JP4857431B2 - Anti-8-nitrocyclic guanosine 3 ', 5'-monophosphate antibody - Google Patents

Description

  The present invention relates to an antibody capable of recognizing 8-nitrocyclic guanosine 3 ′, 5′-monophosphate.

  Cyclic guanosine 3 ', 5'-monophosphate (hereinafter abbreviated as "cGMP") is an intracellular signal transduction molecule that transmits extracellular signals such as nitric oxide (NO) and natiuretic peptides. It is. The existence of cGMP-dependent protein kinase (protein kinase G), cGMP regulatory channel, and cGMP-degrading enzyme (phosphodiesterase) has been elucidated as cGMP receptor proteins, and the initial reaction of cGMP signaling is the protein. It is thought to be activation of kinase G.

Protein kinase G is a serine / threonine kinase and is activated at cGMP concentrations on the order of micromolar. In vascular smooth muscle cells, protein kinase G phosphorylates several parts including Ser695 of MBS (myosin-binding subunit) that constitutes MLCP (myosin light chain phosphatase), thereby relaxing vascular smooth muscle cells. It is considered. In addition, protein kinase G phosphorylates Ser683 and Ser696 (bovine type) of IP ₃ receptor-associated cGKI substrate (IRAG) present in the endoplasmic reticulum and inhibits the release of Ca induced by IP ₃ from the endoplasmic reticulum. To do. In addition, it is known that Ca-dependent K channels, ATP-sensitive K channels, L-, N-, and T-type channels are activated by phosphorylation. It is thought to promote the release of transmitter substances. The activity of phosphodiesterase type 5 (PDE5) is also regulated by protein kinase G. When Ser92 (human type) is phosphorylated, cGMP is decomposed into GMP (guanosine monophosphate).

  Thus, cGMP plays an important role in vivo as an information transmission substance. In addition, nitrating agents such as nitroglycerin that activate soluble guanylate cyclase also increase intracellular cGMP concentrations. This is because 8-nitrocyclic guanosine 3 ', 5'-monophosphate (hereinafter referred to as `` 8-nitro cGMP ”may also be abbreviated as“ cGMP. ”) is also thought to be responsible for the intracellular signal transduction function by activating intracellular protein kinase G through the cell membrane. Furthermore, a large amount of nitric oxide is generated at the inflamed site by inducible nitric oxide synthase, and foreign substances such as invading bacteria are eliminated. At this time, cGMP, which is a signal transmitter, is also active with nitric oxide. Nitrogen peroxide (peroxynitrite) generated by oxygen undergoes a nitration reaction and is thought to change to 8-nitro cGMP. Thus, 8-nitro cGMP is considered not only to play a role as a biomarker for inflammatory diseases, but also to function as a new signaling substance that is similar to or completely unknown to cGMP.

  When detecting released 8-nitro cGMP in cultured cells or tissue samples, it is considered that the effective method is to hydrolyze the cells and proteins and analyze them by reversed-phase HPLC. There are no reports of 8-nitro cGMP detection. In order to analyze by HPLC, there are problems such as complicated protein hydrolysis and removal of contaminants. Furthermore, HPLC analysis is not suitable for multi-sample processing because it is an analysis for each sample. This is probably due to a problem. On the other hand, the enzyme immunoassay method (ELISA method) using an antibody against 8-nitro cGMP is considered advantageous for the rapid analysis and quantification of 8-nitro cGMP generated in blood and urine by multi-sample treatment. It is expected that 8-nitro cGMP, an inflammatory biomarker present in various tissues and cells, can be analyzed by ELISA. By immunohistochemical staining of tissues and cultured cells using an anti-8-nitro cGMP antibody, the localization of 8-nitro cGMP generated in the tissues and cells can be visualized as a stained image, and inflammatory It is expected to be useful not only for elucidating the pathogenesis of disease but also for elucidating the information transmission mechanism of 8-nitro cGMP.

However, an antibody that recognizes 8-nitro cGMP has not been provided so far, and a method for measuring 8-nitro cGMP by ELISA cannot be used. This is due to the lack of antigenicity of 8-nitro cGMP itself. As a means for producing an antibody using such a non-antigenic substance as an antigen, a method is known in which the substance is bound to a serum protein and used as an antigen. However, since 8-nitro cGMP has no reactive functional groups such as amino groups and carboxyl groups, there is a problem that this substance cannot be bound to serum proteins.
An antibody that recognizes 8-nitroguanine and a method for producing the same are known (WO 03/99869), but this antibody does not recognize 8-nitro cGMP.
International Publication WO 03/99869

  An object of the present invention is to provide a means for detecting 8-nitro cGMP with high sensitivity. More specifically, 8-nitro cGMP can be measured easily and reliably, as a means to clarify the localization of 8-nitro cGMP in vivo, and simultaneously in a large number of samples by ELISA. It is an object of the present invention to provide an antibody that recognizes 8-nitro cGMP as a means for measuring the amount of 8-nitro cGMP.

  As a result of intensive studies to solve the above problems, the present inventors have found that an antibody that recognizes 8-nitro cGMP can be produced by using a specific antigen protein containing 8-nitro cGMP. Was successfully used to produce polyclonal and monoclonal antibodies that specifically recognize 8-nitro cGMP, completing the present invention.

That is, the present invention provides an antibody that recognizes 8-nitrocyclic guanosine 3 ′, 5′-monophosphate represented by the following formula.
According to the preferable aspect of the said invention, the said antibody which is a polyclonal antibody and the said antibody which is a monoclonal antibody is provided. Furthermore, the present invention provides a method for measuring 8-nitrocyclic guanosine 3 ′, 5′-monophosphate contained in a biological sample or a cell culture sample using the antibody described above.

Further, according to the present invention, there is provided an antigen used for producing an antibody that recognizes 8-nitrocyclic guanosine 3 ′, 5′-monophosphate, which comprises 8-nitrocyclic guanosine 3 ′, 5′-monophosphate. Provided is an antigen comprising an acid-bound serum protein, wherein the sugar moiety of 8-nitrocyclic guanosine 3 ′, 5′-monophosphate is bound to the serum protein via a carbon chain. According to a preferred embodiment of the invention, the antigen in which the hydroxyl group of the sugar moiety of 8-nitrocyclic guanosine 3 ′, 5′-monophosphate and the serum protein are bonded via a carbon chain, the serum protein is albumin, and more. An antigen as described above, preferably bovine serum albumin, is provided.
Furthermore, the present invention provides a method for producing the above antibody, comprising the step of immunizing a mammal using the above antigen.

  By using the antibody recognizing 8-nitro cGMP provided by the present invention, 8-nitro cGMP contained in a biological sample or a cell culture sample can be easily and reliably measured. By using this antibody, the localization of 8-nitro cGMP in a living body can be clarified, and the amount of 8-nitro cGMP in a large number of samples can be measured simultaneously by ELISA. In addition, an antibody that recognizes 8-nitro cGMP can be easily produced using the antigen provided by the present invention.

  The method for producing the antibody of the present invention is not particularly limited. For example, it is desirable to use an antigen containing serum protein to which 8-nitro cGMP is bound, preferably serum albumin to which 8-nitro cGMP is bound. The method for producing this antigen is not particularly limited, but it can be easily produced by adding a basic substance such as succinic anhydride and triethylamine in an aqueous solution.

  As a protein that binds 8-nitro cGMP, any protein that is generally a non-antigenic serum protein may be used. For example, in addition to human serum albumin (HSA), other mammalian serum albumins such as bovine serum albumin (BSA) and rabbit serum albumin (RSA), ovalbumin (OVA), keyhole rimpet hemocyanin (KLH), etc. However, it is not limited to these. Of these, BSA is most preferred.

  The nucleobase 8-nitro cGMP has a limited number of sites that can be chemically modified due to its structure. The easily reactable amino group at position 3 is considered to play an important role as a recognition site for nucleobases by hydrogen bonding, so it is added to serum albumin via the amino group at position 3 of guanine of 8-nitro cGMP. Even using a bound antigen, it is difficult to produce an antibody that recognizes 8-nitro cGMP. On the other hand, by binding the sugar moiety of 8-nitro cGMP to serum protein, an antigen in which 8-nitroguanosine, the nucleobase of 8-nitro cGMP, and the cyclic phosphate site, which is an important structure of the sugar moiety, are preserved as they are. By immunizing a mammal using this, an anti-8-nitro cGMP antibody can be efficiently obtained.

  The means for binding the sugar moiety of 8-nitro cGMP to the serum protein is not particularly limited, but it is desirable that the sugar moiety is bound to the serum protein via a carbon chain. Examples of the introduction of a carbon chain that acts as a linker include a method of succinylation of a free hydroxyl group (2′-position hydroxyl group) present in the sugar moiety, but it is not limited to this method. . Examples of the carbon chain include an unsubstituted or substituted alkylene group and an unsubstituted or substituted alkenylene group. The carbon chain may be either a straight chain or a branched chain, and has an unsaturated bond. One or two or more may be included. The number of carbon atoms constituting the straight chain portion of the carbon chain (which is the carbon chain connecting the hydroxyl group and the reactive functional group of serum protein with the minimum number) is not particularly limited, but for example, about 1 to 20, The number is preferably about 2 to 10, more preferably about 3 to 6. Particularly preferred is 4.

  One or two or more heteroatoms (oxygen atom, nitrogen atom, sulfur atom, etc.) may be present in the carbon chain. The type of substituent that can be present on the carbon chain is not particularly limited, and examples thereof include, but are not limited to, a halogen atom, an oxo group, and a hydroxyl group. A reactive functional group needs to be present at the end of the carbon chain for reaction with serum protein. For example, an amino group or a carboxyl group can be used as the reactive functional group. For example, when the reactive functional group at the end of the carbon chain is a carboxyl group, a saccharide of 8-nitro cGMP is formed via the carbon chain by forming a bond with a carboxyl group or amino group present in serum protein. The moiety can be bound to a serum protein. The above binding can be efficiently performed by using, for example, a dehydration condensing agent. However, since many dehydration condensing agents used for chemical modification of proteins are known (for example, 1-ethyl -3- (3-dimethylaminopropyl) carbodiimide: WSC, etc.), those skilled in the art can select appropriate reagents according to various factors such as the type of serum protein, reactive functional groups, or reaction conditions. is there.

  The antibody of the present invention can be easily produced by dissolving the antigen in a solvent such as phosphate buffered saline (PBS), administering this solution to an animal and immunizing it. If necessary, an adjuvant may be appropriately added to the solution, and then immunization may be performed using an emulsion. As an adjuvant, in addition to an adjuvant such as a water-in-oil emulsion, a water-in-oil-in-water emulsion, an oil-in-water emulsion, a liposome, or an aluminum hydroxide gel, a protein derived from a biological component or a peptide substance may be used. . For example, Freund's incomplete adjuvant or Freund's complete adjuvant can be preferably used. The administration route, dosage, or administration timing of the adjuvant is not particularly limited, but it is desirable to select appropriately so that a desired immune response can be enhanced.

  The type of animal used for immunization is not particularly limited, and for example, a mouse, rat, cow, rabbit, goat, sheep, etc. can be used, and preferably a Japanese white rabbit (Japanese White Rabbit) can be used. In addition, various animals such as NZW rabbits (New Zealand White Rabbit) such as rabbits used for antibody production can be used. Animal immunization may be carried out according to methods available in the art, for example, by injecting a mammal solution subcutaneously, intradermally, intravenously, or intraperitoneally with an antigen solution, preferably a mixture with an adjuvant. it can. Since the immune response generally varies depending on the type and strain of mammal to be immunized, it is desirable to set the immunization schedule as appropriate according to the animal used. The antigen administration is preferably repeated several times after the first immunization.

  For example, as an initial immunization, an emulsion obtained from a solution obtained by dissolving the antigen in PBS and Freund's complete adjuvant is used, and the second and subsequent immunizations are performed by dissolving the antigen in PBS and Freund's incomplete adjuvant. It is desirable to use an emulsion obtained by mixing the In the first immunization, it is advantageous to use Freund's complete adjuvant containing bacterial cells in order to induce an immune response, and the second and subsequent boosters do not produce a new antibody, but at the time of the first immunization. It is advantageous to use Freund's incomplete adjuvant because it aims to increase the antibodies that are already expressed and stored in B cells. The interval from the first immunization to the booster immunization and the interval between the booster immunization and the booster immunization are not particularly limited. For example, the first immunization is for the production of antibodies. Usually, antibodies appear in the blood 4 to 5 days after the first injection of the antigen, and the amount gradually increases and peaks around 10 days. It is preferable to allow two weeks or more from the first immunization to the first boost.

  The antibody of the present invention can be obtained from the serum of the immunized animal, but the method is not particularly limited, and any method may be used as long as it is a method available to those skilled in the art. The antibody can be purified by appropriately combining, for example, DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method and the like. Whether an antibody recognizes 8-nitro cGMP or whether the reaction is specific can be easily confirmed using methods well known to those skilled in the art. In the examples of the present specification, animal immunization methods, antibody purification methods, and antibody property confirmation methods are specifically described for the polyclonal antibody production method of the present invention. The antibody of the present invention can be easily produced by adding appropriate modifications or alterations to the method as necessary while referring to the description and the specific methods of Examples.

  Examples of the antibodies of the present invention include 8-nitro cGMP and a protein bound thereto, guanine, guanosine, cyclic guanosine 3 ′, 5′-monophosphate (cGMP), guanosine diphosphate (GDP), guanosine Antibodies that do not react with triphosphate (GTP), other nucleobases, and their derivatives, and proteins bound to them, such as nitroguanine isomers with different substitution positions of nitro groups and their 3 ', 5'-sites An antibody capable of discriminating between a click monophosphate derivative and 8-nitro cGMP can be preferably used. The antibody of the present invention specifically recognizes 8-nitro cGMP means that the antibody has partial or complete selectivity for 8-nitro cGMP compared to other substances as shown in the above example. However, this word should not be construed as limiting in any way. As the antibody of the present invention, a monoclonal antibody produced from a hybridoma produced using lymphocytes of an immunized animal may be used as the antibody of the present invention. As the antibody of the present invention, a polyclonal antibody having the above properties can be preferably used, but a monoclonal antibody having the above properties is also preferable.

  Monoclonal antibody production methods are well known in the art and are widely used, and those skilled in the art can easily produce monoclonal antibodies that recognize 8-nitro cGMP by using the above antigens. . (See, for example, Chapter 6 of the Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988)). The type of mammal used for the preparation of antibody-producing cells is not particularly limited, and examples include mice, rats, cows, rabbits, goats, sheep, etc., preferably mice, rats, rabbits, and more preferably Can use a mouse. For example, mice of the BALB / c strain are preferable because myeloma-derived cell lines established in the same strain at the time of hybridoma production can be used.

  After the final immunization, spleen cells are removed from the immunized mammal and hybridomas can be produced by cell fusion with a cell line derived from myeloma. For cell fusion, it is preferable to use an immunogenic cell line having a high proliferation ability, and the cell line derived from myeloma is preferably compatible with a mammal derived from the immune-producing cell to be fused. Cell fusion can be performed according to a method known in the art. For example, a polyethylene glycol method, a method using Sendai virus, a method using an electric current, or the like can be employed. The obtained hybridoma can be grown according to conditions generally used in the art, and a desired hybridoma can be selected while confirming the properties of the antibody produced. The hybridoma can be cloned by a known method such as a limiting dilution method or a soft agar method.

  A hybridoma producing a monoclonal antibody having a desired performance can be confirmed by assaying the binding ability of the produced antibody and 8-nitro cGMP by a method such as ELISA, RIA, or fluorescent antibody. Monoclonal antibodies that react specifically with 8-nitro cGMP can be produced by mass-culturing the hybridomas selected as described above. The method of mass culture is not particularly limited. For example, the hybridoma is cultured in an appropriate medium to produce a monoclonal antibody, or the hybridoma is injected into the abdominal cavity of a mammal to proliferate, and the antibody is injected into the ascites. And the like. The monoclonal antibody can be purified by appropriately combining DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG method, ethanol fractionation method and the like.

As the antibody of the present invention, an antibody fragment or a chimeric antibody can also be used. The antibody fragment is preferably a functional fragment, and examples thereof include F (ab ′) ₂ and Fab ′. Such a fragment can be produced by treating an antibody recognizing 8-nitro cGMP with a proteolytic enzyme (for example, pepsin or papain). The monoclonal antibody of the present invention can also be used as an immobilized antibody immobilized on an insoluble carrier such as a solid phase carrier, or as a labeled antibody labeled with a labeling substance. Any of such immobilized antibodies and labeled antibodies are included in the scope of the present invention.

  For example, an immobilized antibody can be produced by physically adsorbing or chemically binding a monoclonal antibody to an insoluble carrier. As the insoluble carrier, an insoluble carrier made of a polymer substrate such as polystyrene resin, an inorganic substrate such as glass, a polysaccharide substrate such as cellulose or agarose, or the like can be used. The shape of the insoluble carrier is not particularly limited, and any shape such as a plate shape or a bead shape can be selected.

  Examples of the labeling substance for producing the labeled antibody include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, and radioisotopes. As a method for binding the labeling substance and the antibody, methods such as glutaraldehyde method, maleimide method, pyridine disulfide method, and succinic acid active ester method that can be used by those skilled in the art can be used. However, the types of immobilized antibodies and labeled antibodies, and the production methods thereof are not limited to the above examples. For example, methods for detecting signals generated by further reacting enzymes, chemiluminescent substances, biotin, and avidin with one or more other substances for detection of labeled antibodies are well known to those skilled in the art. It is preferably used for detection of antibodies. For example, in the case of an enzyme, a method of measuring enzyme activity using a substrate can be used, and in the case of biotin, at least avidin or enzyme-modified avidin is generally reacted.

  8-Nitro cGMP can be measured using the antibody of the present invention. For example, 8-nitro cGMP produced in a living tissue using the antibody of the present invention can be measured, and a site involved in relaxation of blood vessels and information transmission in a living body where 8-nitro cGMP is present Can be easily identified. In the present specification, the term “measurement” should be interpreted in the broadest sense including detection and quantification, and should not be interpreted in a limited manner in any sense.

  For example, 8-nitro cGMP contained in a biological sample separated and collected from a human using the antibody of the present invention can be measured. The type of biological sample isolated from humans is not particularly limited. For example, blood, serum, plasma, lymphocyte culture supernatant, urine, spinal fluid, saliva, sweat, ascites, amniotic fluid, or cell or organ extract, etc. In addition to liquid samples, tissues and cells removed by surgery can be measured. Measurement is a well-known method (for example, “Special Issue on Clinical Pathology No. 53 Special Issue on Clinical Pathology, Immunoassay Techniques and Applications for Clinical Examination” edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publications, 1983, edited by Yuji Ishikawa et al. Measurement method ", 3rd edition, Medical School, 1987, edited by Tsuneaki Kitagawa et al.," Protein Enzyme Nucleic Acid Separate Volume No.31 Enzyme Immunoassay ", Kyoritsu Shuppan, 1987, etc.). In addition, 8-nitro cGMP can also be measured from a sample containing cultured cells using the antibody of the present invention. However, the measurement method and measurement target of 8-nitro cGMP using the antibody of the present invention are not limited to those exemplified above, and it goes without saying that those skilled in the art can appropriately select depending on the purpose. . Since specific measurement methods are shown in the examples of the present specification, a person skilled in the art can refer to the methods of the examples and add appropriate modifications or alterations to the methods as necessary to obtain biological samples. 8-nitro cGMP contained in can be easily and reliably measured.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
Example 1: Succinylation of 8-nitro cGMP
Triethylamine (0.177 ml, 1.28 mmol) and succinic anhydride (64.0 mg, 0.644 mmol) were sequentially added to an aqueous solution (1.5 ml) of 8-nitro cGMP (15.7 mg, 0.04 mmol) at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and lyophilized. The resulting yellow crude product was dissolved in water and purified by high performance liquid chromatography (Develosil RP AQUEOUS (Nomura Chemical), φ20 mm x 250 mm, methanol: water (0.1% TFA) = 30: 70) 11.5 mg (59% yield) of 2′-succinyl-8-nitrocyclic guanosine 3 ′, 5′-monophosphate (2′-succinyl-8-nitro cGMP) was obtained as a yellow solid.
MS (ESI, negative): Calculated value C ₁₄ H ₁₅ N ₆ O ₁₂ P ([MH] ⁻ ): 489.04, measured value 489.74
¹ H-NMR (400 MHz, DMSO-d ₆ ): δ: 2.50-2.67 (4H, m), 4.09 (1H, ddd, J = 4.6, 9.5 Hz), 4.27 (1H, t, J = 9.5 Hz), 4.49 (1H, m), 5.21 (1H, dd, J = 5.9, 9.5 Hz), 5.93 (1H, d, J = 5.9 Hz), 6.49 (1H, s), 7.11 (2H, br s), 11.4 ( 1H, s)

Example 2: Preparation of 8-nitro cGMP-conjugated bovine serum albumin (8-nitro cGMP-BSA)
10 mg / ml bovine serum albumin (BSA, Sigma) dissolved in 0.1 M 2- (n-morpholino) ethanesulfonic acid (MES; manufactured by Dojindo Chemical) (pH 5.5) and 4.7 mg / ml 2′-succinyl- Add 10 mg / ml of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (WSC; manufactured by Dojindo Chemical Co., Ltd.) 150 μl to the mixed solution of 300 μl of 8-nitro cGMP. And then centrifuged at 15,000 × g for 10 minutes at 4 ° C., and the supernatant was collected. The collected supernatant was dialyzed overnight at 4 ° C. with PBS. The obtained 8-nitro cGMP-BSA was subjected to protein quantification by the BCA method to obtain an antigen.

Example 3: Production of rabbit polyclonal antibody
8-Nitro cGMP-BSA was dissolved in PBS to a protein concentration of 1 mg / ml. An emulsion was prepared by taking 1 ml of this solution into a syringe, taking another 1 ml of Freund's complete adjuvant, connecting it to another syringe, and moving it alternately. Two Japanese white rabbits were employed as animals for antibody production, and this emulsion was injected subcutaneously in the back of the rabbits at several locations (primary immunization). Two weeks later, 1 ml of the antigen of Example 1 dissolved in PBS to 1 mg / ml and 1 ml of Freund's incomplete adjuvant were used, and an emulsion was prepared by moving the syringe alternately as described above. This was then injected for booster immunization. This boost was repeated every 2 weeks. A small amount of blood was collected one week after each of the second and third immunizations, and it was confirmed by dot blotting whether the antibody reacted with 8-nitro cGMP-BSA. The interval from the first immunization to the booster immunity and the interval between the booster immunization and the booster immunization are both 2 weeks in this example, but the booster immunization is not limited to such an interval. However, the first immunization is for the production of antibodies, and usually the antibody appears in the blood 4 to 5 days after the first injection of the antigen, and the amount gradually increases and peaks around 10 days. Therefore, it is preferable that the period from the first immunization to the first booster be 2 weeks or longer. In addition, booster immunization is carried out in order to make a large amount of the antibody. However, since the antibody does not increase immediately after booster immunization, blood is collected after one week after booster immunization, and after one week after blood collection. The next booster immunization was decided.

When the production of the polyclonal antibody becomes constant as a result of the above confirmation (where the change in the production of the polyclonal antibody no longer occurs compared to 2 weeks ago, that is, the production of the antibody confirmed 1 week after a certain booster immunization) , Where the production of antibodies confirmed one week after the next booster immunization performed two weeks after the booster was the same), blood was collected from two rabbits, and serum was obtained from this blood. The polyclonal antibody of the present invention was obtained from the serum.
The step of obtaining the polyclonal antibody of the present invention from the blood of the Japanese white rabbit immunized as described above was performed as follows. First, a large amount of blood was collected from the heart of a Japanese white rabbit, kept at 37 ° C. for 1 hour, and then allowed to stand at 4 ° C. overnight. This was centrifuged at 3,000 rpm for 10 minutes to obtain a supernatant. Thereafter, the antibody was purified using an affinity column described below.

  First, the antibody was fractionated with a saturated ammonium sulfate solution. The antibody solution after ammonium sulfate precipitation was dissolved in 10 mM Tris-HCl (pH 7.0) and 10 mM NaCl, and dialyzed overnight against the same buffer. Antibody protein was purified from 2 ml of this serum solution using melon gel kit (PIERCE). Specifically, 100 μl of the antibody solution was dispensed onto a spin column with 500 μl of the gel, and the flow-through fraction was collected by centrifugation, and 1.6 ml of the 5.4 mg / ml IgG fraction was collected. Furthermore, a BSA binding column in which bovine serum albumin (BSA) was bound to HiTrap NHS-activated HP (Amersham Biosciences) (1 ml volume) was prepared according to the company's protocol, and 1 ml of the IgG fraction was passed through the column. The anti-BSA antibody mixed in the polyclonal antibody was removed by adsorption. In addition, a column with cGMP-BSA was prepared using Activated Thiol Sepharose 4B (Amersham Biosciences) according to the protocol of the company using the SH group of BSA. The anti-cGMP-BSA antibody mixed in was adsorbed and removed.

  Since the polyclonal antibody for detecting 8-nitro cGMP of the present invention is contained in the serum obtained by coagulating the blood of the immunized Japanese white rabbit, the polyclonal antibody of the present invention is selected from the serum. The process of obtaining is not limited to that described above. However, when affinity purification is performed as in the above method, an antibody that strongly recognizes 8-nitro cGMP can be selected from antibodies having various characteristics, and the added value can be further increased. is there. In addition, various components such as albumin contained in the serum other than the antibody can be removed, and there is an advantage that an experimental result with a low background can be obtained.

Example 4: Dot blotting
Dots with cGMP-BSA and 8-nitro cGMP-BSA as the antigen as proof that an antibody that recognizes 8-nitro cGMP (hereinafter sometimes referred to as "anti-8-nitro cGMP antibody") is produced. Blotting was performed. Specifically, cGMP-BSA 0.3 μg / drop or 8-nitro cGMP-BSA 0.3 μg / drop as an antigen protein was adsorbed on a polyvinylidene difluoride (PVDF) membrane (Millipore), 5% skim milk (Becton Dickinson), Blocking was performed at room temperature for 1 hour using 0.1% Tween 20, 0.9% NaCl, 100 mM Tris-HCl (pH 7.5) (hereinafter, blocking buffer). Subsequently, the mixture was reacted overnight at 4 ° C. with 1 ml of anti-8-nitro cGMP antibody diluted to 1 μg / ml with blocking buffer. After the reaction, the membrane was washed 3 times with a blocking buffer, reacted with an HRP-labeled anti-rabbit goat antibody (manufactured by Santa Cruz Biotechnology) diluted 3,000 times with the same buffer at room temperature for 1 hour, and then the membrane was twice with a blocking buffer and further 0.1% Washed twice with Tween20, 0.9% NaCl, 100 mM Tris-HCl (pH 7.5). Detection was performed by chemiluminescence using an ECL plus Western Blotting Detection System (Amersham Biosciences), and exposed to Hyperfilm ECL (Amersham Biosciences) for 2 seconds. The film was developed with FPM800A (manufactured by FUJI FILM). The results are shown in FIG. It was confirmed that the purified antibody was an antibody that specifically recognizes only 8-nitro cGMP.

Example 5: Preparation of mouse monoclonal antibody The antigen obtained in Example 1 was dissolved in PBS so as to be 1 mg / ml. An emulsion was prepared by taking 150 μl of this solution and 350 μl of physiological saline into a syringe, further taking 500 μl of Freund's complete adjuvant, connecting it to another syringe, and moving this alternately. Three female 5-week-old BALB / c mice were employed as antibodies for antibody production, and this emulsion was injected into the back skin of the mice at several locations (primary immunization). Three weeks later, 1 mg / ml, 90 μl of the same antigen, 410 μl of physiological saline and 500 μl of Freund's incomplete adjuvant were used to make an emulsion by alternately moving the syringe as described above. It was. This boost was repeated every week. Seven weeks after the first immunization, a small amount of blood was collected from the tail of the mouse, and the production of the antibody was confirmed by ELISA against 8-nitro cGMP-BSA. In this example, the interval from the first immunization to the booster immunity, and the interval between the booster immunization and the booster immunization was set to two weeks and one week. Absent.

  When the production of the monoclonal antibody became constant by the above confirmation, 50 μg / animal of 8-nitro cGMP-BSA was injected intraperitoneally. Three days later, the spleen of the corresponding mouse was removed. Spleen cells were mixed with mouse myeloma cells P3U1 and cell fusion was performed with polyethylene glycol to prepare hybridomas. The obtained hybridoma was cultured in RPMI medium (HAT medium) containing 10% fetal bovine serum containing aminopterin, hypoxanthine and thymidine. After 7 days, screening was performed using the culture supernatant. As a screening method, 24-cells positive for 8-nitro cGMP-BSA and negative for cGMP-BSA and BSA were selected. Further, the cells were cultured in RPMI medium for 10 days, and the screening was repeated. Subcloning by the limiting dilution method was performed to establish a monoclonal antibody producing cell line. These antibody-producing cells were transplanted into the peritoneal cavity of BALB / c mice that had been previously immunosuppressed with pristane. The obtained ascites was fractionated and purified with 10 mM Tris-HCl (pH 8.0) containing 50 to 100 mM sodium chloride using diethylaminoethyl cellulose ion exchange resin to obtain the monoclonal antibody of the present invention.

Example 6: Direct ELISA using monoclonal antibodies
8-Nitro cGMP-BSA, cGMP-BSA or 8-Nitroguanosine-BSA are each dissolved in 50 mM carbonate buffer (pH 9.7) to a concentration of 5 μg / ml. The plate was placed in each well and allowed to stand at room temperature for 1 hour to immobilize. After washing with 0.4 ml of PBS containing 0.05% Tween 20 (PBS-T) three times, 0.2 ml of 50 mM carbonate buffer (pH 9.7) containing 0.5% gelatin was added to each well and left at room temperature for 1 hour. Blocked to prevent subsequent non-specific adsorption. Each well was washed 3 times with PBS-T, and 0.1 ml of an anti-8-nitro cGMP antibody-producing cell culture supernatant was added and incubated at room temperature for 1 hour. Wash 3 times with PBS-T, add 0.1 ml of HRP-labeled anti-mouse goat IgG antibody diluted 5,000 times with PBS-T and let stand for another 1 hour, then wash wells 4 times with PBS-T Color was developed with 0.1 ml of 1,2-phenylenediamine (0.55 mg / ml) containing 0.6 μl of hydrogen oxide water. Color development was stopped with 0.1 ml of 1 mol / l sulfuric acid, and absorbance at 490 nm was measured with an ELISA plate reader.

  The results are shown in FIG. The monoclonal antibody of the present invention reacted strongly with 8-nitro cGMP and did not react with 8-nitroguanosine and cGMP at all. By this test, the monoclonal antibody of the present invention reacts only with 8-nitro cGMP, does not react with normal nucleobases such as cGMP, and further reacts with 8-nitroguanosine which is only the nucleobase site of 8-nitro cGMP. It was shown not to. In addition, since the monoclonal antibody of the present invention reacts only with 8-nitro cGMP in which a cyclic phosphate structure is bound to 8-nitroguanosine, it recognizes both the 8-nitroguanosine site and the cyclic phosphate structure simultaneously. Was confirmed.

Example 7: Competitive ELISA method using monoclonal antibody Next, the competitive ELISA method was used to examine that the recognition structure (epitope) of this antibody was 8-nitro cGMP by cross-reaction with an 8-nitro cGMP analog. . In the competitive ELISA method, it is not necessary to immobilize the test substance on the protein, and it is possible to detect the presence of antigen-antibody reaction by adding the low-molecular test substance as it is to the ELISA system, so it usually binds to the protein. However, it is possible to examine only a difficult nucleobase structure and to detect the presence or absence of a cross-reaction to a more detailed similar structure. In addition to 8-nitro cGMP, cAMP, nitroxanthine, guanosine, and 8-nitroguanosine were used as competitors as test substances.

  In the same manner as described in Example 6, 100 μl of 8-nitro cGMP-BSA 1 μg / ml solution was added to the ELISA plate and allowed to stand at room temperature for 2 hours to be solid-phased. After washing three times with 0.4 ml of PBS containing 0.05% Tween 20 (PBS-T), 200 μl of 50 mM carbonate buffer (pH 9.7) containing 0.5% gelatin was added to each well and allowed to stand at room temperature for 1 hour. Were blocked to prevent nonspecific antibody adsorption. Each well was washed 3 times with PBS-T, and 0.05 ml of the culture supernatant of anti-8-nitro cGMP antibody-producing cells and 0.05 ml of the competitor dissolved in PBS-T at various concentrations were mixed in advance. After stirring at room temperature for 1 hour, 100 μl each was added and allowed to stand at room temperature for 1 hour. The wells were similarly washed 3 times with PBS-T, 100 μl of HRP-labeled anti-mouse goat IgG antibody diluted 5,000 times with PBS-T was added, and the mixture was allowed to stand for another 1 hour, and then containing 0.6 μl of hydrogen peroxide solution 1 The color was developed with 100 μl of 2-phenylenediamine 0.55 mg / ml citrate-potassium phosphate buffer (pH 5.0). Color development was stopped by adding 100 μl of 1 mol / l sulfuric acid, and absorbance at 490 nm was measured with an ELISA plate reader. As a result, the monoclonal antibody of the present invention did not react at all with cAMP, guanosine, nitroxanthine, and 8-nitroguanosine. By this test, the monoclonal antibody of the present invention reacts only with 8-nitro cGMP, does not react with the existing nucleic acids guanosine and cAMP, and is structurally similar in part to the chemical structure of 8-nitro cGMP. It was confirmed that there was no cross-reactivity with similar nitroxanthine and 8-nitroguanosine.

It is the figure which showed the result of the dot blotting using the polyclonal anti-8-nitro cGMP antibody. (A) cGMP-BSA or (B) 8-nitro cGMP-BSA was immobilized at 0.3 μg / drop on the PVDF membrane, and the reactivity was confirmed using a polyclonal anti-8-nitro cGMP antibody (1 μg / ml). FIG. 5 shows the reactivity of monoclonal anti-8-nitro cGMP antibody. The results of confirming the reactivity using 8-nitro cGMP-BSA, cGMP-BSA, or 8-nitroguanosine-BSA immobilized on a 96-well microtiter plate and using the culture supernatant of monoclonal anti-8-nitro cGMP antibody-producing cells. Indicated.

Claims (7)

It specifically recognizes 8-nitrocyclic guanosine 3 ′, 5′-monophosphate represented by the following formula, but does not recognize 8-nitro-guanosine and cyclic guanosine 3 ′, 5′-monophosphate. antibody.
The antibody according to claim 1, which is a polyclonal antibody. The antibody according to claim 1, which is a monoclonal antibody. A method for enzymatically measuring 8-nitrocyclic guanosine 3 ', 5'-monophosphate contained in a sample using the antibody according to any one of claims 1 to 3. The method includes the step of immunizing a non-human mammal using an antigen in which a hydroxyl group of a sugar moiety of 8-nitrocyclic guanosine 3 ', 5'-monophosphate and a serum protein are bound via a carbon chain. 4. A method for producing the antibody according to any one of items 3 to 3. The method according to claim 5, wherein the serum protein is albumin. The method according to claim 6, wherein the serum protein is bovine serum albumin.