JP3742415B2 - Reagent for immunoassay for detecting autoantibody against RB1 gene product - Google Patents

Description

  The present invention relates to an immunoassay reagent for detecting an autoantibody against an RB1 gene product, an immunoassay reagent kit containing the same, and an immunoassay method for an autoantibody against 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 referred to as RB1 gene product). In various human cancers, it is known that deletion, point mutation, and the like occur frequently in the RB1 gene (Non-patent Documents 1 and 2). On the other hand, in lung cancer patients, it has been confirmed by Western blotting that autoantibodies that react with the RB1 gene product appear in the blood (Non-patent Document 3).

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

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

  As a result of intensive studies to achieve the above object, the present inventors have found that an autoantibody against the RB1 gene product can be detected simply, with high sensitivity and good reproducibility, and have reached the present invention. That is, the immunoassay reagent of the present invention is characterized by an immunoassay reagent for detecting an autoantibody against the RB1 gene product, which is a protein comprising the amino acid sequence contained in the amino acid sequence of SEQ ID NO: (1) as a constituent unit. The point is that the water-insoluble carrier is bound to the water.

  The reagent for immunoassay for detecting an autoantibody against the RB1 gene product of the present invention has few non-specific reactions and is extremely excellent in sensitivity and specificity. That is, the difference between the measured value of the sample from a healthy person who does not have autoantibodies and the measured value of the sample from cancer patients who have autoantibodies is sufficiently large (high sensitivity), and Highly accurate measurement is possible because there is very little non-specific reaction to the specimen. Moreover, since it can measure simply, many specimens can be measured easily in a short time. That is, the reagent for immunoassay of the present invention can detect an autoantibody against the RB1 gene product simply, with high sensitivity and with good reproducibility. Therefore, by using the measurement reagent, measurement reagent kit, and measurement method of the present invention, cancer can be diagnosed extremely quickly and accurately.

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

  The protein (P) having the amino acid sequence contained in the amino acid sequence of SEQ ID NO: (1) as a structural unit is the protein having the amino acid sequence of SEQ ID NO: (1) or a part of the amino acid sequence of SEQ ID NO: (1) as the structural unit (P1 , P2) and the like. The protein (P1) having the amino acid sequence of SEQ ID NO: (1) as a structural unit is difficult to prepare, and it is preferable to use a protein (P2) having a partial amino acid sequence of SEQ ID NO: (1) as a structural 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, more preferably 100 to 360, is a structural unit. For example, a protein having a structural unit of an amino acid sequence of SEQ ID NO: selected from the group consisting of SEQ ID NO: (2) (adenovirus E1A binding region) and (3) to (19) Can be mentioned. Among these, SEQ ID NOs: (2), (3), (5), (6), (7), (9), (11), (12), (13), (14), (15), A protein comprising the amino acid sequence of (17) or (18) as a structural unit is preferred, and more preferably SEQ ID NOs: (2), (3), (5), (6), (7), (9), (15 ), (17) or (18) as a structural unit.

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

  When the number of amino acids in 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 A protein expressed from a protein expression vector may be included. Examples of the protein expressed from the protein expression vector include glutathione transferase, a histidine tag (for example, a peptide in which six histidines are bound), a protein in the ZZ region of protein A, and the like. Among these, a protein expressed from a protein expression vector is preferable, and a glutathione transferase and a protein in the ZZ region of protein A are more preferable.

  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 structural unit is represented by GST (glutathione S- Proteins fused with transferase) are included. When the protein (P) is composed of 100 or more amino acid sequences as constituent units, a fusion protein is preferable from the viewpoint of preparation and the like.

  The protein (P) can be prepared by a conventionally known peptide synthesis method or a gene recombination method. According to the peptide synthesis method, if the number of amino acids exceeds 20, the synthesis of protein (P) is very difficult, whereas the genetic recombination method is easy, so the viewpoint of ease of preparation, etc. Therefore, the genetic recombination method is preferable.

  The peptide synthesis method can be achieved in solution or on a solid support, but 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 synthesis operations, types of side chain protection, and cleavage methods are described in, for example, Solid Phase Peptide Synthesis 2nd Edition, Peace Chemical Co., 1984 (Stewart and Young, “Solid Phase Peptide Synthesis”, No. 1). 2nd edition, Pierce Chemical Company, 1984), and Solid Phase Peptide Synthesis, IRL, 1989 (Atherton and Sheppard, “Solid Phase Peptide Synthesis”, IRL Press, 1989).

  As a gene recombination method, cDNA is prepared from mRNA extracted from human cells such as human vascular endothelial cells or human fibroblasts using primers corresponding to the target amino acid sequence, and this is used for PCR method (polymerase chain). Reaction fragment), and then cleaved with a restriction enzyme, this fragment is incorporated into a vector, and a host such as E. coli is transformed with the obtained expression vector. 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 Experimental Method 45 “Recombinant Protein Production Method”, Academic Publishing 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 RB1 gene amino acid sequence with a DNA synthesizer and then incorporating the gene into a vector for expression.

  The reagent for immunoassay of the present invention only needs to contain at least one kind of such protein (P), but it contains two or more kinds of proteins (P) to react with autoantibodies ( (Specificity and sensitivity) are preferable in terms of higher. In addition, even if it is a protein which uses amino acid sequence (1) as a structural unit, it is preferable that the other kind of protein is included. More preferably, in addition to the protein comprising the amino acid sequence of SEQ ID NO: (2) as a structural 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 includes a protein as a structural unit and / or a protein whose structural unit is the C-terminal region of SEQ ID NO: (1) {the amino acid sequence of SEQ ID NO: (8), (14) or (19)}. Most preferably, a protein having the amino acid sequence of SEQ ID NO: (2) as a structural unit, a protein having the N-terminal region of SEQ ID NO: (1) as a structural unit, and a protein having the C-terminal region of SEQ ID NO: (1) as a structural unit It is to include.

  Examples of preferable protein combinations of the immunoassay reagent of the present invention include 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 No. as a structural unit. That is, (2) + (3) represents a combination of a protein having the amino acid sequence of SEQ ID NO: (2) as a structural unit and a protein having the amino acid sequence of SEQ ID NO: (3) as a structural 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) 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 immunoagglutination. In the present invention, the protein (P) is used as an immunization reagent in the form of binding to the water-insoluble carrier (B). included. The water insolubility 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., and the carrier is any 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), those described in JP-A-2-205774 can be used, and inorganic and organic substances can be used. For example, cellulose, polystyrene, polypropylene, polyolefin, polyurethane, nitrocellulose, cellulose acetate Polyester, epoxy resin, phenol resin, silk, fibroin, lignin, hemicellulose, chitin, ebonite, rubber, glass, quartz, silicon and ceramics. Of these, polystyrene, glass, quartz and silicon are preferable, polystyrene and glass are more preferable, and glass is particularly preferable.

  The shape of the water-insoluble carrier (B) can be freely determined according to the purpose of use, and it is true spherical or disk-shaped beads, plate-shaped or rod-shaped sticks, test tubes, and non-woven fabric or filter strips (strip-shaped thin strips). Piece), fine particles, and the like. Of these, beads and fine particles are preferable, and true spherical beads are more preferable.

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 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 a disk-shaped bead, the diameter (mm) is preferably 1 to 10, more preferably 2 to 8, particularly preferably 3 to 7, and the thickness (mm) is preferably 0.1 to 5, more preferably. Is 0.2 to 2, particularly preferably 0.3 to 1. In the case of a stick, the length (mm) is preferably 2 to 10, more preferably 3 to 8, particularly preferably 4 to 7. Moreover, 1-25 are preferable, as for the cross-sectional area (mm < ² >) of a stick, More preferably, it is 2-16, Most preferably, it is 3-9. In addition, a cross-sectional area means the cross-sectional area of the cut part at the time of cut | disconnecting perpendicularly | vertically with respect to a major 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. Moreover, 5-20 are preferable, as for the internal diameter (mm) of a test tube, More preferably, it is 6-16, Most preferably, it is 8-12. 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. Moreover, 1-20 are preferable, as for the width | variety (mm) of a strip, More preferably, it is 2-16, Most preferably, it is 3-10. The thickness (mm) is preferably from 0.1 to 2, and more preferably from 0.1 to 0.5. The average pore diameter (μm) of the nonwoven fabric or filter is preferably 0.1 to 10, more preferably 0.3 to 5. In the case of fine particles, the average particle size (μm) is preferably 0.01 to 200, more preferably 0.1 to 50, and particularly preferably 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 method for binding the protein (P) and the water-insoluble carrier (B) can be performed by a conventionally known method such as a chemical binding method or a physical adsorption method. The chemical bonding method includes functional groups such as amino groups and / or sulfhydryl groups introduced on the surface of the water-insoluble carrier (B), and functional groups such as amino groups and / or sulfhydryl groups of the protein (P). (Eg, U.S. Pat. Nos. 4,280,992 and 3,565,761) and the like with a binder (glutaraldehyde, succinaldehyde, m-maleimidobenzoyl-N-hydrosuccinimide ester, o-phenylenebismaleimide, etc.) ) 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 0.001 to 0.04% (W / V) carbonate buffer aqueous solution (pH 9.0) of the protein (P). And the like (Bio-Sim Bio-Fids Actor, Vol. 251, 427, 1971). This method can also be applied to substances other than polystyrene, such as polypropylene, silicon, glass and cellulose. In addition, a protein (P) and a water-insoluble carrier (B) are indirectly bound using a specific binding substance (eg, 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 a water-insoluble carrier bound with avidin. As avidin, egg white-derived avidin and streptavidin can be used, and streptavidin is preferred. Among these specific binding substances, antigen-antibody, avidin-biotin, and lectin-sugar chain are those described in [Biochemical Experimental Method 11 “Enzyme Immunoassay”, Tokyo Chemical Dojinsha, 1989], complementary genes Examples include complementary genes described in JP-A-6-186232, such as a combination of polydeoxyadenylic acid and polythymidylic acid.

  Among these, 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 an avidin-biotin bond is particularly preferable. preferable. When the number of amino acids in the protein (P) is 100 or more, a chemical bonding method and a physical adsorption method are preferable, and a chemical bonding method is most preferable. When binding a protein having 20 to 100 amino acids and a protein having 100 or more amino acids, for example, avidin and a protein having 100 or more amino acids are mixed and bound to the water-insoluble carrier (B). Then, it is reacted with a protein modified with biotin.

  The immunoassay reagent of the present invention may be in a state of being immersed in a suitable buffer solution, but after binding the protein (P) and the water-insoluble carrier (B), it is coated with saccharide and / or protein and dried. preferable. As the buffer solution, a phosphate buffer solution, a Good buffer solution, or the like can be used, and it may contain a protein, a salt, and / or a surfactant. Proteins include albumin (bovine serum albumin, rabbit serum albumin, mouse serum albumin, ovalbumin, conalbumin, lactalbumin, etc.), antibodies (antibodies that do not bind to proteins (P) such as normal rabbit IlegG 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 names: Tween 20 and Tween 40, ICI America). Examples of the coating / drying method include a method of dipping in a solution containing at least a saccharide and then drying (Japanese Patent Application Laid-Open No. 09-318628 and Japanese Patent Publication No. 5-41946). As saccharides, disaccharides and monosaccharides are preferable, and as disaccharides, maltose, cellobiose, gentiobiose, melibiose, sucrose, lactose and isosaccharose can be mentioned, and monosaccharides include glucose, fructose, mannose, galactose, Examples thereof include arabinose and xylitol. Of these, lactose, sucrose, glucose and fructose are preferable, lactose and sucrose are more preferable, and sucrose is particularly preferable. When coating, the coating amount (content of saccharides, etc.), the concentration of the solution containing saccharides (coating solution), the kind of the solution, etc. may be within a known range. For example, JP-A-09-318628 and It is described in Japanese Patent Publication No. 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, those prepared by a conventionally known method can be used. For example, human immunoglobulin can be prepared by immunizing a suitable animal (eg, mouse, rabbit, pig, goat, horse, etc.) and purifying the antibody from the obtained antiserum by salting out, ion exchange column chromatography, etc. . 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 labeling compound from the viewpoints of sensitivity and ease of measurement. When the anti-human immunoglobulin antibody is not labeled with a labeling compound, an antibody that recognizes the anti-human immunoglobulin antibody (for example, when the anti-human immunoglobulin antibody is mouse IgG, an anti-mouse IgG antibody prepared in a rabbit) is labeled. A reagent or the like is used. As the labeling compound, conventionally known compounds can be used, and radioisotopes, fluorescent materials, luminescent materials, enzymes and the like are used. Examples of the isotope include ¹²⁵ I, examples of the fluorescent substance include europium complexes, examples of the luminescent substance include N-methyl acridium ester, and examples of the enzyme include horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Etc. Among these labeling compounds, enzymes are preferable, horseradish peroxidase, alkaline phosphatase and β-galactosidase are more preferable, and horseradish peroxidase and alkaline phosphatase are particularly preferable.

As a method for labeling a labeled compound to an anti-human immunoglobulin antibody, a conventionally known method or the like can be applied. “Second Biochemistry Experiment Course 5 Immunobiochemistry Experiment Method” (Edited by the Japanese Biochemical Society, Tokyo Chemical Dojin, 1986, 102 To the method described on page 112, for example, the following methods (1) to (4) can be applied.
(1) A method of introducing radioactive iodine into a tyrosine residue of an anti-human immunoglobulin antibody using chloramine T as an oxidizing agent when the labeled compound is an isotope.
(2) A method in which when the labeling compound is a fluorescent substance, fluorescein isothiocyanate is reacted with an anti-human immunoglobulin antibody in a buffer and bound to a lysine residue of the anti-human immunoglobulin antibody.
(3) When the labeling substance is a luminescent substance, the product name “acridinium derivative-I” (manufactured by Doujin Chemical Laboratory Co., Ltd.) is reacted with an anti-human immunoglobulin antibody in a buffer solution to form an amino group of the anti-human immunoglobulin antibody. How to combine.
(4) A method in which when the labeling substance is an enzyme, the amino group possessed by the enzyme and the thiol group possessed by the anti-human immunoglobulin antibody are bound by a bi-crosslinking reagent such as N-succinimidyl-6-maledohexanoate.

  As long as the immunoassay reagent kit of the present invention includes the immunoassay reagent of the present invention, the reagent dosage form and the configuration of the reagent kit are not limited. That is, the reagent kit of the present invention can be applied to homogeneous immunoassay methods such as latex agglutination method and latex turbidimetric method as well as heterogeneous immunoassay methods. Among these, from the viewpoint of measurement sensitivity, a heterogeneous immunoassay method is preferable, and a reagent kit for a heterogeneous sandwich immunoassay labeled with a labeled compound and containing an anti-human immunoglobulin antibody is most preferable. That is, when the immunoassay reagent of the present invention is reacted with the sample, only the autoantibody against the RB1 gene product specifically binds to the protein (P). Therefore, when an anti-human immunoglobulin antibody is added, this antibody binds to an autoantibody to form an immune complex “protein (P) -autoantibody-anti-human immunoglobulin antibody”. The amount of autoantibodies can be quantified by quantifying the anti-human immunoglobulin antibody in the immune complex. 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 labeling compound can be measured by a conventionally known method or the like depending on the type of the labeling 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 with a photomultiplier tube. When the labeling compound is a chemiluminescent substance, for example, in the case of an acridinium ester, the amount of luminescence generated by adding an alkaline solution is quantified with a photomultiplier tube.

  When the labeling compound is an enzyme, the enzyme activity can be measured as absorbance (absorbance measurement method), fluorescence amount (fluorescence amount measurement method) or luminescence amount (chemiluminescence amount measurement method) by reacting with an appropriate substrate. For example, when the enzyme is peroxidase, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) (absorbance measurement method) and luminol / peroxide (chemiluminescence) are used as substrates. (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 measurement method) and the like can be selected. Absorbance is quantified with a spectrophotometer, and fluorescence and chemiluminescence are quantified with a photomultiplier tube. Of these, the chemiluminescence measurement method is preferred (that is, the chemiluminescence enzyme immunoassay method is preferred as the method for quantifying autoantibodies using the reagent kit of the present invention), more preferably peroxidase and luminol / peroxidation. This is a chemiluminescence measurement method based on a combination of substances or 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 (sodium and potassium etc.) salts, alkaline earth metal (calcium and magnesium etc.) salts and the like can be used. Of these, luminol and luminol metal salts are preferred, luminol metal salts are more preferred, and luminol sodium salts are particularly preferred. As the peroxide, both inorganic peroxides and organic peroxides can be used. Inorganic peroxides include hydrogen peroxide, sodium perborate, potassium perborate, peroxy acids, peroxycarbonic acid, peroxydicarbonate, hypochlorous acid, potassium hypochlorite, chlorous acid, chlorine. Examples thereof include acid, sodium chlorate, perchloric acid, perbromic acid, peroxosulfuric acid and peroxophosphoric acid. Examples of the organic peroxide include peracetic acid, perpropionic acid, dimethyl sulfoxide (DMSO), triethylamine oxide, methyldiethylamine oxide, and phthaloyl peroxide. Among these, from the viewpoint of storage stability and the like, an inorganic peroxide is preferable, and hydrogen peroxide is more preferable.

  In the reagent kit for immunoassay of the present invention, a reagent containing anti-human immunoglobulin is preferably added to the reagent (reagent for immunoassay of the present invention) formed by binding the protein (P) and the water-insoluble carrier (B). In addition, an immune reaction buffer, a B / F separation buffer, a control sample, and the like can be further included. As the buffer solution for immune reaction and the buffer solution for B / F separation, a buffer solution and the like conventionally used for immunoassay can be used, and a phosphate buffer solution containing a protein, salt and / or a surfactant, and Good (Good) ) Buffer solution or the like can be used. Examples of the protein, salt, and surfactant include the same as described above.

  The control sample is used as a comparative sample for determining whether or not an autoantibody against the RB1 gene product is present in the specimen. Usually, a sample not containing an autoantibody (negative control) and a sample containing an autoantibody (positive control) ) Is prepared. As a negative control, a buffer containing the same protein as the immune reaction buffer, a human pool serum that does not have an autoantibody against the RB1 gene product, and the like can be used. As a positive control, a human pool serum having an autoantibody against the RB1 gene product, a buffer solution containing serum containing an autoantibody against the RB1 gene product (containing the same protein as the immune reaction buffer solution), etc. can be used. .

  The specimen 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 examples thereof include blood, urine, saliva, lymph fluid, bile, gastric fluid, pancreatic juice, and the like, and further collected from a living body. A homogenate extract of the prepared tissue can also be used. Of these, blood and urine are preferable, and blood (including whole blood, serum, or plasma) is more preferable.

Specific examples (steps 1 to 6) of a method (sandwich assay) for detecting an autoantibody against the RB1 gene product using the reagent kit of the present invention are shown below.
Step 1. A sample is reacted with an immunoassay reagent (immunoassay reagent of the present invention) in which a protein (P) and a water-insoluble carrier (B) are bound to react a reaction mixture (including the complex 1 in Step 2). obtain.
Step 2. Unreacted substances are removed from the reaction mixture of step 1 (B / F separation) to obtain 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 of step 3 (B / F separation) to obtain complex 2.
Step 5. The amount of the labeled compound of complex 2 is measured.
Step 6. The amount of the labeled compound is used to compare with a reference value (negative control and / or positive control) to determine the presence or absence of autoantibodies.

  In the step of determining the presence or absence of autoantibodies to the RB1 gene product in the sample (step 6 in the above example), the amount of the anti-human immunoglobulin antibody against the blood as the sample (measured as the amount of the labeled compound labeled with the anti-human immunoglobulin) And the reference value set from the amount of anti-human immunoglobulin (measured as the amount of labeled compound labeled with anti-human immunoglobulin) in the blood of a plurality of healthy individuals, It is preferable to determine the presence or absence. The reference value setting method is described in, for example, “Clinical Laboratory Guide 1999-2000, p64-71, Bunkodo (1999)”, “Clinical Laboratory Data Book 1997-1998, p8-13, Medical School (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 to determine the amount of the labeled compound (in the case of chemiluminescent enzyme immunoassay, this is the amount of luminescence). Statistical processing of the amount of labeled compound {parametric method, nonparametric method, etc. [parametric method is used when the population shows a normal distribution (or can be a power transform), nonparametric method is used when the population is not normally distributed used. ] Is set as a reference range for obtaining a range including 95% of the sample group, and an upper limit is set as an upper limit reference value and a lower limit is set as a lower limit reference value. Therefore, a specimen that exceeds the upper reference value is highly likely to have an autoantibody, but may be exceeded by a healthy person with a probability of 5%. In order to prevent this, usually, a cutoff value obtained by further multiplying and / or adding a coefficient to the upper limit reference value is set. That is, a sample that exceeds the cut-off value is determined as having an autoantibody. The cut-off value is set based on the relationship between the upper limit reference value and the lower limit value of the distribution of specimens having autoantibodies against the RB1 gene product, but is usually about 1.5 to 3 times the upper limit reference value. .

  The negative control and the positive control described above are prepared so that the set cutoff value can be easily reproduced. For example, it is possible to set such that the measured value of the negative control (labeled compound amount) is 3 times the cutoff value, or the average value of the negative control and the positive control is the cutoff value.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.

<Preparation Example 1> Production 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) A fusion protein was prepared as follows.

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

(Preparation of AGPC reagent)
(1) Preparation of 1M 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 to pH 7.0, and then distilled water at 25 ° C. To make 100 ml. And it was used after autoclaving (120 degreeC, 20 minutes).
(2) Preparation of solution D GTC (guanidinium thiocyanate) 236.3 g (4 M), sarkosyl (L-Lauroylsarcosine) 2.5 g (0.8 wt%), 1 M sodium citrate buffer (pH 7.0) 12 0.5 ml (25 mM) and 250 ml of distilled water were uniformly dissolved at 85 ° C., then returned to room temperature (25 ° C.), and adjusted to 496 ml with distilled water. It filtered and preserve | saved with the 0.45 micrometer pore diameter bottle top filter. At the time of use, 360 μl (0.1 M) of 2-mercaptoethanol was added to 50 ml of this stock solution to prepare a D solution.
(3) Preparation of 2M sodium acetate (pH 4.0) 27.2 g of sodium acetate trihydrate was dissolved in 10 ml of distilled water, adjusted to pH 4.0 with acetic acid, and distilled water was added at 25 ° C. 100 ml. It was used after being filtered through a bottle top filter having a pore diameter of 0.45 μm.

(Operation of AGPC method)
(1) 0.5 ml of solution D was added to the tube to disperse human umbilical vein endothelial cells and 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 each time one type was added, the tube was shaken 2-3 times and mixed.
(3) After vigorous mixing for 10 seconds, the mixture was ice-cooled for 15 minutes.
(4) After centrifuging at 10 ° C. 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 the intermediate layer containing DNA was not mixed.
(5) To the collected aqueous layer, 0.5 ml of isopropanol was added and cooled at -20 ° C for 1 hour. Next, centrifugation was performed for 10 minutes (10,000 G, 0 ° C.) to precipitate RNA.
(6) The precipitated RNA is dissolved again in 0.5 ml of solution D, 0.5 ml of isopropanol is added, and the mixture is placed at -20 ° C for 1 hour.
(7) The RNA is precipitated by centrifugation, and the precipitate is washed with 1 mL of 80 wt% aqueous ethanol, dried, and then dissolved in water.

  From the RNA thus extracted, cDNA was synthesized and amplified using the 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 the ready-to-go RT-PCR beads, reacted at 42 ° C. for 1 hour, and then heated at 95 ° C. for 5 minutes. After treatment, cDNA was synthesized. Subsequently, a 5 ′ primer is added to the reaction solution containing cDNA, and PCR (polymerase chain reaction) is performed by denaturation reaction (95 ° C., 1 minute), annealing reaction (60 ° C., 1 minute), extension reaction (72 ° C., 1 minute). Min) using a PCR thermal cycler (MP PCR thermal cycler, manufactured by Takara Shuzo Co., Ltd.) in 30 cycles to obtain an amplified cDNA.

2. Preparation and expression of recombinant gene Molecular cloning in the BamHI / EcoRI site of the expression vector pGEX-4T-3 (purchased from Amersham), Laboratory Manual 2nd edition (Molecular Cloning, A Laboratory Manual, 2nd ed ), Incorporated in 1989. A medium containing 0.1 mM isopropylthiobetagalactoside (IPTG) after confirming that the insertion direction of cDNA is correctly integrated by sequencing and then transforming into E. coli BL21 (DE3), LysS (Novagen) Induction was carried out at 37 ° C. for 2 hours. The target fusion protein is purified from the cell lysate of Escherichia coli prepared by ultrasonic disruption by affinity chromatography using a glutathione-binding affinity gel (trade name: glutathione sepharose 4B, manufactured by Amersham) (eluent: 10 mM reduced form) 0.02M Tris (hydroxymethyl) aminomethane / hydrochloric acid buffer containing glutathione) to prepare GST fusion protein GST1 {protein having the amino acid sequence of SEQ ID NO: (2) as a structural unit}.

<Creation examples 2-7>
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 1000 glass beads with a diameter of 3.2 mm (manufactured by Immunochemical) were added to a polyethylene bottle with a lid containing 20 mL of an acetone solution containing 1% by weight γ-aminopropyltriethoxysilane, and the temperature was 25 ° C. for 1 hour. The reaction residue was removed by suction with an aspirator.
Next, 20 mL of deionized water was added, the cap was closed, the polystyrene bottle was slowly inverted and stirred twice, and then the liquid was sucked and removed 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 wt% 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 cap was capped, the polystyrene bottle was slowly inverted and stirred twice, and the liquid was sucked and removed with an aspirator to wash the glass beads. This washing operation was performed three times.
Furthermore, 1000 glass beads after washing were added to a polyethylene bottle with a lid containing 20 mL of 0.02M phosphate buffer (pH 8.7) containing GST1 at a concentration of 20 μg / mL, and allowed to react at 25 ° C. for 1 hour. It was.
After the reaction, the GST1-containing phosphate buffer was removed and immersed in 20 mL of 0.1 wt% bovine serum albumin-containing phosphate buffer (pH 7.2) at 4 ° C. for 8 hours. Thereafter, the buffered aqueous solution is removed by suction with an aspirator, and the beads are washed twice with 20 mL of a phosphate buffer solution (pH 7.2) containing 0.1% by weight of bovine serum albumin, and then phosphate containing 10% by weight of sucrose. After being immersed in a buffer solution (pH 7.2) for 30 minutes, the phosphate buffer solution is removed with an aspirator, the beads are spread on a filter paper and air-dried at room temperature (about 25 ° C.), and the GST fusion protein-bound beads 1 (immunoassay of the present invention) Reagent 1) was prepared and stored refrigerated (2 to 10 ° C.) in a sealed container containing a desiccant (silica gel).

2. Preparation of buffer for immune reaction To 0.02M phosphate buffer (pH 8.0), casein was added to a concentration of 3 g / L and sodium chloride to a concentration of 8.5 g / L, and buffer for immune reaction It was created. 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, Etol; Jay. Biochem, Vol. 92 (1982) 1413-1424], a peroxidase-labeled anti-β2-microglobulin polyclonal antibody [a solution of 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 Using the immune reaction buffer and peroxidase-labeled anti-human immunoglobulin antibody prepared above, an enzyme-labeled antibody solution was prepared as follows. That is, 100 μg of a peroxidase-labeled anti-human immunoglobulin antibody was added to 100 mL of an immune reaction buffer in a protein amount, and the mixture was stirred and mixed to obtain an enzyme-labeled antibody solution.

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

6). Preparation of substrate solution 0.18 g of luminol (manufactured by Tokyo Chemical Industry) and 0.1 g of 4- (cyanomethylthio) phenol (manufactured by Sanshin Chemical Co., Ltd.) at 0.1 M (mol / L), pH 8.5, tris (hydroxymethyl) amino 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) in the same manner as in Example 1 except that GST fusion proteins shown in Table 2 were 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 buffer for immune reaction, peroxidase-labeled anti-human immunoglobulin antibody, enzyme-labeled antibody solution, hydrogen peroxide solution and substrate solution.

<Example 11>
It is the example which measured healthy person pool serum and cancer patient serum.
1. Sample Specimens 1 were prepared by mixing 0.2 mL each of serum collected from 50 healthy volunteers to prepare 10 mL of pooled serum from healthy individuals. In addition, 10 mL each of 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 solution, 10 μL of specimen 1 (pooled serum of a healthy person) 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 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 washed twice in the same manner. The enzyme activity of the washed beads was measured as follows. In addition, enzyme activities were measured in the same manner for specimens 2 to 4.

3. Enzyme activity measurement procedure Set the test tube (12 x 75 mm) containing the washed beads in a sample holder of the luminescence reader BLR-201 manufactured by Aloka, and add chemiluminescence reaction by adding 200 µL of the substrate solution and 200 µL of hydrogen peroxide. Started. Forty seconds after starting the luminescence reaction, the amount of luminescence for 10 seconds was integrated and measured, and this was taken as the amount of luminescence showing enzyme activity.

4). Measurement results Table 2 shows the measurement results of the luminescence. The numerical values in Table 2 indicate the amount of luminescence when the upper row indicates the luminescence amount, and the lower row indicates the relative value of each luminescence amount when the luminescence amount of the healthy human pool serum is 1.0.
In cancer patients, the measured luminescence level is higher than the luminescence level of the healthy human pool serum, and the luminescence level ratio (the value in the lower part of the table divided by the measured luminescence level of the healthy human pool serum) is large. I understand. It can also be seen that the sensitivity of the patient serum is improved by combining a plurality of GST fusion proteins, even if the sera of patients have low reactivity to individual GST fusion proteins. In particular, the combination including the entire region of the E1A binding region has a high light emission amount ratio and improved sensitivity.

<Comparative Example 1>
This comparative example is an example in which measurement is performed by a method according to the prior art.
1. Blotting of GST fusion protein 1 μg 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 Pharmacia's “Fast System”. For electrophoresis, Pharmacia “Fast Gel Homogenous 12.5” was used as a gel, and Pharmacia “Fast Gel Dies Buffer Strips” was used as a running buffer, at a temperature of 15 ° C. and a voltage of 250 V. Electrophoresis was performed in 2 hours. The GST protein was blotted from the electrophoresed gel on a nitrocellulose membrane (Millipore) using “PhstTransfer semi-drying” manufactured by Pharmacia. As the blotting buffer, 25 mM Tris (hydroxymethyl) aminomethane buffer (pH 8.3) containing 192 mM glycine and 10% by volume methanol was used. Blotting was performed at a temperature of 15 ° C., a voltage of 20 V, and electrophoresis for 30 minutes.

2. Detection of autoantibodies in specimen This membrane was immersed in 0.02M phosphate buffer (pH 7.0) containing 5% by weight of nonfat dry milk at room temperature (25 ° C) for 8 hours. The membrane was removed, and specimen 1 (healthy human pool serum) was immersed in a 400-fold diluted solution containing 0.02M phosphate buffer (pH 7.0) containing 5% by weight of nonfat dry milk and reacted at 25 ° C. for 3 hours. . Subsequently, the filter was taken out and left standing in a 0.02M phosphate buffer solution (7.0) containing 0.1% Tween 20 at 25 ° C. for 1 hour, and then the filter was washed by removing the solution 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 washing was performed 6 times in the same manner. 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-described washing was performed 5 times (however, the standing time was 10 minutes), the filter was air-dried at 25 ° C., and the resulting colored band was visually confirmed. Similarly, the colored bands were visually confirmed for Samples 2 to 4 (the sera of cancer patients 1 to 3).

3. Evaluation Compared to the band (normally very weak or extremely weak) in the case of healthy human boulesse serum, the result is expressed as-, slightly strong case +, and strong case + in the same level as the healthy boule serum. Are shown in Table 3. Even if there was a clear S / N difference in Example 11, there was a case where Comparative Example 1 was negative or positive. Further, the determination of Comparative Example 1 is a subjective judgment by visual observation, and is so delicate that there is a possibility that the result may be different depending on the determiner. On the other hand, since the method of the present invention can be clearly measured as a numerical value as shown in Example 11, there is no such problem.

<Example 12>
This is an example in which a plurality of healthy human sera are measured to determine a reference value, a cut-off value is set, serum from 10 cancer patients is measured, and the presence or absence of autoantibodies to the RB1 gene product is determined.

1. Sample Measurement Using the GST fusion protein (GST4, GST5, GST6, GST2, and GST7) binding beads 10 prepared in Example 10, in the same manner as in Example 11, serum samples from 120 healthy volunteers and Specimen 1 of Example 11 (a healthy human pooled serum) was measured.
The distribution of measured values (light emission amount) of 120 healthy volunteers is shown in FIG. In the figure, the horizontal axis represents the light emission amount (cps), and the points represent the distribution of the specimen (one point corresponds to one specimen).

2. Setting of cut-off value Results of analyzing distribution values of serum specimens and specimen 1 measured from 120 healthy volunteers using statistical processing software “STATFLEX v. 4.1” (manufactured by Artec) The light emission amount distribution had a skewness of 1.49 and a kurtosis of 4.90. 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 is set as the cutoff value, the light emission amount is 11321. At this time, the amount of luminescence of specimen 1 (pooled serum of healthy subjects) was 3406, which was 1 / 3.32 of the cutoff value. Therefore, when Specimen 1 (healthy person pool serum) was measured as a negative control, the cut-off value was set as “healthy person pool serum measured luminescence amount” × 3.32.

3. Measurement of Specimens Serum specimens 5 to 14 collected from 10 cancer patients (patients 4 to 13) and specimen 1 (healthy human pool serum) prepared in Example 11 as a negative control were measured. The measurement was performed in the same manner as in Example 11 using the GST fusion protein-bound bead 10 of Example 10.

4. Judgment result Specimen 1 (a healthy human pool serum) was used as a negative control, and a cut-off value (“amount of healthy human pool serum measured luminescence” × coefficient) was determined using the coefficient set in 2 above. A measured value with a luminescence level greater than or equal to the cutoff value (cutoff ratio 1.0 or higher) is positive for autoantibodies, while a measured value with a luminescence level less than the cutoff value (with a cutoff ratio less than 1.0) is negative for autoantibodies. It was judged. These results are shown in Table 4.

  From Table 4, autoantibodies against the RB1 gene product were determined to be 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 cancer diagnosis.

<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 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) in the same manner as in Example 1 except that the GST fusion proteins shown in Table 6 or 7 were used instead of GST1. The measuring reagents 11 to 26) 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 buffer for immune reaction, 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 are shown in Tables 6 and 7 (the numerical values in Tables 6 and 7 indicate the luminescence level at the top and the luminescence level of the healthy human pool serum as 1.0. This is the relative value of each light emission amount.
In cancer patients, the measured luminescence level is higher than the luminescence level of the healthy human pool serum, and the luminescence level ratio (the value in the lower part of the table divided by the measured luminescence level of the healthy human pool serum) is large. I understand. It can also be seen that the sensitivity of the patient serum is improved by combining a plurality of GST fusion proteins, even if the sera of patients have low reactivity to individual GST fusion proteins. In particular, the combination including the entire region of the E1A binding region has a high light emission amount 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. And compared to the band (normally very weak or extremely weak) in the case of healthy human boule serum, the case of the same level as that of the normal boule serum is represented by-, slightly strong case is represented by ±, and strong case is represented by +. The results are shown in Table 8. Even when there was a clear S / N difference in Example 29, in Comparative Example 2, it could be negative or positive. Further, the determination of Comparative Example 2 is a subjective judgment by visual observation, and is so delicate that there is a possibility that the result may be different depending on the determiner. On the other hand, since the method of the present invention can be clearly measured as a numerical value as shown in Example 29, there is no such problem.

It is the graph which showed distribution of the luminescence amount measured about serum of 120 healthy volunteers using the GST fusion protein binding bead (reagent 10 for immunoassay of this invention) (Example 12).

Claims (8)

A reagent for immunoassay for detecting an autoantibody against 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 structural unit and a water-insoluble carrier (B) A reagent for immunoassay characterized by being bound. The reagent for immunoassay 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). A reagent kit for immunoassay comprising the reagent according to claim 1 or 2 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. A method for immunoassay of an autoantibody against an RB1 gene product, wherein the presence or absence of an autoantibody against the RB1 gene product in a sample by applying the reagent kit according to claim 4 or 5 to the sandwich method and quantifying the labeled compound An immunoassay method comprising the step of determining The immunoassay method according to claim 6 by chemiluminescence enzyme immunoassay. In the step of determining the presence or absence of autoantibodies to the RB1 gene product in a sample, blood is used as the sample, and a standard set from the amount of anti-human immunoglobulin for the sample and the amount of anti-human immunoglobulin for the blood of a plurality of healthy individuals The immunoassay method according to claim 7 or 8, comprising comparing the value.