JPH11140098A - Method for producing anti-peptide antibody - Google Patents

Abstract

(57) [Summary] (Corrected) [PROBLEMS] To provide a method for producing an antibody capable of immunochemically distinguishing between a target protein and a protein having an amino acid sequence similar to the amino acid sequence of the protein. (1) An amino acid sequence which is a partial amino acid sequence of a target protein, which is composed of 10 to 15 amino acid residues and which does not contain at least three adjacent amino acid residues which coincide with the amino acid sequence of the protein to be discriminated And select
(2) An amino acid sequence corresponding to a region exposed on the surface of the molecular model of the target protein is selected from the amino acid sequences selected in (1) above, and (3) an amino acid sequence selected in (2) above. Among them, an amino acid sequence having low homology as compared with a known amino acid sequence of a protein derived from an animal to be immunized is selected, and (4) a peptide having the amino acid sequence selected in (3) above and a carrier are selected. A method for producing an anti-peptide antibody, comprising the steps of: preparing a conjugated effective immunization antigen; and administering the effective immunization antigen to immunize an animal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an anti-peptide antibody. More specifically, the present invention relates to a method for producing an anti-peptide antibody capable of immunochemically distinguishing a target protein from a protein having an amino acid sequence similar to the amino acid sequence of the protein.

[0002]

2. Description of the Related Art In the case of a protein having a similar function, for example, an enzyme protein composed of a number of molecular species having overlapping substrate specificities such as cytochrome P450, the amino acid sequence is generally different between the molecular species. Similar. Examining the distribution and abundance of proteins having similar functions and amino acid sequences in living organisms, for example, clarifies the role of each protein in living organisms, and examines the metabolism, effects,
It is important for predicting and evaluating toxicity. In order to examine the distribution and abundance of each of the proteins having similar functions and amino acid sequences, it is necessary to have a means for discriminating between these proteins. For example, specific means for specifically recognizing specific proteins among these proteins Antibodies that can be used are used. In order to produce an antibody capable of specifically recognizing a specific protein among proteins having similar functions and amino acid sequences as described above, a high-purity protein sample containing no contaminating other similar proteins is used as an antigen. Although it is necessary to use it, it is very difficult to separate and purify proteins having similar functions and amino acid sequences to each other to a high degree of purity using ordinary biochemical techniques. Therefore, recombinant DN
A technology is used to express a gene of a target protein in a host cell which does not naturally express a protein having an amino acid sequence similar to the target protein at a high level, and purify the target protein from the host cell, thereby contaminating other similar proteins. By preparing a large amount of high-purity protein preparation without lipase and immunizing animals with this as an antigen,
An antibody that specifically recognizes the target protein can be produced.

[0003]

However, depending on the protein, the efficiency of expression of the target protein gene in the host cell is low, the instability of the target protein in the host cell, the adverse effect of the target protein on the host cell, and the like. In some cases, the above-described recombinant DNA technology cannot always be used, and it has been desired to develop a new method for producing an antibody that specifically recognizes a target protein.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in view of such circumstances, and as a result, have found a method for preparing an antigen used for producing an antibody that specifically recognizes a target protein, leading to the present invention. . That is, the present invention relates to a method for producing an anti-peptide antibody capable of immunochemically discriminating between a target protein and a protein having an amino acid sequence similar to the amino acid sequence of the protein, wherein (1) an amino acid sequence of the target protein; By comparing the amino acid sequence of the protein to be discriminated with the protein, a partial amino acid sequence of the target protein, consisting of 10 to 15 amino acid residues, which matches the amino acid sequence of the protein to be discriminated is determined. An amino acid sequence that does not contain at least three adjacent residues is selected. (2) Among the amino acid sequences selected in (1), an amino acid sequence corresponding to a region exposed on the surface of the molecular model of the target protein is selected. ,
(3) Among the amino acid sequences selected in (2) above, an amino acid sequence having low homology as compared with a known amino acid sequence of a protein derived from an animal to be immunized is selected,
(4) An anti-peptide comprising a step of preparing an effective immunizing antigen comprising a peptide having the amino acid sequence selected in the above (3) and a carrier, and immunizing an animal by administering the effective immunizing antigen. It is intended to provide a method for producing an antibody (hereinafter referred to as the production method of the present invention) and an effective immunizing antigen used in the production method.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

[0006] In the production method of the present invention, first, the amino acid sequence of the protein of interest is compared with the amino acid sequence of the protein to be discriminated from the protein.
An amino acid sequence consisting of 10 to 15 amino acid residues, which is a partial amino acid sequence of the target protein, and adjacent to and containing no more than 3 amino acid residues that match the amino acid sequence of the protein to be discriminated is selected. To compare amino acid sequences, for example, an alignment of the amino acid sequences of the proteins to be compared may be created, and it may be determined whether the corresponding amino acid residues in each amino acid sequence are the same. The alignment of the amino acid sequence can be created using commercially available computer software capable of analyzing the amino acid sequence, such as Genetyx (manufactured by Software Development Co., Ltd.). Based on the results of such comparison, the partial amino acid sequence of the target protein
An amino acid sequence consisting of 10 to 15 amino acid residues and having no adjacent 3 or more amino acid residues that match the amino acid sequence to be determined is selected.

Next, an amino acid sequence corresponding to a region exposed on the surface of the molecular model of the target protein is selected from the amino acid sequences selected in the previous step. To select an amino acid sequence corresponding to a region exposed on the surface of the molecular model of the target protein, for example, a molecular model of the target protein is constructed on a computer using a homology modeling technique, and the constructed molecular model A region exposed on the surface of the above may be specified, and an amino acid sequence included in the specified region may be selected from the amino acid sequences selected in the above step. In order to construct a molecular model on a computer, for example, MSI (molecular
simulations) Quanta / CHARMm Pr
Software such as otein Pkg can be used.

Next, by comparing the amino acid sequence selected in the previous step with the known amino acid sequence of a protein derived from an animal to be immunized, the compared amino acid sequence of the amino acid sequences selected in the previous step is compared with the amino acid sequence selected in the previous step. Select an amino acid sequence with low homology to To perform such a comparison, for example, the homology of the amino acid sequence to be compared may be calculated using a computer program for homology search. Based on this calculation result, an amino acid sequence showing about 50% or less homology with the compared amino acid sequence may be selected. As a homology search program, using commercially available software such as FASTA and BRASTA, for example, EMBL, PIR, DDBJ,
A search can be performed on a protein amino acid sequence database publicly disclosed in SWISS-PROT or NCBI or the like. Here, when an amino acid sequence which does not contain at least 3 residues adjacent to an amino acid residue corresponding to a known amino acid sequence of a protein derived from an animal to be immunized is selected,
It is more advantageous to produce antibodies.

Further, a peptide having the amino acid sequence selected in the preceding step is chemically synthesized to prepare an effective immunizing antigen comprising the synthetic peptide and a carrier bound thereto, and the effective immunizing antigen is administered to immunize an animal. Sensitize. Here, the carrier is a high-molecular substance that can be used to prepare an antigen effective for immunization by binding to a peptide, and specifically, a carrier protein described later, polylysine having a branched structure. Examples include peptides and the like. As a method for chemically synthesizing a peptide having a selected amino acid sequence and preparing an effective immunizing antigen obtained by combining the synthetic peptide with a carrier, for example, chemically synthesizing a peptide having a selected amino acid sequence, A method for binding to a protein, having a selected amino acid sequence at the amino terminus of a polylysine peptide having a branched structure formed by bonding the carboxyl group of the next lysine residue to the α and ε amino groups of the lysine residue Examples include a method of chemically synthesizing a multi-antigen peptide in which the carboxyl terminus of the peptide is bound.
For chemical synthesis of peptides, a solid phase synthesis method (E. Ath.
erton & RC Sheppard "Solid phase peptide synthe
sis: a practical approach "IRL PRESS at Oxford Un
iversity Press 1989 D. Richwood, BD Hames p25
-161) or the like can be used. Also, commercially available peptide synthesizers, for example,
By using Biolynx manufactured by Pharmacia LKB or PSSM-8 type manufactured by Shimadzu Corporation, peptides can be synthesized in a solid phase. In order to prepare an effective immunizing antigen using the synthesized peptide, the synthetic peptide is bound to a carrier protein. As the carrier protein, for example, albumin, myoglobin, hemocyanin and the like can be used. The binding between the synthetic peptide and the carrier protein is described, for example, by Jean-phlippe Defoort et al., (1992) P
Acad. Sci. USA, 89, 3879-3889, and the like. Further, for chemically synthesizing a multi-antigen peptide, for example, Tam JP, (1988) ProcN
atl. Acad. Sci. USA, 85, 5409-5413, David N. Posne
tt et al., (1988) JBC, 263 (4), 1719-1725, James
s P. Tam et al., (1989) J. Immumo. Methods. 124 53
Peptide synthesis can be performed according to the MAP synthesis method described in -61 and the like. Specifically, upon solid-phase synthesis of a peptide, lysine is bound to a resin, and α and ε
A polylysine peptide having a branched structure is synthesized by repeating the step of bonding the carboxyl group of the next lysine to the amino group. For example, when the step of bonding the carboxyl group of lysine to the α and ε amino groups of lysine is repeated twice, four branches are formed, and when the step is repeated three times, eight branched structures are formed. Next, a polyantigen peptide is synthesized by sequentially binding amino acids constituting the selected amino acid sequence to the α- and ε-amino groups of the lysine residues at the amino terminus of the polylysine peptide having the branched structure from the carboxyl terminus side. be able to. For the synthesis of such a multi-antigen peptide, a commercially available MAP resin (having a branched structure) such as TAKO8-WTG (Shimadzu Corporation) may be used. The multi-antigen peptide synthesized in this manner can be used as an effective immunizing antigen without further binding to a carrier protein.

The target protein is cytochrome P derived from human.
In the case of 2C8, which is a molecular species belonging to the 450 2C family, for example, an effective immunizing antigen comprising a peptide having the amino acid sequence represented by SEQ ID NO: 1 and a carrier may be prepared and administered to an animal. Similarly, when the target protein is 2C9 which is a molecular species belonging to the 2C family of human-derived cytochrome P450, for example, an effective immunizing antigen to which a peptide having an amino acid sequence represented by SEQ ID NO: 2 is bound is used as 2C18. In the case of, for example, an effective immunizing antigen to which a peptide having the amino acid sequence represented by SEQ ID NO: 3 is bound, and in the case of 2C19, for example, a peptide having the amino acid sequence represented by SEQ ID NO: 4 is bound An effective immunizing antigen may be prepared and administered to an animal. When the target protein is 1A1 which is a molecular species belonging to the 1A family of human-derived cytochrome P450, for example, an effective peptide having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOS: 5 to 7 is bound. An immunizing antigen may be prepared. Either an effective immunizing antigen obtained by binding a peptide having the amino acid sequence represented by SEQ ID NO: 5, 6 or 7 to a carrier may be administered to an animal alone, or a mixture of these may be administered as appropriate. Good. In addition, 2 having an amino acid sequence selected from the amino acid sequences represented by SEQ ID NOS: 5 to 7
An effective immunizing antigen obtained by combining a species or three kinds of peptides with a carrier may be prepared and administered to an animal.

The desired antibody can be produced by immunizing an animal using the thus prepared effective immunizing antigen as an antigen. Examples of the animal to be immunized include mammals such as mice, hamsters, guinea pigs, rats, rabbits, dogs, goats, and birds such as chickens. To immunize these animals, for example, J .
The antigen is administered one or more times using the usual method described in ASSOC.OFF.ANAL.CHEM 70 (6) 1025-1027 (1987).
For example, every 2 to 7 to 30 days, preferably 10 to 21 days
Administer ~ 3 times. Each dose is, for example, about 0.05 to about 2 mg of the antigen. The administration route can be selected from subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like. Intravenous, intraperitoneal or subcutaneous injection is a preferable administration form. In this case, the antigen is prepared using an appropriate buffer such as CFA (Complete Freu).
nd's Adjuvant), RAS (MPL (Monophosphoryl Lipid
A) + TDM (Synthetic Trehalose Dicorynomycolate) + CWS (C
ell Wall Skeleton) adjuvant system], sodium phosphate buffer containing one kind of commonly used adjuvants such as aluminum hydroxide, dissolved in physiological saline, etc., and used depending on the administration route and conditions. Adjuvants such as those described above may not be used. Here, an adjuvant means a substance that, when administered together with an antigen, nonspecifically enhances an immune response to the antigen. After breeding the animal to which the antigen has been administered for 0.5 to 3 months, a small amount of blood of the animal is sampled from a vein or the like, and the antibody titer is measured. When the antibody titer increases, the antigen is further administered an appropriate number of times depending on the situation. For example, 0.1 μg to 1 μg, preferably 0.05 μg
The antigen is administered in a dose of g-0.5 mg. One to two months after the last dose, blood is collected from the animal by conventional methods, and the blood is subjected to, for example, centrifugation, precipitation by using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography, An antibody can be obtained as a polyclonal antiserum by separating and purifying by ordinary methods such as chromatography such as affinity chromatography. The serum may, for example, be treated at 56 ° C. for 30 minutes to effect inactivation of the complement system. In addition, immunocompetent B cells are isolated from the immunized animal, the immunocompetent B cells are fused with tumor cells capable of continuous cell division, the resulting fusion is isolated, and after selection, An anti-monoclonal antibody having a high degree of specificity and affinity by cloning the hybridoma cells producing the desired peptide antibody and culturing the hybridoma cells in vitro or in vivo to produce a monoclonal antibody Can also be manufactured.

The antibody thus produced (hereinafter referred to as the present antibody) can immunochemically discriminate a target protein from a protein having an amino acid sequence similar to the amino acid sequence of the protein. For a protein having an amino acid sequence similar to the amino acid sequence of the protein of interest, selective recognition such that the cross-reactivity is less than about 1/200 is substantially possible. Here, the degree of cross-reactivity is determined, for example, assuming that the lowest protein concentration of a target protein that can be detected by an immunoblot method described later is 1, and the lowest concentration at which a similar protein is detected by the same method is C. If there is, it is determined as 1 / C.

Use of the present antibody includes a method for immunologically detecting a target protein, for example, a method for detecting the present antibody bound to the target protein or an antibody against the antibody using a label of the antibody. Can be. Specific examples include a method of analyzing a target protein by immunoblotting, separation by immunoprecipitation, ELISA, immunological staining of cells or tissues, and the like. These methods can be performed, for example, by the following procedure.

I. Immunoblot method This is a method in which a target protein present in a sample bound to a solid support material is recognized by the present antibody, and the present antibody bound to the target protein is detected. Nitrocellulose in the form of a membrane, sheet, filter, etc. is generally used as the solid support material to bind the sample, but it absorbs the sample well and loses the antigenicity of the target protein present in the sample There is no particular limitation as long as it is not allowed. When nitrocellulose is used, to bind the sample to nitrocellulose, a solution obtained by dissolving the sample in an appropriate buffer such as phosphate buffered saline is spotted on the nitrocellulose. The amount of the spot for quantification is desirably such that the amount of the present antibody with respect to the target protein as the antigen becomes excessive, and the solution volume is 1 μl / 3 m
About m square is good. Spotted in this manner, and the nitrocellulose is incubated for about 1 minute to about 24 hours under appropriate humidity conditions. After the incubation, in order to prevent non-specific adsorption to sites other than the spotted site, the solid support material to which the sample is attached is a high-molecular-weight carrier molecule that is not recognized by the antibody, that is, a high-molecular-weight carrier molecule that is not used in the production of the antibody. A solution containing molecular weight carrier molecules (serum albumin or skim milk of another animal such as goat, cow, etc.) and about 2
Cover the surface of the solid support with the high molecular weight carrier molecule by incubating at room temperature for 0 minutes to about 24 hours. After incubation, the resulting solid support is thoroughly washed to remove the free high molecular weight carrier molecules. After mixing the solid support thus prepared with the preparation containing the present antibody, the mixture is incubated with shaking for about 10 minutes to about 3 hours. The preparation containing the antibody is
It refers to a preparation solution in which the present antibody can be present in a free state in a solution such as distilled water, buffer solution, or physiological saline. Thus, the present antibody is bound to the target protein contained in the sample. Next, a method for detecting the present antibody bound to the target protein will be described. The present antibody is labeled in advance with an enzyme such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase. In this case, after incubating the preparation solution containing the present antibody and the solid support material, the free antibody is removed by washing, and then the substrate of the above-mentioned labeling enzyme is allowed to act on the solid support material. By measuring the reaction by color development or the like, the present antibody bound to the solid support via the target protein can be detected. For example, when the present antibody is labeled with peroxidase, it becomes brown or yellow by using hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a coloring reagent. When the present antibody is labeled with glucose oxidase, the substrate may be, for example, 2,2′-
Acid-di- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or the like is used. For detection of the present antibody, a second antibody labeled with an enzyme or a radioisotope that recognizes the present antibody (first antibody) and binds to the present antibody is used.
When an antibody is used, the solid solution to which the sample is bound and the preparation solution containing the present antibody is incubated, and then the free present antibody (first antibody) is removed by washing. The material is incubated with the second antibody. Examples of the second antibody labeled with this enzyme or the like include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase and the like. And an antibody capable of recognizing the present antibody (first antibody) to which the above enzyme has been bound. As a specific example, the present antibody (first antibody)
When using rabbit serum as the second antibody,
Anti-rabbit immunoglobulin (IgG) and goat immunoglobulin (IgG) conjugated with alkaline phosphatase can be used. The anti-rabbit IgG goat IgG is commercially available. The detection of the second antibody labeled with an enzyme or the like can be performed in the same manner as in the case of the labeled present antibody (first antibody).

II. Separation method by immunoprecipitation The present antibody is bound to the target protein present in the sample to form an immune complex composed of the present antibody and the target protein, and the immunoprotein is separated from the sample to separate the target protein from the sample. The target protein in the sample is detected by separating and detecting the separated target protein using a method such as gel electrophoresis, enzyme activity measurement, or immunoblot method. First, an immune complex is formed by mixing a sample and the present antibody against the target protein with stirring at 4 ° C. for, for example, about 1 hour to about 24 hours. The mixing ratio between the sample and the present antibody at this time can be, for example, about 1: 8, and is appropriately increased or decreased depending on the amount of the target protein present in the sample and the antibody titer of the present antibody. Next, to the formed immune complex, a secondary reagent capable of specifically binding to the antibody and separating the immune complex consisting of the antibody and the target protein bound to the antibody from the solution is added, By incubating this mixture, a complex consisting of the immune complex and the secondary reagent is formed and collected.
Here, examples of the secondary reagent include bacterial cell wall proteins that bind to antibodies such as protein A and protein G, and anti-immunoglobulin antibodies. Note that these 2
If a secondary reagent is used which is bound to an insoluble support material, it is extremely easy to recover and wash the complex comprising the immune complex and the secondary reagent. Insoluble supports have a very wide range of designs and can have very different shapes depending on the particular purpose intended in use.
For example, beads, dishes, spheres, plates, small rods, cells, small bottles, small tubes, fibers, nets and the like can be mentioned. Specific examples include beads made of polysaccharides such as agarose (eg, sepharose, biogel, etc.) and transparent plastic materials, such as microtiter plates made of polyvinyl chloride or polystyrene, small spheres made of polystyrene and polystyrene latex, and tubes. Alternatively, a rod or the like can be used. Next, the target protein is released by subjecting the recovered complex to an operation such as heat treatment. The target protein in a free state may be detected by a method such as gel electrophoresis, enzyme activity measurement, or immunoblot.

III. ELISA method For example, the amount of free target protein contained in a sample is determined based on the competition of the target protein sample bound to the solid support material with the free target protein to be measured contained in the sample for the present antibody. Can be measured. The target protein is directly or indirectly bound to the solid support. Solid supports include, for example, plastic surfaces of microtiter plates or test tubes, polystyrene,
Surfaces of beads made of polypropylene, polyvinyl chloride, glass or plastic, etc., surfaces of filter paper, strips of dextran, cellulose or nitrocellulose or other similar materials, and the like can be mentioned. A specific example of the solid support material is a 96-well microtiter plate made of polystyrene. In order to directly or indirectly bind the target protein sample to these solid supports (hereinafter referred to as coating), for example, the solid supports are preliminarily prepared by a usual method using glutaraldehyde or cyanogen bromide. Is activated. Next, the activated solid support material is incubated with a solution containing the target protein (hereinafter, referred to as a coating solution) for a certain period of time to perform coating. Here, examples of the coating liquid include about 10 mM phosphate buffer (pH 7.4) containing 140 mM sodium chloride. As the conditions for coating, for example, the concentration of the target protein in the coating solution is preferably about 0.05 μg / ml to about 1 μg / ml, and the coating time is, for example, about 6 hours to 24 hours.
I can give time. When the present antibody is indirectly bound to the solid support, the solid support and the present antibody may be bound via a spacer and / or a high molecular weight carrier molecule that is not recognized by the antibody. Here, the high molecular weight carrier molecule that is not recognized by the present antibody is a high molecular weight carrier molecule excluding the carrier protein used in the production of the present antibody. The solid support to which the target protein thus obtained is directly or indirectly bound is mixed with a sample containing the target protein to be measured and a test solution containing the present antibody, and the mixture is incubated. After incubating for about 10 minutes to about 2 hours, the test solution is removed and the solid support is washed, and the solid support is then washed with a second antibody that recognizes the present antibody (first antibody) and is labeled with an enzyme or the like. Incubate for about 10 minutes to about 2 hours. Examples of the second antibody that recognizes this antibody (first antibody) and is labeled with an enzyme include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, An antibody that recognizes the present antibody (first antibody), to which an enzyme such as rate dehydrogenase or glucose-6-phosphate dehydrogenase has been bound, can be mentioned. As a specific example, when rabbit serum is used as the present antibody (first antibody), peroxidase-conjugated anti-rabbit immunoglobulin (IgG) and goat immunoglobulin (IgG) can be used as the second antibody. . The anti-rabbit IgG goat IgG is commercially available. After the incubation, the second antibody is removed, and the solid support is washed. Then, the solid antibody is reacted with a coloring reagent to detect the second antibody bound to the solid support. When the second antibody is labeled with peroxidase, it reacts with hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a coloring reagent to give a brown or yellow color. When the second antibody is labeled with glucose oxidase, the substrate may be, for example, 2,
2'-Acid-di- (3-ethylbenzothiazoline-6
-Sulfonic acid) (ABTS) or the like. The method for measuring the amount of free target protein contained in a sample based on the competition between the target protein bound to the solid support material and the free target protein contained in the sample for the present antibody comprises the steps of: The higher the concentration of the protein of interest to be detected therein, the less the amount of the second antibody bound to the solid support. In order to measure the concentration of the target protein to be detected in the sample, a calibration curve may be prepared by performing a similar test in advance using a purified sample of the target protein. As an embodiment of the above method using the present antibody, a kit for immunological detection / analysis of a target protein can be mentioned. The test kit can contain, for example, the following components: (1) a solid support that binds the protein of interest directly or indirectly, (2) a reagent containing the antibody, (3) a label for the antibody. A reagent containing the second antibody, which is labeled with an enzyme or the like that recognizes and binds to the present antibody (first antibody). In addition, (4)
Standard solution of target protein, (5) Buffer solution, (6) Additives such as polypeptide and surfactant to prevent non-specific adsorption and formation of aggregates, and (7) Pipette, reaction vessel, calculation curve, etc. Should be included. The solid supports described above have a very wide range of designs and can have very different shapes depending on the particular purpose intended in use. For example, a dish, a sphere, a plate, a small rod, a cell, a small bottle, a small tube, a fiber, a net and the like can be mentioned. As a specific example, a microtiter plate made of a transparent plastic material such as polyvinyl chloride or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod, or the like can be used. On the other hand, the concentration of the target protein contained in the sample is measured based on the competition between the target protein to be measured contained in the sample and the target protein sample pre-labeled with the enzyme for a certain amount of the present antibody. Can also. When the amount of the target protein to be measured increases, the binding of the target protein labeled with the enzyme to the antibody is inhibited. In this case, a sample containing the target protein which is the antigen to be measured and the target protein labeled with the enzyme are simultaneously reacted with the present antibody bound to the solid support material. Next, the solid protein is washed to remove the target protein in a free state, and the activity of the labeling enzyme bound to the solid material is measured. The higher the concentration of the target protein to be measured in the sample, the lower the activity of the enzyme bound to the solid support. In another embodiment of this method, a target protein to be measured contained in a sample, a target protein sample previously labeled with an enzyme, and the present antibody are reacted in a solution, and the reaction product is bound to a solid support material. By reacting with the antibody (second antibody) recognizing the present antibody, the complex of the labeled target protein and the present antibody is bound to the second antibody bound to the solid support material,
After washing the solid support, there is also a method of measuring the activity of the label enzyme bound to the solid support.

IV. Immunological staining method for cells or tissues This is a method for recognizing target proteins present in fixed cells or tissue specimens with the present antibody and detecting the antibodies. Useful for checking your status. A cell or tissue to be examined is fixed, and a specimen that can be observed with an optical microscope or an electron microscope is prepared. The reagent used for fixing cells or tissues is not particularly limited as long as it is suitable for observation under a microscope and does not destroy the ability of the present antibody to recognize the target protein as an antigen. Examples include a 7% formalin solution, 100% methanol, 100% ethanol, a 4% paraformaldehyde solution, and a 1% glutaraldehyde solution. Then, the fixed cells or tissue specimen and the present antibody are allowed to stand at 4 ° C. for about 30 minutes to about 24 hours.
To bind the present antibody to the target protein present in the fixed cell or tissue specimen. Next, a method for detecting the present antibody bound to the target protein will be described. When the antibody is labeled with an enzyme such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase. After the incubation, the antibody of the present invention in a free state is removed by washing, and then the substrate of the above-mentioned labeling enzyme is allowed to act, and the antibody is detected by measuring the reaction by color development or the like. For example, when the present antibody is labeled with peroxidase, it becomes brown or yellow by using hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a coloring reagent. When the present antibody is labeled with glucose oxidase, as a substrate, for example, 2,
2'-Acid-di- (3-ethylbenzothiazoline-6
-Sulfonic acid) (ABTS) or the like. When a second antibody labeled with an enzyme or the like that recognizes and binds to the present antibody (first antibody) is used, the free antibody (first antibody) is removed by washing after incubation. And then incubate with the second antibody. Examples of the second antibody labeled with this enzyme or the like include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase and the like. And the antibody to the present antibody (first antibody) to which the above enzyme is bound. As a specific example, when rabbit serum is used as the present antibody (first antibody), peroxidase-conjugated anti-rabbit immunoglobulin (IgG) and goat immunoglobulin (IgG) can be used as the second antibody. . The anti-rabbit IgG goat IgG is commercially available. As the detection of the second antibody, the above labeled present antibody (first antibody)
The same method as in the above case can be used.

[0018]

Hereinafter, the present invention will be described with reference to examples.
The present invention is not limited to the following examples.

Example 1 2C8, 2C9, 2C18 and 2C, which are molecular species belonging to the human-derived cytochrome P4502C subfamily
A multiple alignment of the 19 amino acid sequences was made. Genetyx alignment software
Clastal V (manufactured by Software Development Co., Ltd.) was used. By comparing the amino acid sequences of the above molecular species,
For each molecular species, adjacent amino acid residues consisting of 10 to 15 amino acid residues and matching the amino acid sequence of other molecular species
A partial amino acid sequence containing no more than 3 residues was selected (Fig.
1). The amino acid sequences selected for each molecular species are listed below. 2C8: 1) Asn Leu Asn Thr Thr Ala Val Thr Lys Gly Ile Val Ser Leu Pro Pro Tyr Gln Ile Cys Phe, 2) Arg Gly Asn Ser Pro Ile Ser Gln Arg Ile Thr Lys Gly 2C9: 1) Ser Leu Glu Asn Thr Ala Val Asp Leu Phe Gly 2C18: 1) Ser Leu Ile Ala Thr Val Thr Asp Met Phe Gly, 2) Asn Lys Ile Ala Glu Asn Phe Ala Tyr Ile Lys Ser Tyr Val Leu Glu, 3) Lys Asp Ile Asp Ile Thr Pro Leu Pro Met His Leu Val Gly Val Pro 2C19: 1) Asn Leu Ala Phe Met Glu Ser Asp Ile Leu Glu, Asn Leu Val Ile Thr Ala Ala Asp Leu Leu Gly Human-derived cytochrome P4501A is a molecular species belonging to the subfamily. A multiple alignment of the amino acid sequences of 1A1 and 1A2 was made. As the alignment preparation software, clastal V of Genetyx (manufactured by Software Development Co., Ltd.) was used. By comparing the amino acid sequences of both molecular species, a partial amino acid sequence of 1A1 consisting of 10 to 15 amino acid residues and containing no more than 3 adjacent amino acid residues identical to the amino acid sequence of 1A2 was selected ( (Fig. 2). The selected amino acid sequences are listed below. 1A1: 1) Phe Gly Arg Arg Tyr Asp His Asn His Gln Glu Leu Leu, 2) Asp Ser Leu Ile Glu His Cys Gln Glu Lys Gln Leu Asp Glu Asn Ala Asn Val Gln Leu Ser Asp, 3) Leu Thr Pro Asp Gly Ala Ile Asp Lys Val Leu Ser, 4) Ser Leu Asn Ala Phe Lys Asp Leu Asn Glu Lys Phe Tyr Ser Phe Met Gln Lys Met Val Lys, 5) Asn Leu Asn Asn Asn Phe Gly Glu Val Val Gly Ser Gly

Example 2 Each cytochrome P whose amino acid sequence was compared in Example 1
X-ray crystal structure analysis of 450 molecular species has been performed and the molecular coordinates have been determined. Based on the structure of cytochrome P450cam (Protein Data Bank ID No. 2CPP), a molecular model is constructed on a computer using a homology modeling method. did. The software for molecular modeling for model building is Quanta / CHARMm's Protein Pkg (MSI)
It was used. A region exposed on the surface of the molecular model was identified from the constructed molecular model. Then, by selecting an amino acid sequence corresponding to the specified region from the amino acid sequences selected in Example 1, the amino acid sequence represented by SEQ ID NO: 1 in the case of cytochrome P4502C8, and the amino acid sequence represented by SEQ ID NO: 1 in the case of cytochrome P4502C9 Is the amino acid sequence represented by SEQ ID NO: 2, cytochrome P45
In the case of 02C18, the amino acid sequence represented by SEQ ID NO: 3, and in the case of cytochrome P4502C19, the amino acid sequence represented by SEQ ID NO: 4
And the cytochrome P4501
In the case of A1, the amino acid sequences represented by SEQ ID NOs: 5, 6, and 7 were selected.

Example 3 The partial amino acid sequence of each of the cytochrome P450 molecular species selected in Example 2 is subjected to immunization using the NCBI protein amino acid sequence database as a search target and using the homology search program FASTA. Mammals (rabbits)
The homology of the protein constitutively expressed with the known amino acid sequence was examined. As a result, no amino acid sequence of a rabbit protein showing more than 50% homology was found for any of the amino acid sequences. Thus, the peptide having the amino acid sequence selected in Example 2 was used for preparing an effective immunizing antigen.

Example 4 Synthesis of Effective Immune Antigen and Preparation of Antibody Using a Shimadzu PSSM-8 type peptide synthesizer, TAKO8-WTG (branched structure consisting of seven lysine residues) sold by Shimadzu Corporation as a MAP resin. And a total of 8 units of the antigen peptide can be introduced by solid-phase synthesis.) The amino acid of the third candidate of the antigenic antigen region in each cytochrome P450 molecular species determined in Example 3 as an effective immunizing antigen A multi-antigen peptide comprising eight peptides having the same amino acid sequence as the sequence was prepared. For preparing an antibody against human-derived cytochrome P4502C8, a multi-antigen peptide comprising a peptide having the amino acid sequence represented by SEQ ID NO: 1 is bound, and for preparing an antibody against 2C9, a peptide having the amino acid sequence represented by SEQ ID NO: 2 Is used to prepare an antibody against 2C18. A polyantigen peptide to which a peptide having the amino acid sequence represented by SEQ ID NO: 3 is bound is used to prepare an antibody against 2C18.
For the production of an antibody against C19, a multi-antigen peptide comprising a peptide having the amino acid sequence represented by SEQ ID NO: 4 bound thereto, and for the production of an antibody against 1A1, SEQ ID NO: 5,
Polyantigen peptides each having a peptide having the amino acid sequence shown in 6 or 7 bound thereto were synthesized. The synthesized multi-antigen peptide was applied to column PD-10 (Pharma
(cia Biotech) using simple gel filtration chromatography eluting with 2M acetic acid. The polyantigen peptide (effective immunizing antigen) of each cytochrome P450 molecular species thus prepared was dissolved in physiological saline to a concentration of 1 mg / ml. RAS [MPL (MoL) which was pre-incubated at 42 ° C to 43 ° C in 2 ml of this aqueous solution.
nophosphoryl Lipid A) + TDM (Synthetic Trehalose Dico
(rynomycolate) + CWS (CellWall Skeleton) adjuvant system] (Sigma) was added and mixed well. The resulting mixture was administered at a rate of 1 ml per New Zealand White rabbit (female, 14 weeks old, average 2.4 kg). The details were subcutaneously administered to 10 places on the back in an amount of 100 μl. Three and five weeks after the administration, the first half of the antigen was similarly administered. During this time, a small amount of blood was sampled from the rabbit ear vein and the antibody titer was measured. Since the antibody titer increased after the third administration, about 2 weeks after the third administration, whole blood was collected from the carotid artery of the immunized rabbit, and the obtained blood was placed in a separapit tube (manufactured by Sekisui Chemical Co., Ltd.). , And centrifuged (3000 rpm, 20 min, room temperature), and the supernatant was collected to obtain an antiserum. In addition, the antiserum was
Treatment was performed at 6 ° C. for 30 minutes to inactivate the complement system.

Test Example Analysis of Target Protein by Immunoblot Method Ten kinds of human cytochrome P450 molecular species (1A
2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C
19, 2D6, 2E1 and 3A4) versus Example 4
Immunoblotting was performed using the four types of antibodies 2C8, 2C9, 2C18, and 2C19 obtained in the above. In addition, 11 human cytochrome P450 molecular species including 1A1 (1A1, 1A2, 2A)
6, 2B6, 2C8, 2C9, 2C18, 2C19, 2
D6, 2E1 and 3A4) were subjected to immunoblotting using the 1A1 antibody obtained in Example 4. First, human cytochrome P4501A2, 2A6, 2
B6, 2C8, 2C9, 2C18, 2C19, 2D6,
0.5 pmol of each of 2E1 and 3A4 was electrophoresed on SDS-polyacrylamide gel (40 mA, 1 hour).
The gel was overlaid with a nitrocellulose membrane, immersed in a transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol), and treated with a Bio-Rad blotting device at 4 ° C, 20 mA for 15 hours. The electrophoresed protein was transferred to a nitrocellulose membrane. Four nitrocellulose membranes prepared by electrophoresing and transferring the same sample were prepared, and TBS + Tween20
Solution (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.05% Twee
n20), then wash with TBS + Tween20 containing 3% gelatin.
The solution was incubated at 37 ° C. for 40 minutes. Then
Each of the nitrocellulose membranes is TB containing 1% gelatin.
Reaction was carried out with an anti-2C8 antibody diluted 1000-fold, an anti-2C9 antibody diluted 200-fold, an anti-2C18 antibody diluted 400-fold or an anti-2C19 antibody diluted 400-fold with the S + Tween20 solution for 1 hour at 37 ° C. Also, 0.5 pmol of each of the above 10 molecular species and 1A1 was electrophoresed on an SDS-polyacrylamide gel (40 mA, 1 hour), and the electrophoresed protein was transferred to a nitrocellulose membrane in the same manner as described above. The membrane was reacted with a 400-fold diluted anti-1A1 antibody. After the reaction,
Each nitrocellulose membrane was washed four times with a TBS + Tween20 solution, and then washed in a TBS + Tween20 solution containing 3% gelatin.
Incubated at ℃ 20 for 20 minutes, and then ¹²⁵ I-labeled protein diluted 2500-fold with TBS + Tween 20 solution containing 1% gelatin
The reaction was carried out at 37 ° C. for 1 hour in A (Amersham). After the reaction, each nitrocellulose membrane was washed four times with TBS + Tween20, air-dried, and exposed to an imaging plate manufactured by Fuji Film for 15 hours.
A signal was detected in the system. The antibody against each molecular species obtained in Example 4 was a human-derived cytochrome P
Of the 450, only the target molecular species was specifically recognized (FIGS. 3 to 7).

[0024]

According to the present invention, it is possible to produce an antibody capable of immunochemically discriminating a target protein from a protein having an amino acid sequence similar to the amino acid sequence of the protein.

[0025]

[Sequence list]

<110> Sumitomo Chemical Co. Ltd. <120> Process for producing anti-peptide antibody <130> P149512 <150> JP 09/0218458 <151> 1997-08-13 <160> 7 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <210> 2 <211> 10 <212> PRT <213> Homo sapiens <210> 3 <211> 14 <212> PRT <213> Homo sapiens <400> 3 Asp Ile Asp Ile Thr Pro Leu Pro Met His Leu Val Gly Val 5 10 <210> 4 <211> 11 <212> PRT <213> Homo sapiens <210> 5 <211> 13 <212> PRT <213> Homo sapiens <400> 5 Phe Gly Arg Arg Tyr Asp His Asn His Gln Glu Leu Leu 5 10 <210> 6 <211> 15 <212> PRT <213 > Homo sapiens <400> 6 His Cys Gln Glu Lys Gln Leu Asp Glu Asn Ala Asn Val Gln Leu 5 10 15 <210> 7 <211> 12 <212> PRT <213> Homo sapiens

[Brief description of the drawings]

FIG. 1. Molecular species belonging to the human-derived cytochrome P4502C subfamily (2C9, 2C19, 2C8, 2C
18) shows a multiple alignment of the amino acid sequences of cytochrome P450cam (CAM). 1 amino acid
Expressed in character notation. Human-derived cytochrome P45
Of the amino acid sequences of each of the molecular species 02C8, 2C9, 2C18 and 2C19, the single or double underlined region was selected in Example 1 and the double underlined region was selected in Example 2. 1 shows the amino acid sequence obtained.

FIG. 2 shows molecular species (1A1, 1A2) belonging to the human-derived cytochrome P4501A subfamily and cytochrome P
FIG. 4 shows a multiple alignment of the amino acid sequence of 450 cam (CAM). Amino acids are represented in single letter notation. In the amino acid sequence of the human-derived cytochrome P4501A1 molecular species, a single or double underlined region is the amino acid sequence selected in Example 1, and a double underlined region is the amino acid sequence selected in Example 2. Show.

FIG. 3 shows that human-derived P4 is obtained by immunoblotting using an antibody (anti-2C8 antibody) produced by the production method of the present invention.
The results of analyzing 50 molecular species are shown. The samples migrated in each lane are as follows. Lane 1: P450
1A2, 2: P4502A6, 3: P4502B6,
4: P4502D6, 5: P4502E1, 6: P45
03A4, 7: P4502C8, 8: P4502C9,
9: P4502C18, 10: P4502C19

FIG. 4 shows that human-derived P4 is obtained by immunoblotting using an antibody (anti-2C9 antibody) produced by the production method of the present invention.
The results of analyzing 50 molecular species are shown. The samples migrated in each lane are as follows. Lane 1: P450
1A2, 2: P4502A6, 3: P4502B6,
4: P4502D6, 5: P4502E1, 6: P45
03A4, 7: P4502C8, 8: P4502C9,
9: P4502C18, 10: P4502C19

FIG. 5 shows that human-derived P4 is obtained by immunoblotting using an antibody (anti-2C19 antibody) produced by the production method of the present invention.
The results of analyzing 50 molecular species are shown. The samples migrated in each lane are as follows. Lane 1: P450
1A2, 2: P4502A6, 3: P4502B6,
4: P4502D6, 5: P4502E1, 6: P45
03A4, 7: P4502C8, 8: P4502C9,
9: P4502C18, 10: P4502C19

FIG. 6 shows that human-derived P4 is obtained by immunoblotting using an antibody (anti-2C18 antibody) produced by the production method of the present invention.
The results of analyzing 50 molecular species are shown. The samples migrated in each lane are as follows. Lane 1: P450
1A2, 2: P4502A6, 3: P4502B6,
4: P4502C8, 5: P4502C9, 6: P45
02C18, 7: P4502C19, 8: P4502D
6, 9: P4502E1, 10: P4503A4

FIG. 7 shows that human-derived P4 was obtained by an immunoblot method using an antibody (anti-1A1 antibody) produced by the production method of the present invention.
The results of analyzing 50 molecular species are shown. The samples migrated in each lane are as follows. Lane 1: P450
1A1, 2: 1A2, 3: P4502A6, 4: P45
02B6, 5: P4502C8, 6: P4502C9,
7: P4502C18, 8: P4502C19, 9: P
4502D6, 10: P4502E1, 11: P450
3A4

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 15/02 C12P 21/08 C12P 21/08 C12N 15/00 C

Claims (14)

[Claims] 1. A method for producing an anti-peptide antibody capable of immunochemically discriminating between a target protein and a protein having an amino acid sequence similar to the amino acid sequence of the protein, comprising: (1) an amino acid sequence of the target protein; By comparing the amino acid sequence of the protein to be discriminated with the protein, a partial amino acid sequence of the target protein, consisting of 10 to 15 amino acid residues, which matches the amino acid sequence of the protein to be discriminated is determined. Select an amino acid sequence that does not contain three or more adjacent residues,
(2) An amino acid sequence corresponding to a region exposed on the surface of the molecular model of the target protein is selected from the amino acid sequences selected in (1) above, and (3) an amino acid sequence selected in (2) above. Among them, an amino acid sequence having low homology as compared with a known amino acid sequence of a protein derived from an animal to be immunized is selected, and (4) a peptide having the amino acid sequence selected in (3) above and a carrier are selected. A method for producing an anti-peptide antibody, comprising the steps of: preparing a conjugated effective immunizing antigen; and administering the effective immunizing antigen to immunize an animal. 2. The method for producing an anti-peptide antibody according to claim 1, wherein the target protein is cytochrome P450. 3. The protein of interest is cytochrome P4502C.
3. A protein belonging to the family.
A method for producing the anti-peptide antibody according to the above. 4. The protein of interest is cytochrome P4501A.
3. A protein belonging to the family.
A method for producing the anti-peptide antibody according to the above. 5. The method for producing an anti-peptide antibody according to claim 1, wherein the effective immunizing antigen is an effective immunizing antigen obtained by binding a peptide having the amino acid sequence represented by SEQ ID NO: 1 to a carrier. 6. The method for producing an anti-peptide antibody according to claim 1, wherein the effective immunizing antigen is an effective immunizing antigen obtained by binding a peptide having the amino acid sequence represented by SEQ ID NO: 2 to a carrier. 7. The method for producing an anti-peptide antibody according to claim 1, wherein the effective immunizing antigen is an effective immunizing antigen obtained by binding a peptide having the amino acid sequence represented by SEQ ID NO: 3 to a carrier. 8. The method for producing an anti-peptide antibody according to claim 1, wherein the effective immunizing antigen is an effective immunizing antigen obtained by binding a peptide having the amino acid sequence represented by SEQ ID NO: 4 to a carrier. 9. The antibody according to claim 1, 2 or 4, wherein the effective immunizing antigen is an effective immunizing antigen obtained by binding a peptide having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 5 to 7 to a carrier. A method for producing a peptide antibody. 10. An effective immunizing antigen for producing a human-derived cytochrome P4502C8-specific anti-peptide antibody, comprising a peptide having the amino acid sequence represented by SEQ ID NO: 1 and a carrier. 11. An effective immunizing antigen for producing a human-derived cytochrome P4502C9-specific anti-peptide antibody comprising a peptide having the amino acid sequence represented by SEQ ID NO: 2 and a carrier. 12. An effective immunizing antigen for producing a human-derived cytochrome P4502C18-specific anti-peptide antibody comprising a peptide having the amino acid sequence represented by SEQ ID NO: 3 and a carrier bonded thereto. 13. An effective immunizing antigen for producing a human-derived cytochrome P4502C19-specific anti-peptide antibody, which is obtained by combining a peptide having the amino acid sequence represented by SEQ ID NO: 4 with a carrier. 14. A human-derived cytochrome P4501A1 in which a peptide having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 5 to 7 is combined with a carrier.
Effective immunizing antigen for producing specific anti-peptide antibodies.