JP2004264294A - Reagent for immunity measurement for detecting autoantibody to rb1 genetic product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent for immunity measurement capable of simply, sensitively detecting an autoantibody to an RB1 genetic product with high reproducibility. <P>SOLUTION: The reagent for immunity measurement is used for detecting autoantibody with respect to the RB1 genetic product. The reagent for immunity measurement, uses protein (P) bonded with an amino acid sequence contained in the amino acid sequence of an array number (1) as a constitutional unit bonded to a water-insoluble carrier (B). Preferably, the amino acid sequence is contained in an adenovirus E1A bonding region, expressed by the amino acid sequence of a sequence number (2). <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to an immunoassay reagent for detecting an autoantibody to an RB1 gene product, an immunoassay reagent kit containing the same, and an immunoassay method for an autoantibody to an anti-RB1 gene product.

  The RB1 gene is a tumor suppressor gene that encodes a protein having a molecular weight of about 110 kilodalton (hereinafter, RB1 gene product). It is known that deletion and point mutation occur frequently in the RB1 gene in various human cancers (Non-Patent Documents 1 and 2). On the other hand, in a lung cancer patient, the appearance of autoantibodies that react with the RB1 gene product in blood has been confirmed by Western blotting (Non-Patent Document 3).

Science, 243, 937, 1989 Oncogene, 8, 1913, 1993. International Journal of Oncology (Int. J. Oncology), 19, 1035, 2001

  Conventionally, the presence of an autoantibody to the RB1 gene product has been confirmed by Western blotting. The western blotting method requires complicated and long-time operations such as electrophoresis or blotting, and when measuring a large number of samples, it is difficult to adapt it to a clinical test agent or the like. Further, the Western blot method has problems in sensitivity, reproducibility, and the like. That is, an object of the present invention is to provide an immunoassay reagent capable of detecting autoantibodies against an RB1 gene product simply, with high sensitivity, and with good reproducibility.

  As a result of intensive studies to achieve the above object, the present inventors have found that autoantibodies to the RB1 gene product can be detected simply, with high sensitivity, and with good reproducibility, and have reached the present invention. That is, a feature of the immunoassay reagent of the present invention is an immunoassay reagent for detecting an autoantibody to the RB1 gene product, wherein the protein comprises an amino acid sequence contained in the amino acid sequence of SEQ ID NO: 1 as a constituent unit. The gist is that the and the water-insoluble carrier are combined.

  The immunoassay reagent for detecting an autoantibody to the RB1 gene product of the present invention has few nonspecific reactions and is extremely excellent in sensitivity and specificity. That is, the difference between the measured value of a sample from a healthy person without an autoantibody and the measured value of a sample from a cancer patient with an autoantibody is sufficiently large (high sensitivity), Since there is very little non-specific reaction to the sample, highly accurate measurement can be performed. In addition, since measurement can be performed easily, many samples can be easily measured in a short time. That is, the reagent for immunoassay of the present invention can detect autoantibodies against the RB1 gene product simply, with high sensitivity, and with good reproducibility. Therefore, the use of the measurement reagent, the measurement reagent kit and the measurement method of the present invention enables extremely rapid and accurate diagnosis of cancer.

  The RB1 gene product, which is an expression product from the RB1 gene, is a protein of about 110 kilodaltons {a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1}.

  As the protein (P) having an amino acid sequence contained in the amino acid sequence of SEQ ID NO: 1 as a constituent unit, the protein (P1) having SEQ ID NO: 1 or a partial amino acid sequence of SEQ ID NO: 1 as a constituent unit , P2) and the like can be used. It is difficult to prepare a protein (P1) having the amino acid sequence of SEQ ID NO: 1 as a constitutional unit, and it is preferable to use a protein (P2) having a partial amino acid sequence of SEQ ID NO: 1 as a constitutional unit.

  As the protein (P2), a fragment (amino acid sequence) obtained by dividing the amino acid sequence of SEQ ID NO: 1 into 20 to 600 amino acids, preferably 50 to 500 amino acids, and more preferably 100 to 360 amino acids, is a structural unit. For example, a protein having SEQ ID NO: (2) (adenovirus E1A binding region) and a protein having an amino acid sequence of SEQ ID NO: selected from the group consisting of (3) to (19) as a structural unit, etc. No. Among these, SEQ ID NOs: (2), (3), (5), (6), (7), (9), (11), (12), (13), (14), (15), Proteins comprising the amino acid sequence of (17) or (18) as a constitutional unit are preferred, and more preferably SEQ ID NOs: (2), (3), (5), (6), (7), (9), (15) ), (17) or (18).

  When the number of amino acids of the protein (P) is 20 to 100 (more preferably, 20 to 50), the protein (P) may be composed of only the amino acid sequence, The N-terminus and / or C-terminus may be modified with biotin, thioglycolic acid, and / or amino acids (cysteine, lysine, tyrosine, glutamic acid, aspartic acid, etc.) used as a linker. Further, the N-terminal may be acetylated with acetic anhydride or N-acetylimidazole, and the C-terminal may be water-soluble carbodiimide (for example, 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride) or isoxazolium. (For example, N-methyl-5-phenylisoxazolium fluoroborate) or the like. Also, these modifications can be used in combination. When the N-terminus and / or the C-terminus are modified with an amino acid, the number of amino acids is preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 to 2, per N-terminus or C-terminus. . When the N-terminal and / or the C-terminal are modified with biotin and / or thioglycolic acid, the number of biotin or thioglycolic acid is preferably 1 to 5, more preferably 1 to 3, per molecule of protein (P). And particularly preferably 1 to 2. Among these, it is preferable to be modified with biotin or thioglycolic acid, and from the viewpoint of simplicity in binding the protein (P) and the water-insoluble carrier (B), more preferably, the N-terminal and / or That is, the C-terminal is modified with biotin.

  When the number of amino acids of the protein (P) is 100 or more, the protein (P) may be composed only of the amino acid sequences of SEQ ID NOS: (1) to (19), or may be modified as described above. And a protein expressed from a protein expression vector. Examples of proteins expressed from the protein expression vector include glutathione transferase, a histidine tag (for example, a peptide to which six histidines are bound), and a protein in the ZZ region of protein A. Of these, preferred are proteins expressed from protein expression vectors, and more preferred are glutathione transferase and proteins in the ZZ region of protein A.

  The protein (P) may be a fusion protein prepared by genetic recombination or the like. For example, a polypeptide having any one of the amino acid sequences of SEQ ID NOs: (1) to (19) as a constituent unit may be obtained by using GST (glutathione S- Transferase) and the like. When the protein (P) has a constituent unit of 100 or more amino acid sequences, a fusion protein is preferred from the viewpoint of preparation and the like.

  The protein (P) can be prepared by a conventionally known peptide synthesis method, a gene recombination method, or the like. According to the peptide synthesis method, the synthesis of protein (P) is extremely difficult when the number of amino acids exceeds 20. On the other hand, the genetic recombination method makes it easy to synthesize protein (P). Therefore, the genetic recombination method is preferred.

  Although the peptide synthesis method can be achieved in a solution or on a solid support, a solid phase synthesis method using a solid support is preferable, and a solid phase synthesis method using an automatic peptide synthesizer is more preferable. Peptide synthesis methods generally use activated amino acids protected with t-butyloxycarbonyl (BOC group) or 9-fluorenylmethoxy-carbonyl (Fmoc group). In addition, specific synthetic procedures, types of side chain protection, and cleavage methods are described in, for example, Solid Phase Peptide Synthesis, 2nd Edition, Peace Chemical Company, 1984 (Stewart and Young, “Solid Phase Peptide Synthesis”, eds. Second Edition, Pierce Chemical Company, 1984), and Solid Phase Peptide Synthesis, IR, 1989 (Atherton and Sheppard, "Solid Phase Peptide Synthesis", IRL Press, 1989).

  As the gene recombination method, cDNA is prepared from mRNA extracted from human cells, for example, human vascular endothelial cells or human fibroblasts, using primers corresponding to the amino acid sequence of interest, and is prepared by PCR (polymerase chain). After amplification by the reaction method, the fragment is digested with a restriction enzyme, this fragment is incorporated into a vector, a host such as Escherichia coli is transformed with the obtained expression vector, induction treatment is performed if necessary, and the host is cultured. Extract and purify the target fusion protein from the lysate. In this way, a fusion protein containing the protein (P) can be prepared (for example, Biochemical Experiment Method 45 “Recombinant Protein Production Method”, Gakkai Shuppan Center, 2001). In addition to the above, a protein (P) can be prepared by synthesizing a gene corresponding to the target amino acid sequence from the amino acid sequence of the RB1 gene using a DNA synthesizer, and incorporating and expressing the gene in a vector.

  The reagent for immunoassay of the present invention only needs to contain at least one kind of such protein (P), and the presence of two or more kinds of protein (P) means that the reactivity with autoantibodies ( (Specificity and sensitivity) are preferred. In addition, even if it is a protein which has an amino acid sequence (1) as a structural unit, it is preferable to contain another kind of protein. More preferably, in addition to the protein having the amino acid sequence of SEQ ID NO: (2) as a constituent unit, the N-terminal region of the amino acid sequence (1) {the amino acid sequence of SEQ ID NO: (3), (9) or (15), etc.} It is intended to include a protein as a structural unit and / or a C-terminal region of SEQ ID NO: (1) {amino acid sequence of SEQ ID NO: (8), (14) or (19) or the like} as a structural unit. Most preferably, a protein having the amino acid sequence of SEQ ID NO: 2 as a constitutional unit, a protein having an N-terminal region of SEQ ID NO: 1 as a constitutional unit, and a protein having the C-terminal region of SEQ ID NO: 1 as a constitutional unit It is to include.

  Preferred examples of protein combinations of the immunoassay reagent of the present invention include, for example, the following combinations. The numbers in parentheses correspond to the SEQ ID NOs, and represent proteins having the amino acid sequence of the SEQ ID NOs as a constituent unit. That is, (2) + (3) represents a combination of a protein having the amino acid sequence of SEQ ID NO: 2 as a constituent unit and a protein having the amino acid sequence of SEQ ID NO: 3 as a constituent unit.

  That is, (5) + (6) + (7), (17) + (18), (5) + (6) + (18), (5) + (6) + (13), (5) + (12) + (7), (5) + (12) + (18), (5) + (12) + (13), (11) + (6) + (7), (11) + (6) ) + (18), (11) + (6) + (13), (11) + (12) + (7), (11) + (12) + (18), (17) + (6) + (7), (17) + (6) + (13), (17) + (12) + (7), (17) + (12) + (13), (2) + (3), (2) ) + (9), (2) + (15), (2) + (8), (2) + (14), (2) + (19), (2) + (3) + (8), (2) + (3) + (14), (2) + (3) + (19), (2) + (9) + (8), (2) + (9) + (14) (2) + (9) + (19), (2) + (15) + (8), (2) + (15) + (14), (2) + (15) + (19), (5) ) + (6) + (7) + (3), (5) + (6) + (7) + (9), (5) + (6) + (7) + (15), (5) + (6) + (7) + (8), (5) + (6) + (7) + (14), (5) + (6) + (7) + (19), (5) + (6) ) + (7) + (3) + (8), (5) + (6) + (7) + (3) + (14), (5) + (6) + (7) + (3) + (19), (5) + (6) + (7) + (9) + (8), (5) + (6) + (7) + (9) + (14), (5) + (6) ) + (7) + (9) + (19), (5) + (6) + (7) + (15) + (8), (5) + (6) + (7) + (15) + (14), (5) + (6) + (7) + 15) + (19), (9) + (11) + (12) + (13) + (14), (11) + (12) + (13), (17) + (18) + (3) , (17) + (18) + (9), (17) + (18) + (15), (17) + (18) + (8), (17) + (18) + (14), ( 17) + (18) + (19), (17) + (18) + (3) + (8), (17) + (18) + (3) + (14), (17) + (18) + (3) + (19), (17) + (18) + (9) + (8), (17) + (18) + (9) + (14), (17) + (18) + ( 9) + (19), (17) + (18) + (15) + (8), (17) + (18) + (15) + (14) and (17) + (18) + (15) + (19).

  Of these, (2) + (3) + (14), (2) + (3) + (19), (2) + (9) + (8), (2) + (9) + (14) ), (2) + (9) + (19), (2) + (15) + (8), (2) + (15) + (14), (2) + (15) + (19), (5) + (6) + (7) + (3) + (8), (5) + (6) + (7) + (3) + (14), (5) + (6) + (7) ) + (3) + (19), (5) + (6) + (7) + (9) + (8), (5) + (6) + (7) + (9) + (14), (5) + (6) + (7) + (9) + (19), (5) + (6) + (7) + (15) + (8), (5) + (6) + (7) ) + (15) + (14), (5) + (6) + (7) + (15) + (19), (9) + (11) + (12) + (13) + (14), (11) + (12 + (13), (17) + (18) + (3) + (8), (17) + (18) + (3) + (14), (17) + (18) + (3) + ( 19), (17) + (18) + (9) + (8), (17) + (18) + (9) + (14), (17) + (18) + (9) + (19) , (17) + (18) + (15) + (8), (17) + (18) + (15) + (14), and (17) + (18) + (15) + (19) Preferably, more preferably (5) + (6) + (7) + (3) + (8), (5) + (6) + (7) + (3) + (14), (5) + ( 6) + (7) + (3) + (19), (5) + (6) + (7) + (9) + (8), (5) + (6) + (7) + (9) + (14), (5) + (6) + (7) + (9) + (19), (5) + (6) + (7) + (15) (8), (5) + (6) + (7) + (15) + (14), (5) + (6) + (7) + (15) + (19), (17) + (18) ) + (3) + (8), (17) + (18) + (3) + (14), (17) + (18) + (3) + (19), (17) + (18) + (9) + (8), (17) + (18) + (9) + (14), (17) + (18) + (9) + (19), (17) + (18) + (15) ) + (8), (17) + (18) + (15) + (14), and (17) + (18) + (15) + (19).

  The protein (P) itself can be used as a reagent for immunoassay such as an immunoagglutination method. However, in the present invention, the protein (P) is combined with the water-insoluble carrier (B) to form an immunological reagent. included. The water-insoluble of the water-insoluble carrier means that the solubility in water is 0.01 g / 100 g or less at any temperature of 1 to 50 ° C. It means a substance that is solid (including gel) even at temperature and can bind to the RB1 gene product. As the water-insoluble carrier (B), carriers described in JP-A-2-205774 can be used, and inorganic and organic substances can be used. Examples thereof include cellulose, polystyrene, polypropylene, polyolefin, polyurethane, nitrocellulose, and cellulose acetate. , Polyester, epoxy resin, phenol resin, silk, fibroin, lignin, hemicellulose, chitin, ebonite, rubber, glass, quartz, silicon and ceramics. Among them, polystyrene, glass, quartz and silicon are preferred, and polystyrene and glass are more preferred, and glass is particularly preferred.

  The shape of the water-insoluble carrier (B) can be freely determined according to the purpose of use, and can be true spherical or disk-shaped beads, plate- or stick-shaped sticks, test tubes, and strips of nonwoven fabrics or filters (strip-shaped strips). Fragment), fine particles and the like. Of these, beads and microparticles are preferred, and more preferably spherical beads.

The size of the water-insoluble carrier (B) can be freely determined according to the purpose of use, but is usually a size that can be charged into a cylindrical reaction vessel (beaker or the like) having an inner diameter of about 4 to 10 mm and a depth of about 10 to 20 mm. (Except when the water-insoluble carrier is a test tube.) In the case of true spherical beads, the diameter (mm) is preferably 1 to 10, more preferably 2 to 8, and particularly preferably 3 to 7. In the case of disc-shaped beads, the diameter (mm) is preferably from 1 to 10, more preferably from 2 to 8, particularly preferably from 3 to 7, and the thickness (mm) is preferably from 0.1 to 5, more preferably. Is from 0.2 to 2, particularly preferably from 0.3 to 1. In the case of a stick, the length (mm) is preferably 2 to 10, more preferably 3 to 8, and particularly preferably 4 to 7. The cross-sectional area (mm ² ) of the stick is preferably 1 to 25, more preferably 2 to 16, and particularly preferably 3 to 9. The cross-sectional area means a cross-sectional area of a cut portion when cut perpendicular to the long axis direction. In the case of a test tube, the length (mm) is preferably 5 to 100, more preferably 8 to 80, and particularly preferably 10 to 20. Further, the inner diameter (mm) of the test tube is preferably 5 to 20, more preferably 6 to 16, and particularly preferably 8 to 12. In the case of a strip, the length (mm) is preferably 5 to 100, more preferably 10 to 80, and particularly preferably 10 to 50. Further, the width (mm) of the strip is preferably 1 to 20, more preferably 2 to 16, and particularly preferably 3 to 10. The thickness (mm) is preferably from 0.1 to 2, more preferably from 0.1 to 0.5. The average pore size (μm) of the nonwoven fabric or the filter is preferably from 0.1 to 10, more preferably from 0.3 to 5. In the case of fine particles, the average particle diameter (μm) is preferably from 0.01 to 200, more preferably from 0.1 to 50, particularly preferably from 0.2 to 10. The average particle diameter can be measured by transmission electron microscopy, alignment measurement using an optical microscope, or the like.

  The protein (P) and the water-insoluble carrier (B) can be bound by a conventionally known method such as a method of binding chemically or a method of binding by physical adsorption. As a method of chemically bonding, a functional group such as an amino group and / or a sulfhydryl group introduced on the surface of the water-insoluble carrier (B) and a functional group such as an amino group and / or a sulfhydryl group of the protein (P) are used. And the like with a binder (glutaraldehyde, succinaldehyde, m-maleimidobenzoyl-N-hydrosuccinimide ester, o-phenylenebismaleimide, etc.) (US Pat. Nos. 4,280,992 and 365,2761). ) And the like. As a method of binding by physical adsorption, when the water-insoluble carrier (B) is polystyrene, the water-insoluble carrier is added to a 0.001 to 0.04% (W / V) carbonate buffer aqueous solution (pH 9.0) of the protein (P). (Biosim Biofiz Acta, Vol. 251, 427, 1971). This method can be applied to the case where the carrier is a substance other than polystyrene, such as polypropylene, silicon, glass, and cellulose. Further, the protein (P) is indirectly bound to the water-insoluble carrier (B) using a specific binding substance (for example, antigen-antibody, avidin-biotin, lectin-sugar chain, complementary gene chain, etc.). You can also. For example, the protein (P) can be bound to the water-insoluble carrier (B) by modifying the protein (P) with biotin and reacting with the water-insoluble carrier bound with avidin. As avidin, egg white-derived avidin and streptavidin can be used, and streptavidin is preferable. Among these specific binding substances, antigen-antibody, avidin-biotin, and lectin-sugar chains are described in [Biochemical Experimental Method 11 “Enzyme Immunoassay”, Tokyo Kagaku Dojinsha, 1989]. Examples thereof include a complementary gene described in JP-A-6-186232, such as a combination of polydeoxyadenylic acid and polythymidylic acid.

  Among them, when the number of amino acids of the protein (P) is 20 to 100, a method using a specific binding substance is preferably used, and among the methods using a specific binding substance, a method using avidin-biotin bond is particularly preferable. preferable. When the number of amino acids in the protein (P) is 100 or more, a method of chemically bonding and a method of physical adsorption are preferable, and a method of chemically bonding is most preferable. When binding a protein having 20 to 100 amino acids to a protein having 100 or more amino acids, for example, avitin and a protein having 100 or more amino acids are mixed and bound to the water-insoluble carrier (B). After that, it is reacted with the protein modified with biotin.

  The immunoassay reagent of the present invention may be in a state of being immersed in an appropriate buffer, but after binding the protein (P) and the water-insoluble carrier (B), coated with a saccharide and / or a protein and dried. preferable. As the buffer, a phosphate buffer, a Good's buffer, or the like can be used, and may contain a protein, a salt, and / or a surfactant. Examples of the protein include albumin (bovine serum albumin, rabbit serum albumin, mouse serum albumin, ovalbumin, conalbumin and lactalbumin), antibody (antibody having no binding to protein (P) such as normal rabbit IlgG and normal mouse IgG). And gelatin. Examples of the salt include sodium chloride, potassium chloride and lithium bromide. Examples of the surfactant include nonionic surfactants such as sorbitan lauric acid monoester ethylene oxide adduct (trade name: Tween 20, Tween 40, ICI America). Examples of the coating and drying method include a method of dipping in a solution containing at least a saccharide and then drying (JP-A-09-318628 and JP-B-5-41946). As saccharides, disaccharides and monosaccharides are preferable.Examples of disaccharides include maltose, cellobiose, gentiobiose, melibiose, sucrose, lactose and isosaccharose, and monosaccharides include glucose, fructose, mannose, galactose, Arabinose, xylitolose and the like. Of these, lactose, sucrose, glucose and fructose are preferred, lactose and sucrose are more preferred, and sucrose is particularly preferred. When coating, the coating amount (content of saccharides and the like), the concentration of a solution containing saccharides and the like (coating solution), the type of the solution, and the like may be in known ranges. For example, JP-A-09-318628 and This is described in, for example, Japanese Patent Publication No. Hei 5-41946.

The immunoassay reagent kit of the present invention preferably contains an anti-human immunoglobulin antibody together with the immunoassay reagent of the present invention. As the anti-human immunoglobulin antibody, an antibody prepared by a conventionally known method can be used. For example, it can be prepared by immunizing a suitable animal (eg, mouse, rabbit, pig, goat, horse, etc.) with human immunoglobulin and purifying the antibody from the obtained antiserum by salting out, ion exchange column chromatography or the like. . The anti-human immunoglobulin antibody may be a polyclonal antibody or a monoclonal antibody, or may be an antibody fragment such as (Fab ') ² or Fab'. The human immunoglobulin includes all immunoglobulins such as IgG, IgA, and IgM, but is usually IgG and IgM, and is mainly IgG.

The anti-human immunoglobulin antibody may not be labeled, but is preferably labeled with a labeled compound in terms of sensitivity, simplicity of measurement, and the like. When the anti-human immunoglobulin antibody was not labeled with a labeling compound, an antibody recognizing the anti-human globulin antibody (for example, when the anti-human immunoglobulin antibody was mouse IgG, an anti-mouse IgG antibody prepared in rabbits) was labeled. Reagents and the like are used. Conventionally known compounds can be used as the labeling compound, and radioisotopes, fluorescent substances, luminescent substances, enzymes and the like are used. The isotopes, ¹²⁵ I and the like, and the fluorescent substance include europium complex, etc., as the light-emitting material include N- methyl acridinium ester, etc., as the enzyme horseradish peroxidase, alkaline phosphatase and β- galactosidase And the like. Of these labeled compounds, enzymes are preferred, more preferably horseradish peroxidase, alkaline phosphatase and β-galactosidase, particularly preferably horseradish peroxidase and alkaline phosphatase.

As a method of labeling a labeled compound with an anti-human immunoglobulin antibody, conventionally known methods and the like can be applied, and “Seizoku Chemistry Experiment Course 5 Immunobiochemical Experiment Method” (edited by The Japanese Biochemical Society, Tokyo Chemical Dojin, 1986, 102 To 112, and the following methods (1) to (4) can be applied.
(1) When the labeled compound is an isotope, a method of introducing radioactive iodine into a tyrosine residue of an anti-human immunoglobulin antibody using chloramine T as an oxidizing agent.
(2) When the labeling compound is a fluorescent substance, a method of reacting fluorescein isothiocyanate with an anti-human immunoglobulin antibody in a buffer to bind to a lysine residue of the anti-human immunoglobulin antibody.
(3) When the labeling substance is a luminescent substance, a trade name “Acridinium Derivative-I” (manufactured by Dojindo Laboratories) is reacted with an anti-human immunoglobulin antibody in a buffer solution to react with an amino group of the anti-human immunoglobulin antibody. How to combine.
(4) When the labeling substance is an enzyme, a method in which the amino group of the enzyme and the thiol group of the anti-human immunoglobulin antibody are bound with a di-crosslinkable reagent such as N-succinimidyl-6-maleidohexanoate.

  The reagent kit for immunoassay of the present invention is not limited in the form of the reagent, the configuration of the reagent kit, and the like as long as the reagent for immunoassay of the present invention is included. That is, the reagent kit of the present invention can be applied to a homogeneous immunoassay such as a latex agglutination method and a latex nephelometry, as well as to a heterogeneous immunoassay. Among these, a heterogeneous immunoassay is preferable in terms of measurement sensitivity, and a reagent kit for heterogeneous sandwich immunoassay containing a labeled compound and an anti-human immunoglobulin antibody is most preferable. That is, when the immunoassay reagent of the present invention is reacted with a sample, only the autoantibody to the RB1 gene product specifically binds to the protein (P). Then, when an anti-human immunoglobulin antibody is added, this antibody binds to the autoantibody to form an immune complex "protein (P) -autoantibody-anti-human immunoglobulin antibody". By quantifying the amount of the anti-human immunoglobulin antibody in the immune complex, the amount of the autoantibody can be quantified. When the anti-human immunoglobulin antibody is labeled with a labeling compound, the amount of the anti-human immunoglobulin antibody can be quantified by measuring the amount of the labeling compound.

  The measurement of the labeled compound can be performed by a conventionally known method or the like depending on the type of the labeled compound. When the labeling compound is a fluorescent substance, for example, the amount of fluorescence generated by irradiation with excitation light having an appropriate wavelength is quantified using a photomultiplier. When the labeling compound is a chemiluminescent substance, for example, in the case of acridinium ester, the amount of luminescence generated by adding an alkaline solution is quantified by a photomultiplier.

  When the labeled compound is an enzyme, the enzyme activity can be measured as an absorbance (absorbance measurement method), a fluorescence amount (fluorescence measurement method) or a luminescence amount (chemiluminescence measurement method) by reacting an appropriate substrate. For example, when the enzyme is peroxidase, the substrate is 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) (absorbance measurement method) and luminol / peroxide (chemiluminescence). Quantity measurement method) can be selected. When the enzyme is alkaline phosphatase, p-nitrophenyl phosphate (absorbance measurement method), 4-methylumbelliferyl phosphate (4-MUP) (fluorescence measurement method) and 3- (2′-spiroadamantane) are used as substrates. ) -4-methoxy-4- (3 ″ phosphoryloxy) phenyl-1,2-dioxetane disodium (AMPPD) (chemiluminescence assay) and the like. The absorbance is determined by a spectrophotometer, and the amount of fluorescence and the amount of chemiluminescence are determined by a photomultiplier. Of these, a chemiluminescence assay is preferable (ie, a method for quantifying autoantibodies using the reagent kit of the present invention is a chemiluminescent enzyme immunoassay), and more preferably peroxidase and luminol / peroxidase. This is a method for measuring the amount of chemiluminescence based on a combination of a substance and a combination of alkaline phosphatase and AMPPD.

  Luminol includes luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI), and metal salts thereof. As these metal salts, alkali metal (such as sodium and potassium) salts and alkaline earth metal (such as calcium and magnesium) salts can be used. Of these, luminol and a metal salt of luminol are preferred, more preferably a metal salt of luminol, and particularly preferably a sodium salt of luminol. As the peroxide, either an inorganic peroxide or an organic peroxide can be used. Inorganic peroxides include hydrogen peroxide, sodium perborate, potassium perborate, peroxide, carbon dioxide, dicarbonate, hypochlorous acid, potassium hypochlorite, chlorite, chlorine Acids, sodium chlorate, perchloric acid, perbronic acid, peroxosulfuric acid, peroxophosphoric acid, and the like. Examples of the organic peroxide include peracetic acid, perpropionic acid, dimethylsulfoxide (DMSO), triethylamine oxide, methyldiethylamine oxide, and phthaloyl peroxide. Among these, from the viewpoint of storage stability and the like, inorganic peroxides are preferable, and hydrogen peroxide is more preferable.

  To the reagent kit for immunoassay of the present invention, a reagent containing an anti-human immunoglobulin, preferably an anti-human immunoglobulin, is added to a reagent (reagent for immunoassay of the present invention) comprising a protein (P) and a water-insoluble carrier (B) bound thereto. And a buffer for immunoreaction, a buffer for B / F separation, a control sample, and the like. As the buffer for immunoreaction and the buffer for B / F separation, buffers and the like conventionally used for immunoassays can be used. Phosphate buffer containing proteins, salts and / or surfactants and Good (Good) ) Can be used. Examples of the protein, salt and surfactant include the same as described above.

  The control sample is used as a comparison sample to determine whether or not an autoantibody against the RB1 gene product exists in the sample, and is usually a sample containing no autoantibody (negative control) and a sample containing the autoantibody (positive control). ) Is prepared. As a negative control, a buffer containing a protein or the like similar to the buffer for an immune reaction, a human pooled serum having no autoantibody to the RB1 gene product, or the like can be used. As a positive control, a human pool serum containing an autoantibody to the RB1 gene product, a buffer solution containing a serum containing an autoantibody to the RB1 gene product (containing a protein similar to the buffer solution for immunoreaction), and the like can be used. .

  The sample to be measured by the immunoassay reagent kit of the present invention is not particularly limited as long as it is a human-derived body fluid, and includes, for example, blood, urine, saliva, lymph, bile, gastric juice, pancreatic juice, etc. A homogenate extract of the obtained tissue can also be used. Of these, blood and urine are preferred, and blood (including whole blood, serum or plasma) is more preferred.

Specific examples (steps 1 to 6) of a method for detecting an autoantibody against the RB1 gene product using the reagent kit of the present invention (sandwich measurement method) are shown below.
Step 1. A sample is reacted with an immunoassay reagent (the immunoassay reagent of the present invention) in which the protein (P) and the water-insoluble carrier (B) are bound to form a reaction mixture (including the complex 1 in step 2). obtain.
Step 2. The unreacted material is removed from the reaction mixture of Step 1 (B / F separation) to obtain a complex 1.
Step 3. The complex 1 obtained in step 2 is reacted with an anti-human immunoglobulin antibody labeled with a labeling compound to obtain a reaction mixture (including complex 2 in step 4).
Step 4. Unreacted substances are removed from the reaction mixture obtained in Step 3 (B / F separation) to obtain a complex 2.
Step 5. The amount of the labeled compound of the complex 2 is measured.
Step 6. Using the amount of the labeled compound, the presence or absence of an autoantibody is determined by comparing with a reference value (negative control and / or positive control).

  In the step of determining the presence or absence of an autoantibody to the RB1 gene product in the sample (Step 6 in the above example), the amount of the anti-human immunoglobulin antibody to the blood sample (measured as the amount of the labeled compound labeled with the anti-human immunoglobulin) Is compared with a reference value set from the amount of anti-human immunoglobulin (measured as the amount of the labeled compound labeled with anti-human immunoglobulin) in the blood of a plurality of healthy persons. It is preferable to determine the presence or absence. The method of setting the reference value is described in, for example, "Clinical Laboratory Drug Guide 1999-2000, p64-71, Bunkodo (1999)", "Clinical Laboratory Data Book 1997-1998, p8-13, Medical Shoin (1997)". Can be done in the manner described. That is, a plurality of healthy individuals (at least 50 or more, preferably 120 or more) are measured, and the amount of the labeled compound is determined (in the case of a chemiluminescent enzyme immunoassay, the luminescence is obtained). Statistical processing of the amount of labeled compounds ｛Parametric method, non-parametric method, etc. [The parametric method is used when the population shows a normal distribution (power transformation is possible). The non-parametric method is used when the population does not have a normal distribution. used. Then, a range including 95% of the sample group is set as a reference range, the upper limit is set as an upper reference value, and the lower limit is set as a lower reference value. Therefore, a sample exceeding the upper reference value has a high possibility of having an autoantibody, but may have a probability of 5% even in a healthy person. In order to prevent this, it is usual to multiply the upper limit reference value by a coefficient and / or set an added cutoff value. That is, a sample exceeding the cutoff value is determined as having an autoantibody. The cutoff value is set based on the relationship between the upper limit reference value and the lower limit value of the distribution of a sample having an autoantibody to the RB1 gene product, and is usually about 1.5 to 3 times the upper limit value. .

  The negative control and the positive control described above were prepared so that the set cutoff value could be easily reproduced. For example, it is possible to set such that the cutoff value is three times the measured value (the amount of the labeled compound) of the negative control, or the average value of the negative control and the positive control is the cutoff value.

  Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.

<Preparation Example 1> Preparation of RB1 gene product (GST fusion protein) According to the method described in International Journal of Oncology (Int. J. Oncology), 19, 1035, 2001, GST (glutathione S transferase) was used. The fusion protein was made as follows.

1. Preparation of RB1 gene RNA was extracted from human umbilical vein endothelial cells {Human Umbilical Vein Endothelial Cell (purchased from Funakoshi)}. The extraction was performed as follows using the AGPC (acetate buffer, guanidinium thiocyanate, phenol, chloroform) method.

(Preparation of AGPC method reagent)
(1) Preparation of 1 M sodium citrate (pH 7.0) After dissolving 29.4 g of trisodium citrate in 80 ml of distilled water, citric acid was added to adjust the pH to 7.0, and then distilled water was added at 25 ° C. Was added to make up to 100 ml. Then, it was used after autoclaving (120 ° C., 20 minutes).
(2) Preparation of solution D 236.3 g (4 M) of GTC (guanidium thiocyanate), 2.5 g (0.8 wt%) of sarkosyl (L-Lauroylsarcosine), 1 M sodium citrate buffer (pH 7.0) 12 After uniformly dissolving 0.5 ml (25 mM) and 250 ml of distilled water at 85 ° C., the mixture was returned to room temperature (25 ° C.) and adjusted to 496 ml with distilled water. The solution was filtered through a 0.45 μm bottle top filter and stored. At the time of use, 360 μl (0.1 M) of 2-mercaptoethanol was added to 50 ml of this stock solution to prepare solution D.
(3) Preparation of 2M sodium acetate (pH 4.0) After dissolving 27.2 g of sodium acetate trihydrate in 10 ml of distilled water, the pH was adjusted to 4.0 with acetic acid, and distilled water was added at 25 ° C. The volume was 100 ml. It was used after being filtered with a 0.45 μm bottle top filter.

(Operation of AGPC method)
(1) 0.5 ml of solution D was added to a tube, and human umbilical vein vascular endothelial cells were dispersed to destroy the cells.
(2) Further, 50 μl of 2M sodium acetate, 0.5 ml of phenol, and 100 μl of chloroform / isoamyl alcohol (volume ratio 49 / l) were sequentially added, and the tube was shaken 2-3 times for each type, and mixed.
(3) After mixing vigorously for 10 seconds, the mixture was ice-cooled for 15 minutes.
(4) After centrifugation at 10,000 G for 20 minutes at a centrifugal acceleration of 10,000 G, the aqueous layer separated at the bottom was separated into another tube so that an intermediate layer containing DNA was not mixed.
(5) 0.5 ml of isopropanol was added to the separated aqueous layer, and the mixture was cooled at -20 ° C for 1 hour. Next, it was centrifuged (10,000 G, 0 ° C.) for 10 minutes to precipitate RNA.
(6) The precipitated RNA is redissolved in 0.5 ml of solution D, 0.5 ml of isopropanol is added, and the mixture is left at -20 ° C for 1 hour.
(7) The RNA is precipitated by centrifugation, and the precipitate is washed with 1 mL of an 80% by weight aqueous ethanol solution, dried, and then dissolved in water.

  From the RNA thus extracted, cDNA synthesis and amplification were performed using a trade name “Ready-To-Go RT-PCR Beads” (Ready-To-Go RT-PCR Beads, Amersham). That is, RNA and a 3 ′ primer solution corresponding to the amino acid sequence described in Preparation Example 1 in Table 1 were added to ready-to-go RT-PCR beads, reacted at 42 ° C. for 1 hour, and then heated at 95 ° C. for 5 minutes. Then, cDNA was synthesized. Subsequently, 5 ′ primer was added to the reaction solution containing cDNA, and PCR (polymerase chain reaction) was performed for denaturation reaction (95 ° C., 1 minute), annealing reaction (60 ° C., 1 minute), extension reaction (72 ° C., 1 minute). ) Using a thermal cycler for PCR (MP PCR thermal cycler, manufactured by Takara Shuzo) for 30 cycles to obtain amplified cDNA.

2. Preparation and Expression of Recombinant Gene The obtained cDNA was placed at the BamHI / EcoRI site of the expression vector pGEX-4T-3 (purchased from Amersham) using Molecular Cloning, Laboratory Manual, 2nd edition (Molecular Cloning, A Laboratory Manual, 2nd edd.). ), Incorporated by the method described in 1989. After confirming that the insertion direction of the cDNA is correctly incorporated by the sequencing method, the transformant is transformed into Escherichia coli BL21 (DE3), LysS (Novagen), and a medium containing 0.1 mM isopropylthiobetagalactoside (IPTG). Induction was performed at 37 ° C. for 2 hours. Purification of the desired fusion protein from an E. coli cell lysate prepared by sonication by affinity chromatography using a glutathione-binding affinity gel (trade name: Glutathione Sepharose 4B, manufactured by Amersham) (eluate: 10 mM reduced form) GST fusion protein GST1 {protein having the amino acid sequence of SEQ ID NO: (2) as a constitutional unit} was prepared using 0.02 M tris (hydroxymethyl) aminomethane / hydrochloric acid buffer containing glutathione.

<Creation examples 2 to 7>
GST fusion proteins (GST2-7) were prepared in the same manner as in Preparation Example 1, except that the 3 ′ primer and 5 ′ primer shown in Table 1 were used.

<Example 1>
1. Preparation of GST1-Binding Beads To a polyethylene bottle with a lid containing 20 mL of an acetone solution containing 1% by weight of γ-aminopropyltriethoxysilane, 1000 glass beads of 3.2 mm in diameter (manufactured by Immunochemical Co., Ltd.) were added, and the mixture was added at 25 ° C. for 1 hour. And the reaction residue was removed by suction with an aspirator.
Then, 20 mL of deionized water was added thereto, the lid was closed, and the polystyrene bottle was slowly inverted twice and stirred, and then the liquid was suctioned off with an aspirator to wash the glass beads. This washing operation was performed three times.
Next, 1000 glass beads after washing were added to a polyethylene bottle with a lid containing 20 mL of a 2% by weight glutaraldehyde-containing aqueous solution, reacted for 1 hour at 25 ° C., and the reaction residue was removed by suction with an aspirator. Then, 20 mL of deionized water was added, the lid was closed, the polystyrene bottle was slowly inverted twice and stirred, and the liquid was suctioned off with an aspirator to wash the glass beads. This washing operation was performed three times.
Further, 1000 glass beads after washing are added to a polyethylene bottle with a lid containing 20 mL of 0.02 M phosphate buffer (pH 8.7) containing GST1 at a concentration of 20 μg / mL, and reacted at 25 ° C. for 1 hour. Was.
After the reaction, the phosphate buffer containing GST1 was removed, and the plate was immersed in 20 mL of a phosphate buffer containing 0.1% by weight of bovine serum albumin (pH 7.2) at 4 ° C. for 8 hours. Thereafter, the buffer aqueous solution was removed by suction using an aspirator, and the beads were washed twice with 20 mL of a phosphate buffer solution (pH 7.2) containing 0.1% by weight of bovine serum albumin. After immersion in a buffer solution (pH 7.2) for 30 minutes, the phosphate buffer solution was removed with an aspirator, the beads were spread on filter paper, air-dried at room temperature (about 25 ° C.), and GST fusion protein-bound beads 1 (immunoassay of the present invention) Was prepared and stored refrigerated (2 to 10 ° C.) in a sealed container containing a desiccant (silica gel).

2. Preparation of Immune Reaction Buffer Solution Casein was added to a 0.02 M phosphate buffer (pH 8.0) at a concentration of 3 g / L and sodium chloride at a concentration of 8.5 g / L. It was created. Stored refrigerated (2-10 ° C) until use.

3. Preparation of Peroxidase-Labeled Anti-Human Immunoglobulin Antibody Using anti-human immunoglobulin polyclonal antibody (manufactured by Dako Japan) and horseradish-derived peroxidase (manufactured by Toyobo Co., Ltd.), literature [S Yoshitake, M. Imagawa, E. Ishikawa, ethol; Jay. Biochem, Vol. 92 (1982) pp. 1413-1424], using a peroxidase-labeled anti-β2-microglobulin polyclonal antibody [a solution of a 0.02 M phosphate buffer (pH 6.0) having a protein concentration of 1 mg / mL]. Prepared and stored frozen (-30 ° C).

4. Preparation of Enzyme-Labeled Antibody Solution An enzyme-labeled antibody solution was prepared as follows using the immune reaction buffer solution prepared above and a peroxidase-labeled anti-human immunoglobulin antibody. That is, peroxidase-labeled anti-human immunoglobulin antibody (100 μg in protein amount) was added to 100 mL of the immune reaction buffer, and the mixture was stirred and mixed to obtain an enzyme-labeled antibody solution.

5. Preparation of Hydrogen Peroxide Solution 200 μl of 35% by weight hydrogen peroxide solution was dissolved in 1 liter of deionized water to obtain a hydrogen peroxide solution. Stored refrigerated (2-10 ° C) until use.

6. Preparation of Substrate Solution 0.18 g of luminol (manufactured by Tokyo Kasei) and 0.1 g (manufactured by Sanshin Chemical) of 4- (cyanomethylthio) phenol were mixed with 0.1 M (mol / L) of tris (hydroxymethyl) amino acid having a pH of 8.5. Dissolved in 1 liter of methane / hydrochloric acid buffer. Stored in the dark and refrigerated (2-10 ° C) until use.

<Examples 2 to 10>
1. Preparation of RB1 Gene Product (GST Fusion Protein) -Binding Beads GST fusion protein-binding beads 2 to 10 (measurement of the present invention) were carried out in the same manner as in Example 1 except that GST1 shown in Table 2 was used instead of GST1. Reagents 2 to 10) were prepared and stored refrigerated (2 to 10 ° C.) in a sealed container containing a desiccant (silica gel).
When a plurality of GST fusion proteins were used in combination (Examples 8 to 10), the amount of each GST fusion protein was the same, and the total amount was 20 μg / mL. In addition, the buffers prepared in Example 1 were used for the immunoreaction buffer, peroxidase-labeled anti-human immunoglobulin antibody, enzyme-labeled antibody solution, hydrogen peroxide solution, and substrate solution.

<Example 11>
This is an example in which the serum of a healthy person and the serum of a cancer patient were measured.
1. Specimen Serum collected from 50 healthy volunteers was mixed with 0.2 mL each, and 10 mL of pooled healthy serum was prepared as Sample 1. In addition, 10 mL of each serum (patients 1 to 3) obtained from cancer patients was prepared and used as specimens 2 to 4.

2. Immune Reaction Operation In a 12 × 75 mm test tube, 300 μL of an immunoassay buffer, 10 μL of a sample 1 (pool serum from a healthy individual), and one GST fusion protein-bound bead were added, and reacted at 37 ° C. for 10 minutes. After removing the reaction residue with an aspirator, 2 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. Further, 2 mL of physiological saline was added and washed in the same manner. Next, 300 μL of the enzyme-labeled antibody solution was added to the washed beads, and reacted at 37 ° C. for 10 minutes. The reaction solution was removed with an aspirator, 2 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. Further, 2 mL of physiological saline was added, and the mixture was washed twice in the same manner. The enzymatic activity of the washed beads was measured as follows. In addition, the enzyme activities of Samples 2 to 4 were measured in the same manner.

3. Enzyme activity measurement operation A test tube (12 x 75 mm) containing the washed beads was set in a sample holder of a luminescence reader BLR-201 type manufactured by Aloka, and 200 µL of a substrate solution and 200 µL of a hydrogen peroxide solution were added to perform a chemiluminescence reaction. Started. The luminescence amount for 10 seconds from 40 seconds after the start of the luminescence reaction was integrated and measured, and this was defined as the luminescence amount showing the enzyme activity.

4. Measurement results Table 2 shows the measurement results of the light emission amount. In addition, the numerical value in Table 2 shows the light emission amount in the upper part, and the lower part is a relative value of each light emission amount when the light emission amount of the pool serum of healthy volunteers is set to 1.0.
In cancer patients, the measured luminescence is higher than the luminescence of the pool serum of healthy individuals, and the luminescence ratio (the value in the lower part of the table divided by the measured luminescence of the pool sera of healthy individuals) is large. I understand. In addition, even if the serum of the patient has low reactivity to individual GST fusion proteins, the sensitivity is improved by combining a plurality of GST fusion proteins. In particular, the combination including the entire region of the E1A binding region has a high light emission ratio and improved sensitivity.

<Comparative Example 1>
This comparative example is an example in which measurement was performed by a method according to the related art.
1. GST fusion protein blotting 1 μg of each of GST1 to GST7 prepared in Preparation Examples 1 to 7 was subjected to SDS polyacrylamide gel electrophoresis and blotting to a nitrocellulose filter using “PastSystem” manufactured by Pharmacia. The electrophoresis was performed using "PhastGel Homogenous 12.5" manufactured by Pharmacia as a gel, and "PhastGel SDS Buffer Strips" manufactured by Pharmacia as a migration buffer, at a temperature of 15C and a voltage of 250V. Electrophoresis for 2 hours. The GST protein was blotted from the electrophoresed gel onto a nitrocellulose membrane (Millipore) using "PhastTransfer Semi-dry blotting" manufactured by Pharmacia. The blotting buffer used was a 25 mM tris (hydroxymethyl) aminomethane buffer (pH 8.3) containing 192 mM glycine and 10% by volume methanol. The blotting was performed at a temperature of 15 ° C., a voltage of 20 V, and a migration of 30 minutes.

2. Detection of Autoantibodies in Samples This membrane was immersed in a 0.02 M phosphate buffer (pH 7.0) containing 5% by weight of skim milk at room temperature (25 ° C.) for 8 hours. The membrane was taken out, sample 1 (pool serum of a healthy person) was immersed in a solution 400-fold diluted with 0.02 M phosphate buffer (pH 7.0) containing 5% by weight of skim milk powder, and reacted at 25 ° C. for 3 hours. . Next, the filter was taken out, left to stand in a 0.02 M phosphate buffer (7.0) containing 0.1% Tween 20 at 25 ° C. for 1 hour while shaking, and then the filter was washed by removing the liquid with an aspirator. This washing was performed three times. The washed filter was immersed in the enzyme-labeled antibody solution prepared in Example 1, and reacted at 25 ° C. for 1 hour. The above-mentioned washing was similarly performed six times. The washed filter was immersed in 100 mL of 0.05 M tris (hydroxymethyl) aminomethane / hydrochloric acid buffer (pH 7.6) containing 50 mg of 3,3′-diaminobenzidine, and reacted at 25 ° C. for 10 minutes. After the above-mentioned washing was performed 5 times (however, the standing time was 10 minutes), the filter was air-dried at 25 ° C., and the resulting color band was visually observed. In the same manner, for the samples 2 to 4 (the sera of the cancer patients 1 to 3), the coloring bands were visually confirmed.

3. Evaluation Compared to the band of the Boolean serum of a healthy subject (rarely occurring or extremely weak), the result is expressed as-when the Boolean serum is the same as that of the healthy human, ± when it is slightly strong, and + when it is strong. Are shown in Table 3. Even in the case where there is a clear S / N difference in the eleventh embodiment, in the comparative example 1,-or ± was determined in some cases. In addition, the judgment of Comparative Example 1 was a subjective judgment by visual observation, and was so delicate that there was a possibility that the result would differ depending on the judge. On the other hand, the method of the present invention does not have such a problem because it can be clearly measured as a numerical value as shown in the eleventh embodiment.

<Example 12>
This is an example in which a plurality of healthy human sera were measured to obtain a reference value, a cutoff value was set, sera from 10 cancer patients were measured, and the presence or absence of an autoantibody to the RB1 gene product was determined.

1. Using the GST fusion protein (GST4, GST5, GST6, GST2 and GST7) -bound beads 10 prepared in Example 10, serum samples from 120 healthy volunteers were prepared in the same manner as in Example 11. Specimen 1 of Example 11 (pool serum of a healthy person) was measured.
FIG. 1 shows the distribution of measured values (light emission amount) of 120 healthy volunteers. The horizontal axis in the figure is the light emission amount (cps), and the points indicate the distribution of the specimen (one point corresponds to one specimen).

2. Setting of cut-off value Results of analyzing the distribution type of the serum sample and the measurement value (emission amount) of sample 1 from 120 healthy volunteers using statistical processing software “STATFLEX v.4.1” (manufactured by Artec). And the luminescence amount distribution was 1.49 in skewness and 4.90 in kurtosis. Note that this distribution was not a normal distribution. When a 95% confidence interval was determined by the non-parametric method, the reference value was a light emission amount of 1606 to 7547. Here, when the light emission amount 1.5 times the upper limit light emission amount was set as the cutoff value, the light emission amount was 11321. At this time, the luminescence amount of the sample 1 (pool serum of a healthy person) was 3406, which was 1 / 3.32 of the cutoff value. Therefore, when the sample 1 (pool serum of healthy volunteers) was measured as a negative control, the cutoff value was set to “the measured luminescence of the pool serum of healthy volunteers” × 3.32.

3. Measurement of Samples Serum samples 5 to 14 collected from 10 cancer patients (patients 4 to 13) and sample 1 (a healthy person pooled serum) prepared in Example 11 as a negative control were measured. The measurement was carried out in the same manner as in Example 11 using the GST fusion protein-bound beads 10 of Example 10.

4. Judgment Result Using Sample 1 (pool serum of healthy volunteers) as a negative control, the cutoff value (“measured luminescence of pool serum of healthy volunteers” × coefficient) was determined using the coefficient set in the above (2). A measured value whose luminescence amount is equal to or greater than the cutoff value (cutoff ratio 1.0 or more) is positive for the autoantibody, while a measured value whose luminescence amount is less than the cutoff value (less than 1.0) is regarded as the autoantibody negative. It was judged. Table 4 shows the results.

  From Table 4, it was determined that autoantibodies to the RB1 gene product were positive in cancer patients at a high frequency (8 out of 10). It has been shown that the method of the present invention may be useful for diagnosing cancer.

<Creation examples 8 to 15>
GST fusion proteins (GST8 to 15) were prepared in the same manner as in Preparation Example 1, except that the 3 ′ primer and the 5 ′ primer shown in Table 5 were used.

<Examples 13 to 28>
1. Preparation of RB1 Gene Product (GST Fusion Protein) -Binding Beads GST fusion protein-binding beads 11 to 26 (the present invention) were prepared in the same manner as in Example 1 except that GST1 shown in Table 6 or 7 was used instead of GST1. Were prepared and stored refrigerated (2 to 10 ° C.) in a sealed container containing a desiccant (silica gel).
When a plurality of GST fusion proteins were used in combination (Examples 19 to 26), the amount of each GST fusion protein was the same, and the total amount was 20 μg / mL. In addition, the buffers prepared in Example 1 were used for the immunoreaction buffer, peroxidase-labeled anti-human immunoglobulin antibody, enzyme-labeled antibody solution, hydrogen peroxide solution, and substrate solution.

<Example 29>
Samples were measured in the same manner as in Example 11, and the results were shown in Tables 6 and 7 (in the numerical values in Tables 6 and 7, the upper row shows the luminescence level, and the lower row shows the luminescence level of the pool serum of healthy individuals as 1.0. It is a relative value of each light emission amount when performing.
In cancer patients, the measured luminescence is higher than the luminescence of the pool serum of healthy individuals, and the luminescence ratio (the value in the lower part of the table divided by the measured luminescence of the pool sera of healthy individuals) is large. I understand. In addition, even if the serum of the patient has low reactivity to individual GST fusion proteins, the sensitivity is improved by combining a plurality of GST fusion proteins. In particular, the combination including the entire region of the E1A binding region has a high light emission ratio and improved sensitivity.

<Comparative Example 2>
Samples were measured in the same manner as in Comparative Example 1, except that GST1 to GST7 prepared in Preparation Examples 1 to 7 were changed to GST8 to 15 prepared in Preparation Examples 8 to 15. Compared with the band of the Boolean serum of a healthy subject (rarely occurring or extremely weak), the case of the same level as the Boolean serum of a healthy subject was represented by-, the case of a slightly strong case was represented by ±, and the case of a strong case was represented by + Table 8 shows the results. Even in the case where there is a clear S / N difference in Example 29, in Comparative Example 2,-or ± was determined in some cases. In addition, the judgment of Comparative Example 2 was a subjective judgment by visual observation, and was so delicate that the result might be different depending on the judge. On the other hand, the method of the present invention does not have such a problem because it can be clearly measured as a numerical value as shown in Example 29.

It is a graph which showed the distribution of the luminescence amount measured about 120 normal volunteers' serum using GST fusion protein binding beads (reagent 10 for immunoassay of the present invention) (Example 12).

Claims (8)

An immunoassay reagent for detecting an autoantibody to the RB1 gene product, comprising: a protein (P) having an amino acid sequence contained in the amino acid sequence of SEQ ID NO: 1 as a constituent unit; and a water-insoluble carrier (B). An immunoassay reagent characterized by being bound. The immunoassay reagent according to claim 1, wherein the amino acid sequence is an amino acid sequence contained in the adenovirus E1A binding region represented by the amino acid sequence of SEQ ID NO: (2). An immunoassay reagent kit comprising the reagent according to claim 1 and an anti-human immunoglobulin antibody. The reagent kit according to claim 3, wherein the anti-human immunoglobulin antibody is labeled with a labeling compound. The reagent kit according to claim 4, wherein the labeling compound is an enzyme. An immunoassay method for an autoantibody to the RB1 gene product, wherein the reagent kit according to claim 4 or 5 is applied to a sandwich method, and the presence of the autoantibody to the RB1 gene product in the sample is determined by quantifying a labeled compound. An immunoassay method comprising the step of determining The immunoassay according to claim 6, which is performed by a chemiluminescent enzyme immunoassay. In the step of determining the presence or absence of an autoantibody to the RB1 gene product in a sample, blood is used as the sample, and a standard set based on the amount of anti-human immunoglobulin for this sample and the amount of anti-human immunoglobulin for the blood of a plurality of healthy persons The immunoassay according to claim 7 or 8, which comprises comparing with a value.