Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
  • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
  • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
  • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/961—Chemistry: molecular biology and microbiology including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/815—Test for named compound or class of compounds
  • Y10S436/817—Steroids or hormones
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/825—Pretreatment for removal of interfering factors from sample

Definitions

• the present invention relates to a method for measuring an endocrine substance in a specimen, particularly a specimen such as cat blood. More specifically, the present invention relates to a method for measuring an endocrine substance in a specimen such as cat blood for measuring an endocrine substance such as insulin in order to diagnose and treat a disease such as diabetes in a cat.
• the disease is first identified by identifying the causative substance or the related substance of the disease, and measuring the quantitative change of the active substance. It is necessary to check the possibility of illness or illness.
• power lifestyle-related diseases such as lack of exercise due to changes in the living environment are a major social problem in addition to high calorie intake due to diets with a high protein content due to changes in lifestyle habits including dietary habits. It has become.
• lifestyle-related diseases diabetes is increasing not only in patients but also in the reserve army! / Being a serious problem.
• it is necessary to measure the amount of human insulin, which is an endocrine substance of diabetes-related substances in the blood. It has become.
• mammals including cats and dogs such as cats which are not only humans, are also increasing diseases equivalent to lifestyle-related diseases due to changes in the breeding environment.
• high protein-centered and high-calorie diets such as pet food
• diseases such as diabetes due to lack of exercise due to rearing in small rooms are increasing. Therefore, for example, it is needless to say that for the diagnosis and treatment of feline diabetes, it is necessary to measure feline insulin in the feline blood.
• the current diagnosis method has a serious problem in that it cannot grasp the exact cause of diabetic cats, so that appropriate treatment cannot be performed. Therefore, especially in the diagnosis and treatment of animal diseases, establishment of a method for directly measuring the endocrine substances of the animal itself is desired.
• a cat is described as an example
• a cat insulin is described as an example
• the present invention interprets a sample as a cat or an endocrine substance as a cat insulin.
• any sample and any endocrine substance can be applied as long as they are suitable for the present invention.
• an immunoassay As a method for detecting an antigenic substance in a body fluid such as serum or measuring the concentration of the antigenic substance, an immunoassay has been increasingly used in recent years. Such a method is a well-known method, for example, by labeling an antigenic substance or antibody with a radioactive substance, forming a complex between the strong antigenic substance and the antibody, and measuring the labeling component. Thus, the antigenic substance to be measured can be detected and its concentration can be measured.
• Patent Document 1 Japanese Patent Laid-Open No. 11-326322
• the present invention eliminates autoantibodies bound to endocrine substances such as feline insulin present in blood samples of animals such as cats, thereby accurately and rapidly accelerating endocrine substances such as feline thrin in specimens. It is an object of the present invention to provide a method for measuring an endocrine substance in a specimen that can be measured in a simple manner.
• Another object of the present invention is to provide an autoantibody removal method comprising adding an acid to a specimen and separating and removing autoantibodies bound to an endocrine substance in the specimen by subjecting the specimen to force separation.
• Another object of the present invention is to provide a method for measuring an autoantibody titer, which comprises measuring the antibody titer of an autoantibody bound to an endocrine product in a specimen.
• Another object of the present invention is to provide an evaluation method comprising evaluating the diagnostic method or the treatment method by the autoantibody titer measuring method. Furthermore, another object of the present invention is to provide a diagnostic method and a therapeutic method comprising a combination of the autoantibody titer measuring method and the endocrine substance measuring method.
• Another object of the present invention is to provide an endocrine substance measurement kit capable of measuring an endocrine substance, preferably cat insulin.
• the present invention removes autoantibodies bound to endocrine substances such as cat insulin present in blood samples of animals such as cats.
• a method for measuring an endocrine substance in a specimen which can accurately and rapidly measure an endocrine substance such as cat insulin in the sample.
• the present invention provides a sample pretreatment method comprising removing autoantibodies bound to endocrine substances in a sample from the sample.
• the present invention provides an autoantibody removal method comprising adding an acid to a specimen and separating and removing autoantibodies bound to an endocrine substance in the specimen by subjecting the specimen to force separation.
• an autoantibody titer measuring method comprising measuring an antibody titer of an autoantibody binding an endocrine product in a specimen.
• the present invention provides an endocrine substance measuring method comprising measuring an endocrine substance in a specimen pretreated by the specimen pretreatment method.
• the present invention further provides a diagnostic method and a therapeutic method comprising diagnosing the endocrine substance-related disease by measuring the autoantibody titer of the endocrine substance in the specimen by the above autoantibody titer measurement method. provide.
• this invention provides the evaluation method which consists of evaluating the said diagnostic method or the said treatment method by the said autoantibody titer measuring method as another form.
• the present invention further provides a diagnostic method and a therapeutic method comprising a combination of the autoantibody titer measuring method and the endocrine substance measuring method.
• the present invention further provides an endocrine substance measurement kit capable of measuring an endocrine substance, preferably cat insulin.
• the measurement method of substances such as human insulin has been applied mutatis mutandis. It has a great effect that it can measure itself and endocrine substances such as cat insulin can be measured more accurately and quickly.
• an enzyme immunoassay method or the like commonly used in the technical field without using a radioactive substance can be used, and the present invention is extremely simple. If you can handle it!
• FIG. 1 is a flowchart showing a method for confirming anti-insulin autoantibodies in cat blood.
• FIG. 2 is a graph showing the measurement results of feline insulin autoantibody (IgG-insulin complex).
• FIG. 3 is a graph showing measurement results of insulin-binding insulin autoantibody titer and anti-mouse IgG antibody titer.
• FIG. 4 is a graph showing the measurement results of feline serum mono-insulin-sepharose chromatography.
• FIG. 5 is a graph showing measurement results of insulin autoantibody titer.
• FIG. 6 is a graph showing the measurement results of mouse rheumatoid factor (RF) antibody titer.
• FIG. 7 is a graph showing the results of measuring the anti-cat insulin calorie value of the chromatographic eluate.
• FIG. 8 is a graph showing a cat insulin standard curve.
• FIG. 9 is a graph showing the correlation between a cat insulin standard product and a rat insulin standard product.
• Autoantibodies against endocrine substances such as cat insulin are produced in the blood of cats and the like, and some of them exist in a state bound to the endocrine substances.
• the endocrine substance can be measured.
• the insulin-binding insulin autoantibody titer and the anti-mouse-one IgG autoantibody titer are measured to detect the anti-insulin cat antibody and the anti-mouse IgG (rheumatic factor) antibody.
• some autoantibodies against endocrine substances such as feline insulin produced in the blood are also present in a non-bonded state with these endocrine substances.
• adsorb and remove the autoantibodies on a suitable adsorbent such as an endocrine-binding chromatography column, and measure the eluted endocrine unbound autoantibody titer. Is good.
• a method for selecting a treatment method such as a hyperglycemic cat or determining its therapeutic effect a method for selecting a treatment method such as a cat for autoimmune disease, or a method for determining its therapeutic effect, etc. Can be applied.
• the autoantibody titer against endocrine substances such as insulin when the autoantibody titer against endocrine substances such as insulin is measured, it is necessary to remove these autoantibodies. In other words, it is necessary to perform a pretreatment for removing the self-antibodies from the specimen sample to be measured.
• an insulin autoantibody titer should be established to confirm that insulin autoantibodies have been removed. After removing the insulin autoantibodies in this way, the amount of insulin in the feline blood sample is measured.
• the insulin-binding insulin autoantibody before administration of the insulin preparation it can be applied to the selection of the insulin preparation and its administration method. Furthermore, by determining the insulin-binding insulin autoantibody of the cat individual, it can be applied to the determination of the therapeutic effect after administration of the insulin preparation.
• specimens that can be used in the present invention include specimens collected from pets such as cats such as cats, for example, body fluids such as blood collected from pets such as cats, pancreas, etc. Organ samples or powerful tissues.
• a sample such as serum or plasma, whether it is whole blood or serum or plasma separated from a blood sample, can be prepared from a blood sample by a conventional method. it can.
• the endocrine substances that are the subject of this invention are, for example, organs and tissues of pets such as cats. It is a substance that is secreted into throat and body fluids and can act as a marker useful for diagnosing the pathology of a specific disease or treating a disease.
• endocrine substances include feline insulin useful for diagnosing and treating diabetes.
• the endocrine substance derived from the animal species being measured can be measured by comparing the amino acid sequence of the endocrine substance derived from the animal species being measured with the amino acid sequence of the endocrine substance derived from another animal species.
• the endocrine substance to be measured is feline insulin
• feline insulin it has been determined that feline insulin can be measured using insulin of a rat, which is another animal species.
• a sandwich method using a feline insulin A chain antibody and a rat insulin B chain antibody may be used.
• Table 1 (insulin A chain) and Table 2 (insulin B chain) compare the amino acid sequences of cat insulin and rat insulin.
• A alanine
• C cystine
• D asparagine acid
• E glutamic acid
• F ferulalanin
• G glycine
• H histidine
• I isoleucine
• K lysine
• L leucine
• M methionine
• N asparagine
• P Proline
• Q Glutamine
• R Arginine
• S Serine
• T Threonine
• V Palin
• Y Tyrosine
• feline insulin can measure feline insulin using rat insulin. It can be determined that measurement is possible.
• autoantibodies against endocrine substances are produced in the blood of animals such as cats, and in the blood, the autoantibodies are not bound to the endocrine substances. It exists even in a state where it is combined with endocrine substances. Therefore, in order to measure this endocrine substance, it is difficult to measure if the endocrine substance-binding autoantibodies are present in the blood sample. Therefore, the endocrine substance is another substance in the blood. It is a condition that it is not combined with. Therefore, first, it is necessary to measure whether or not there is a substance bound to the endocrine substance, and to measure the value of the autoantibody against the endocrine substance. As a result of this measurement, if autoantibodies in a state of being bound to endocrine substances are present, the strong endocrine substance-bound autoantibodies must be removed from the blood sample.
• feline serum using insulin-binding insulin autoantibodies or anti-mouse IgG autoantibodies is conventionally used in the art.
• An example of measurement according to the ELISA method will be described.
• Insulin-bound insulin autoantibodies can be measured, for example, by adding an anti-insulin antibody to a feline serum sample and reacting the anti-insulin antibody with the antigen in the sample by an antigen-antibody reaction, followed by enzyme-labeled goat anti-cat IgG React with. After completion of the reaction, the color can be developed using an enzyme substrate, and a predetermined absorbance can be measured.
• the measurement of anti-mouse IgG autoantibodies can be performed in substantially the same manner as the measurement of insulin-bound insulin autoantibodies.
• any conventional enzyme used in ELISA can be used.
• peroxidase alkaline phosphatase, ⁇ D galactosidase, glucose oxidase, luciferase, esterase , ⁇ D dalc mouth-dase and the like.
• Peroxidase or alkaline phosphatase is desirable from the viewpoint that it is possible to achieve more sensitive and stable detection.
• the enzyme substrate can be appropriately selected depending on the enzyme used. For example, 3,3 ′, 5,5′-tetramethylbenzidine or the like is used in the case of peroxidase, and para-trophe-in the case of alkaline phosphatase. Sodium luphosphate or the like is used.
• the detection and quantification of the product produced by the enzyme as described above can be performed by measuring the absorbance of the product. For example, if 3, 3 ', 5, 5'-tetramethylbenzidine is used as the enzyme substrate, the absorbance at 450 nm should be measured.
• a purified endocrine substance can be obtained by preparing a homogenate by adding a hydrochloric acid solution to a specimen such as a feline pancreas and purifying the resulting homogenate according to a conventional method.
• Cat insulin can be purified according to known human insulin purification methods.
• feline insulin is obtained by centrifuging a homogenate prepared by adding a hydrochloric acid solution to a specimen such as a feline pancreas, separating the supernatant, and purifying the separated supernatant by a conventional method such as chromatography. can do.
• a protein purification carrier Flatato gel which is known as a biopolymer purification carrier for proteins and nucleic acids. Since this carrier has a higher protein binding capacity than that of an ordinary protein purification carrier, it is more preferable for the purification of a large amount of proteins, antibodies and the like.
• Purified feline insulin can be obtained by fractionating a fraction having a predetermined peak from a fraction obtained by further purifying the eluate obtained as described above by a conventional method such as chromatography.
• chromatography it is convenient to use, for example, a high performance reverse phase chromatography column for biomolecule purification.
• the purified cat insulin was confirmed by SDS-PAGE and the like, and the purified insulin was lyophilized, weighed and used as the first standard of the measurement kit.
• Anti-insulin autoantibodies in cat blood can be confirmed by the method shown in FIG.
• two bands can be confirmed around molecular weights of 50,000 and 25,000, confirming that it is an IgG type antibody.
• measurement of samples such as feline serum / plasma samples will be described.
• feline insulin can be measured using the rat insulin ELISA kit based on the determination that it can be measured by using rat insulin as a result of estimating its amino acid sequence. Insulin can be measured.
• insulin autoantibodies can be confirmed by measuring according to the method described above using rat insulin monoclonal antibodies.
• the endocrine-binding autoantibody when it is confirmed that an endocrine-binding autoantibody such as insulin is present in the sample as described above, the endocrine-binding autoantibody is removed from the sample.
• the endocrine-binding autoantibody By removing the endocrine substance-binding autoantibodies from the sample in this way, it is possible to extract the endocrine substance and measure it accurately and quickly.
• a method for removing the endocrine-binding autoantibodies in the blood sample when they are present will be described.
• the sample is acidified to stop the binding between the endocrine material and the autoantibody due to the antigen-antibody reaction, thereby separating the endocrine material. It can be so.
• an inorganic acid such as hydrochloric acid or sulfuric acid or an organic acid such as acetic acid can be used, and an inorganic acid that is preferred as the acid is, for example, 0. Preference is given to using 5M to 2M hydrochloric acid or 0.25M to 1M hydrochloric acid.
• the amount of acid added depends on the amount of the sample, but is not particularly limited. For example, the amount is about 1/50 to 10 times, preferably about 1/20 to 1 times the amount of the sample. It is good to use.
• the autoantibodies can be precipitated by making the analyte alkaline.
• an alkaline substance such as saturated ammonium sulfate, saturated sodium sulfate, or polyethylene glycol is preferably added.
• saturated ammonium sulfate when saturated ammonium sulfate is used as a strong alkaline substance, about 30% to 60%, or about 33% to 55% saturated ammonium sulfate is preferably used.
• the amount of the alkaline substance added is not particularly limited as long as it can cause the precipitation of the protein, but the final concentration of alkaline substance is about 30% to It is recommended to add 70%, preferably about 40% to 60%, and the sample should be alkaline at about pH 3.0 to pH 2.0.
• the co-precipitated endocrine substance can be recovered by adding an ethanol solution to the alkalinized specimen as described above.
• an ethanol solution for example, ethanol having a concentration of about 60% to 95%, preferably about 65% to 80% may be used.
• the final concentration of ethanol is, for example, about 60% to 80%, preferably about 65% to 75%.
• the ethanol solution is preferably added as a mixed solution with an acid such as hydrochloric acid.
• the insoluble matter is separated from the specimen thus prepared.
• This separation can be performed according to a conventional method.
• insoluble substances such as autoantibodies including autoantibodies bound to endocrine substances
• centrifugation under a predetermined condition or filtration.
• a clear filtrate can be prepared.
• the supernatant and filtrate obtained as described above can be used as a measurement sample. In this way, if the sample pretreated by removing the autoantibodies bound to the endocrine substance in the sample is used as the measurement sample, the endocrine substance to be measured present in the sample can be accurately and quickly detected. Can be measured.
• the autoantibody titer is measured again, and the endocrine-bound autoantibodies remain in the pretreated samples. It is preferable to check whether it exists. If endocrine-binding autoantibodies remain, it is preferable to remove the endocrine-binding autoantibodies by repeating the above autoantibody removal method. On the other hand, when it is confirmed that the endocrine substance-binding autoantibodies are not present in the specimen sample, the endocrine substance can be detected by measuring the sample. According to this invention, endocrine substances such as insulin contained in the pretreated specimen sample can be measured and detected according to, for example, a sandwich ELISA method commonly used in the art.
• an endocrine substance to be measured such as insulin can be measured by adding enzyme-linked avidin such as peroxidase, etc., developing color using the enzyme substrate, and measuring the absorbance.
• endocrine substances such as cat insulin in a sample sample such as cat blood pretreated by the specimen pretreatment method can be quantitatively measured quickly, easily and accurately.
• an endocrine substance-related disease such as diabetes
• an endocrine substance-related disease such as diabetes
• an endocrine substance-related disease such as diabetes
• measuring the autoantibody titer of an endocrine substance such as cat insulin in a specimen such as cat blood
• diagnosis and treatment of endocrine substance-related diseases can be performed.
• kits for measuring endocrine substances that can measure endocrine substances such as cat insulin.
• a kit for measuring feline insulin includes, for example, an antibody-immobilized plate, feline insulin standard solution, buffer, piotin-conjugated insulin antibody, avidin-conjugated peroxidase, color developing solution (for example, TMB), reaction terminator (for example, sulfuric acid). ), The power of the cleaning liquid etc. should also be configured.
• the feline insulin purified solution purified in Example 1 was qualitatively tested by the dot plot method as follows. Saline 3 1 on the left side of the membrane 0.5 cm in length and 1.5 cm in width, cat insulin purified solution (1.5 1) in the center, and cat spleen homogenate supernatant (3 ⁇ 1) on the right side. Phased and blocked with 25% Block Ace® for 2 hours at room temperature. This membrane was washed 3 times with 0.05% Tween® 20—lOmMPBS and then reacted with anti-insulin monoclonal (D3E7) antibody piotin conjugate (rat insulin ELISA kit) at 4 ° C for 15-18 hours. I let you.
• Tween (registered trademark) 20— Washed 3 times with lOmMPBS, reacted with avidin-HRP (horse radish peroxidase) for 30 minutes at room temperature, and 0.05% Tween (registered trademark). Washed 3 times with 20—lOmMPBS. Thereafter, color was developed for 90 minutes at room temperature using a diethylene glycol solution of 4-naphthol and 1-naphthol. As a result, saline is a force that does not react It was confirmed that the purified solution and the cat spleen homogenate supernatant reacted.
• Blood was collected 10 minutes before feeding the standard feed to the cat and 10 minutes after feeding, and separated into serum or plasma by a conventional method to obtain a feline serum sample or plasma sample.
• Rat insulin monoclonal antibody immobilized on 96 well plate Each cat blood sample was diluted 10-fold to 7-step diluted at 50 zl, reacted at room temperature for 1 hour, 0.05% Tween ( (Registered trademark) 20— Washed with lOmMPBS. Each well of the plate thus prepared was added with HRP-labeled goat anti-cat IgG50 1 and allowed to react at room temperature for 1 hour, and then added with 0.05% Tween® 20—lOmMPBS. Washed. To each well, 50 1 each of TMB as a color former was added and allowed to react at room temperature for 10 minutes, and 1 M sulfuric acid 50 1 was added to stop the reaction. The results of measuring the absorbance of each well at 450 nm (620 nm) are shown in Table 4 and FIG.
• a plate (plate A) coated with anti-insulin antibodies in the Ueru, plates mouse I g G and co scratch (plate B) were prepared in each Ueru.
• 50 1 untreated feline serum specimens (10 specimens) were added to each well of the plate, left at room temperature for 1 hour, and washed 3 times with a buffer solution (lOmMPBS).
• 50 HRP-conjugated goat anti-cat IgG per well leave at room temperature for 1 hour, wash 3 times with buffer (lOmMPBS), 3, 3 ', 5, 5, Monotetramethylbenzidine (TMB) was added at 50 ⁇ ⁇ / well.
• 1 M sulfuric acid was added at 50 1 / well to stop the reaction, and the absorbance was 450 Measured at 620 nm.
• a plate (plate A) in which each well was coated with an anti-insulin antibody and a plate (plate B) in which each well was coated with mouse IgG were prepared.
• the plate was allowed to stand at room temperature for 1 hour and washed 3 times with a buffer solution (lOmMPBS).
• HRP-conjugated rabbit anti-cat IgG was added at 50 1 per well, left at room temperature for 1 hour, washed 3 times with buffer (1 OmMPBS), 3, 3 ', 5, 5 , Tetramethylbenzidine (TMB) was added at 50 ⁇ 1 / well.
• 1 M sulfuric acid was added in 50 // 1 / well to stop the reaction, and the absorbance was measured at 450 Z620 nm. The results are shown in Table 5 and Figure 3.
• Each specimen 1 was diluted with a buffer solution (10 mM PBS) to 10 ml. Chromatography was performed at a rate of lml / cycle using 1OmMPBS as the binding buffer and 0.1M glycine monohydrochloride solution (pH 2.5) as the binding buffer for each of these solutions on a sucrose-binding Sepharose column. did. Pass fractions Z elution fractions were collected and the pH was neutral. After preparation, anti-insulin antibody titer and RF-IgG were measured for each fraction. The results are shown in Table 6-1 (Sample No. 5), Table 6-2 (Sample No. 7) and Table 6-3 (Sample No. 8), respectively.
• Figure 4 shows the measurement results in a graph. In Figure 4, specimen number 5 is represented as 264, specimen number 7 is represented as R-1, and specimen number 8 is represented as R-2.
• FIGS. 5 and 6 respectively.
• sample number 5 is represented as sample 264
• sample number 7 is represented as sample R-1
• sample number 8 is represented as sample R-2.
• Usi insulin was added to each well of the plate 501, left at room temperature for 2 hours, and then washed 3 times with buffer. Next, 250% of Block Ace (registered trademark) 250 1 was added to each well, and then washed 3 times with a buffer solution to be immobilized. Subsequently, the three samples of feline serum insulin-Sepharose eluate (Sample Nos. 5, 7, and 8) used in Examples 5 and 6 were diluted with buffer to a predetermined ratio, and 50 // 1 was added to each well. After the addition, it was washed 3 times with buffer.
• Block Ace registered trademark
• each well was supplemented with 501 goat anti-cat IgG-HRP, washed three times with buffer, and each well was supplemented with 50 1 TMB and allowed to stand at room temperature for 30 minutes. Next, 50 M of 1 M sulfuric acid was added to each well, and the absorbance was measured at 450 Z620 nm. As a control, a sample (blank) to which the eluate was not added was used. The results are shown in Table 7 1 (Sample No. 5), Table 7-2 (Sample No. 7), Table 7-3 (Sample No. 8) and FIG. In the figure, sample number 5 is represented as sample 264, sample number 7 is represented as sample R-1, and sample number 8 is represented as sample R-2! /
• VZV 1M10 volume
• PH 2.5 saturated ammonium sulfate solution
• the sample was stirred at room temperature for 10 minutes, and then an ethanol-10 mM hydrochloric acid solution was added so that the final ethanol concentration was 70%.
• This solution was stirred at room temperature for 10 minutes, and then centrifuged at 4 ° C, 2800 rpm for 15 minutes or an insoluble material was removed using a filter. This supernatant or filtrate was obtained as a sample for measurement.
• insulin autoantibodies could be removed from the feline blood sample.
• Insulin extracted and purified from the feline spleen was added to the feline serum specimen, and the specimen was pretreated to determine the recovery rate (Table 8).
• the measured value is the value after liquid volume conversion, and the unit is pg / ml.
• Cat power Serum was separated from the collected blood according to a conventional method to obtain a cat serum solution.
• the feline serum solution was diluted with lOmMPBS to a ratio of 2500, 1250, 625, 313, 156, 78, and 39 ng / ml, respectively.
• each of the wells on the plate was immobilized by adding a piotin-conjugated anti-insulin antibody to each well, and a standard insulin solution ( ⁇ A specimen diluted with a predetermined ratio of g / ml) and the above-mentioned feline specimen (Specimen No. 1 to LO) were added together. The absorbance of each of these specimens was measured at 450Z620nm. The results are shown in Table 91 (standard insulin solution). The results of feline insulin measurement are shown in Table 92.
• the measurement method of substances such as human insulin has been applied mutatis mutandis. It has a great effect that it can measure itself and endocrine substances such as cat insulin can be measured more accurately and quickly.
• an enzyme immunoassay method or the like commonly used in the technical field without using a radioactive substance can be used, and the present invention is extremely simple. If you can handle it!
• the method of measuring endocrine substances such as cat insulin according to the present invention is particularly accurate, quick and simple for endocrine substances such as insulin present in specimens such as cat blood in order to diagnose and treat diseases such as feline diabetes. Can be measured.
• the measurement method of the present invention can be used to measure endocrine substances such as insulin present in specimens such as cat blood using a measurement method commonly used in the technical field. So it is very convenient.

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