3% hydrogen peroxide solution for 30 minutes, blocked with 10% normal sheep serum, and then treated with the anti-STC of the present invention prepared in Example 3.
An antibody (monoclonal antibody) (3 μg / mL) in PBS solution was added and reacted at room temperature overnight. After the reaction, the monoclonal antibody was removed and reacted with biotinylated anti-mouse IgG for 3 hours at room temperature. After washing, avidin-peroxidase was added and reacted for 30 minutes at room temperature. After washing, diaminobenzidine was added to develop color.

Photographs showing the results of this immunostaining are shown [see FIGS. 1 and 2: FIG.
FIG. 2 is an immunostaining image obtained by performing immunostaining using an antibody (monoclonal antibody). FIG.
TC antibody (monoclonal antibody) in excess of recombinant STC (recombinant STC is available from HENRIK S.OLSE
N et al., Proc. Natl. Acad. Sci. USA., Vol. 93, pp1792-179
6 (March 1996) ".
The same applies to the following.) These immunostaining images reveal that STC is localized in the distal tubule in humans, and that STC can be detected by immunostaining using the anti-STC antibody of the present invention. Was.

Example 6 Monoclonal Antibody (Primary)
A polyclonal antibody (secondary) sandwich ELISA plate was added with the anti-STC antibody of the present invention (monoclonal antibody) of Example 3 adjusted to 1 μg / mL in 0.1 M sodium bicarbonate, and allowed to stand at room temperature for 3 hours. Was placed. 0.05%
After washing with a PBS wash solution containing Tween20, wash with a blocking solution containing 1% bovine serum albumin and 0.05% Tween20 at room temperature.
Let stand for hours. The blocking solution was discarded, and the recombinant STC was diluted with the blocking solution, seeded, and allowed to stand at room temperature for 1 hour. After standing, the plate was washed with a washing solution, and then a biotin-labeled anti-STC antibody (polyclonal antibody) of the present invention, which was adjusted to 1 μg / mL in the blocking solution and inoculated with the example, was incubated at room temperature for 3 hours. After washing again, 0.2
Add μg / mL avidin-peroxidase and add
Let stand for hours. After washing again, the color was developed with OPD and the absorbance at 492 nm was measured.

FIG. 3 is a calibration curve obtained by this measurement system. From this calibration curve, it was clarified that by performing sandwich ELISA with the anti-STC antibody of the present invention, it is possible to quantify up to the order of 10 ng.

Example 7 Solution by Flow Cytometer
The analyzed K562 cells were washed with PBS and treated with cold 4% paraformamide for 10 minutes to prepare a 1 × 10 ⁶ /0.5 mL cell solution. The cells are washed twice with 0.1% savonin in Hanks buffer, suspended in 0.1% savonin-Hanks buffer, and non-specific antibodies are added to a final concentration of 500 μg / mL.
And left at room temperature for 30 minutes. Next, the biotin-labeled anti-STC antibody of the present invention (monoclonal antibody) of Example 3 was added to a final concentration of 1 μg / mL.
The reaction was performed at room temperature for 30 minutes. Then, after washing twice with PBS, streptavidin peroxidase was added, reacted for 30 minutes at room temperature, and analyzed with a flow cytometer.

FIG. 4 shows the results. In FIG. 4, the horizontal axis represents the fluorescence intensity (logarithmic display), and the vertical axis represents the number of cells. 4. In FIG. 4, the anti-STC antibody of the present invention (monoclonal antibody) was compared with the fluorescence intensity distribution of the control group in which normal mouse serum was added to K562 cells as a control.
The fluorescence distribution of the fluorescence intensity of the experimental group to which was added was clearly biased to the stronger one. This has revealed that the STC protein can be detected by the anti-STC antibody of the present invention.

[0064]

According to the present invention, a means for producing an antibody against stanniocalcin derived from human is provided.
A means for identifying human-derived stanniocalcin using the resulting antibody to stanniocalcin is provided.

[0065]

[Sequence Table] SEQ ID NO: 1 Sequence length: 19 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Tyr Asn Arg Leu Val Arg Ser Leu Leu Glu Cys Asp Gln Asp Thr Val 16 Ser Thr Ile 19

SEQ ID NO: 2 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Cys Tyr Asn Arg Leu Val Arg Ser Leu Leu Glu Ala Asp Gln Asp Thr 16 Val Ser Thr Ile 20

[Brief description of the drawings]

FIG. 1 is a drawing showing a tissue-stained image of a human kidney using the anti-STC antibody of the present invention.

FIG. 2 is a view showing a tissue-stained image of a human kidney using an anti-STC antibody-absorbed antibody of the present invention.

FIG. 3 is a drawing showing a calibration curve in ELISA using the anti-STC antibody of the present invention.

FIG. 4 is a view showing the results of analysis by flow cytometry using the anti-STC antibody of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/02 C12N 5/00 B 21/08 15/00 C G01N 33/577 ZNAA // (C12N 5 / 10 C12R 1:91) (72) Inventor Hu Jianguo 1361-1 Matoba, Kawagoe-shi, Saitama Prefecture Within BM Research Institute, Inc. (72) Inventor Nobuhiro Sakuma 2-chome, Yawata, Aoba-ku, Sendai, Miyagi −19 Maison Ishida 201 (72) Inventor: Shinobu Shibata 3-65-65, Itohashi, Wakabayashi-ku, Sendai City, Miyagi Prefecture (72) Inventor: Takeshi Sasaki 4-30-407 F-term, Hanadadan, Aoba-ku, Sendai City, Miyagi Prefecture (Reference) 4B024 AA11 BA58 BA61 HA01 HA15 4B064 AG26 AG27 CA10 CA20 DA13 4B065 AA90X AA91Y AA93Y AB04 BA08 BD50 CA25 CA26 CA46 4H045 AA11 AA30 BA10 BA42 CA40 DA76 DA86 EA50 FA72

Claims (12)

[Claims] 1. A stanniocalcin which comprises, as an immunizing antigen, a modified peptide obtained by modifying an amino acid constituting a peptide containing all or a part of the amino acid sequence represented by SEQ ID NO: 1 within a range that does not lose its identity as a whole. Antibodies against. 2. An amino acid constituting a peptide containing the whole or a part of the amino acid sequence represented by SEQ ID NO: 1 as long as the amino acid constituting the peptide is modified within a range not to lose the identity as a whole. The antibody against stanniocalcin according to claim 1, which is one of the modifications. 3. The antibody against stanniocalcin according to claim 2, wherein the amino acid to be modified is cysteine at position 11 from the N-terminus in the peptide having the amino acid sequence represented by SEQ ID NO: 1. 4. The antibody against stanniocalcin according to claim 3, wherein the cysteine at position 11 counted from the N-terminus in the peptide having the amino acid sequence represented by SEQ ID NO: 1 is substituted with alanine. 5. The antibody according to claim 1, wherein the immunizing antigen of the antibody is a hapten-bound immunizing antigen. 6. The antibody according to claim 5, wherein the hapten is KLH. 7. The antibody according to claim 1, wherein the antibody is a monoclonal antibody. 8. The antibody according to claim 1, wherein the antibody is a polyclonal antibody. 9. A method for detecting human-derived stanniocalcin, wherein an antigen-antibody reaction between the antibody according to any one of claims 1 to 8 and human-derived stanniocalcin is used as a detection principle. 10. A method for quantifying human-derived stanniocalcin, wherein the antigen-antibody reaction between the antibody according to any one of claims 1 to 8 and human-derived stanniocalcin is used as a quantification principle. 11. A kit for detecting human-derived stanniocalcin, comprising at least the antibody according to any one of claims 1 to 8. A kit for quantifying human-derived stanniocalcin, comprising at least the antibody according to any one of claims 1 to 8.