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WO2014123131A1 - Procédé d'essai hautement sensible pour un anticorps gad en tant que marqueur pour le diagnostic précoce du diabète sucré de type 1 - Google Patents

Procédé d'essai hautement sensible pour un anticorps gad en tant que marqueur pour le diagnostic précoce du diabète sucré de type 1 Download PDF

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WO2014123131A1
WO2014123131A1 PCT/JP2014/052600 JP2014052600W WO2014123131A1 WO 2014123131 A1 WO2014123131 A1 WO 2014123131A1 JP 2014052600 W JP2014052600 W JP 2014052600W WO 2014123131 A1 WO2014123131 A1 WO 2014123131A1
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gad
antibody
eia
ict
immune complex
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誠一 橋田
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    • 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
    • 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

Definitions

  • the present invention relates to a highly sensitive measurement method for a GAD antibody, which is an evaluation marker for accurately evaluating and diagnosing initial symptoms of type 1 diabetes.
  • Diabetes mellitus is a group of diseases mainly characterized by chronic hyperglycemia due to insufficient insulin action and various characteristic metabolic abnormalities. Both genetic factors and environmental factors are involved in the onset. The long-term persistence is prone to specific complications and also promotes arteriosclerosis, and depending on the degree of metabolic abnormalities, it exhibits a wide range of conditions ranging from asymptomatic to ketoacidosis and coma. " (Non-Patent Document 1). In Japan, it is confirmed that there are about 22.1 million people, including those who are strongly suspected of having diabetes and those whose possibility cannot be denied (preliminary group). (Non-Patent Document 2).
  • Diabetes is classified into 4 groups according to the cause: type 1 diabetes, type 2 diabetes, other diabetes caused by specific causes, and gestational diabetes. Among them, type 1 diabetic patients are 5-10%, type 2 diabetes patients Occupy 90-95%, and most diabetes is type 2 diabetic patients. Although the cause of the onset of diabetes varies depending on each classification, type 1 diabetes is thought to develop due to the absolute deficiency of insulin due to destruction of ⁇ cells of the pancreas that secrete insulin. Although the mechanism of pancreatic ⁇ -cell destruction has not been clearly elucidated yet, it is thought that an autoimmune reaction is involved, and insulin that is specifically expressed in pancreatic ⁇ -cells is regarded as an important antigen. Has been.
  • Type 1 diabetes is considered to be one of the autoimmune diseases, and several islet-related autoantibodies (insulin autoantibodies, GAD (glutamic acid decarboxylase) antibodies, IA (insulinoma-associated antigens)) have been reported from patients with type 1 diabetes. ) -2 antibody etc.) have been detected. Of these, the GAD antibody has recently attracted attention.
  • the GAD antibody is an autoantibody against glutamic acid decarboxylase (GAD). Baekkeskov et al. Reported that an antibody that reacts with the 64 kDa protein of pancreatic islets is present in the serum of type 1 diabetic patients, and it has been clarified that this 64 kDa protein is GAD (Non-patent Document 3).
  • Non-patent Document 4 There are two isoforms of GAD, 65 kDa GAD65 and 67 kDa GAD67, but the GAD antibody of type 1 diabetic patients is an autoantibody against GAD65 (Non-patent Document 4).
  • the positive rate of GAD antibody shows a high positive rate of 70 to 80% at the early onset. However, the positive rate becomes 50% or less after 5 years from the onset, and decreases to 20% after 10 years (Non-Patent Document 5).
  • the GAD antibody has a low positive rate of 70% when the onset is less than 9 years old compared to insulin autoantibodies and IA-2 antibodies, but the positive rate is high at 90% at 10 years of age and older, and 85% for adults. The positive rate is high (Non Patent Literature 6).
  • islet-related autoantibodies When islet-related autoantibodies are detected from diabetic patients, type 1 diabetes is diagnosed. However, in patients with lean type 2 diabetes, islet-related autoantibodies are not detected at the time of onset, indicating that they can be treated with meals and oral hypoglycemic drugs, but the insulin secretion ability gradually declines. In addition, there was an example of transition to insulin-dependent diabetes (type 1 diabetes) in an average of 3 years. In this patient's serum, islet-related autoantibodies are positively or singly positive, and Kobayashi et al. Referred to this type of subtype 1 diabetes as slowly progressive type 1 diabetes mellitus (Slowly progressive IDDM; SPIDDM). (Non-Patent Document 7).
  • Non-patent Document 8 Although it has not yet been established as a diagnostic standard for SPIDDM, it can be treated with diet therapy or an oral hypoglycemic agent for 6 to 12 months after the onset of diabetes, and GAD antibody is positive (Non-patent Document 8). Alternatively, any one of GAD antibody, IA-2 antibody, and insulin autoantibody is positive (Non-patent Document 9).
  • the positive rate of SPIDDM islet-related autoantibodies was 69% for GAD antibody, 39% for IA-2 antibody, and 29% for insulin autoantibody, and GAD antibody was reported to be significantly higher than IA-2 antibody and insulin autoantibody. (Non-patent document 9).
  • the GAD antibody has the highest positive rate in SPIDDM and is regarded as a very important diagnostic marker for diagnosing type 1 diabetes.
  • the conventional enzyme immunoassay ELISA method
  • detection sensitivity is low, it was difficult to detect early detection or autoantibodies at a low concentration. Therefore, the development of a new highly sensitive enzyme immunoassay for GAD antibody is expected, and early SPIDDM discovery and early treatment with insulin therapy have been desired.
  • An object of the present invention is to provide a highly sensitive enzyme immunoassay method for GAD antibody for early detection of onset of type 1 diabetes, particularly SPIDDM, and appropriate living guidance and treatment.
  • ELISA kits for GAD antibodies that are generally commercially available are third generation ELISA methods.
  • a GAD antibody is captured on a solid phase in which a GAD antigen is insolubilized, then biotin-labeled GAD is bound, and finally a peroxidase-labeled avidin is reacted to measure enzyme activity.
  • a non-specific signal of peroxidase-labeled avidin remains, and high sensitivity can still be achieved.
  • there remains a problem that all substances (immunoglobulins and binding proteins other than IgG) that bind to GAD are measured.
  • ICT-EIA method Immunune Complex Transfer Enzyme Immunoassay
  • an anti-GAD IgG antibody ICT-EIA-2 method has been developed to prevent steric hindrance of antigen epitopes.
  • the GAD antibody can be measured with a sensitivity about 33 times that of a normal ELISA kit.
  • the present inventors examined the measurement of IA-2 antibody and insulin autoantibodies, which are diagnostic markers for type 1 diabetes and SPIDDM, using the ICT-EIA-2 method.
  • the present inventors create a highly sensitive detection method for GAD antibody and insulin autoantibodies using ICT-EIA-1 method and ICT-EIA-2 method, which are improved ICT-EIA methods of the present invention.
  • the present inventors have completed the present invention based on these findings. That is, the gist of the present invention is as follows.
  • ICT-EIA method Two-point binding immune complex transferase immunoassay method (ICT-EIA method) of GAD antibody, which comprises the following five reaction steps (method a: ICT-EIA-1 method) or six reaction steps ( b method: ICT-EIA-2 method), a GAD antibody measuring method, a)
  • Five reaction steps 1) An immune complex is formed with a GAD antibody, a DNP-biotin-labeled GAD antigen, and an enzyme-labeled GAD antigen.
  • the concentration of the immune complex is measured by reacting with the substrate using the labeled enzyme contained in the immune complex and measuring the fluorescence of the product.
  • a method for evaluating the progress of diabetes in a subject by measuring the concentration of GAD antibody in serum using the GAD antibody measurement method according to any one of (1) to (3) above.
  • the GAD antibody measurement method according to any one of (1) to (3) above is used. Of measuring the concentration of GAD antibody.
  • SPIDDM slowly progressive IDDM
  • ICT-EIA-2 method of (2) above In order to evaluate subjects with slowly progressive IDDM (SPIDDM) who share GAD antibody and insulin autoantibodies in the serum, in the subject serum by the ICT-EIA-2 method of (2) above And measuring insulin autoantibodies separately by the same ICT-EIA-2 method.
  • SPIDDM slowly progressive IDDM
  • the serum GAD antibody concentration which can be measured by the improved ICT-EIA method (ICT-EIA-1 method and ICT-EIA-2 method) of the present invention was 33 times more sensitive than the conventional ELISA method. Further, by increasing the affinity of the GAD label that reacts with the GAD antibody, the sensitivity can be increased by a factor of about 10 times, so that the sensitivity can be increased by several hundred times compared with the conventional ELISA method. . As a result, the GAD antibody measurement method of the present invention has made it possible to detect the onset of type 1 diabetes and SPIDDM at an early stage, and perform appropriate life guidance and treatment. Furthermore, by measuring insulin autoantibodies separately, highly accurate diagnosis of type 1 diabetes and SPIDDM becomes possible.
  • the improved ICT-EIA method of the present invention can further improve the diagnostic accuracy of type 1 diabetes and SPIDDM.
  • FIG. 1 is a diagram showing the procedure of a two-point binding immune complex transferase immunoassay method (ICT-EIA-1 method and ICT-EIA-2 method) used in the present invention.
  • FIG. 2 is a diagram showing a comparison of detection sensitivity of each ICT-EIA method and commercially available ELISA kit for GAD antibodies.
  • the serum of type 1 diabetic patients diluted with the serum of healthy individuals was measured using the ICT-EIA-1 method, the ICT-EIA-2 method, and a commercially available ELISA kit, and the detection sensitivity of the GAD antibody was compared.
  • ⁇ represents the ICT-EIA-1 method
  • ⁇ represents the ICT-EIA-2 method
  • ⁇ represents the measurement by ELISA kit.
  • FIG. 3 is a diagram showing the measurement result of the GAD antibody level in the serum of a subject by the ICT-EIA-1 method.
  • the GAD antibody in each subject serum was measured by the ICT-EIA-1 method.
  • Dotted line indicates cut-off value (fluorescence intensity; 1.8), n. d represents non-detection.
  • ⁇ is non-diabetic subject, ⁇ is type 2 diabetic patient without insulin treatment, ⁇ is type 2 diabetic patient under insulin treatment, ⁇ is type 2 diabetic patient with unknown insulin treatment, ⁇ is type 1 diabetic patient under insulin treatment , ⁇ represents a type 1 diabetic patient whose insulin treatment is unknown.
  • Black markers represent serum that was inhibited by the addition of excess GAD.
  • FIG. 4 is a diagram showing the measurement result of the GAD antibody level in the serum of a subject by the ICT-EIA-2 method.
  • the GAD antibody in each subject serum was measured by the ICT-EIA-2 method.
  • Dotted line indicates cut-off value (fluorescence intensity; 8.1), n. d represents non-detection.
  • ⁇ is non-diabetic subject, ⁇ is type 2 diabetic patient without insulin treatment, ⁇ is type 2 diabetic patient under insulin treatment, ⁇ is type 2 diabetic patient with unknown insulin treatment, ⁇ is type 1 diabetic patient under insulin treatment , ⁇ represents a type 1 diabetic patient whose insulin treatment is unknown.
  • Black markers represent serum that was inhibited by the addition of excess GAD.
  • FIG. 5 is a diagram showing the correlation between the ELISA method and the ICT-EIA-1 method for GAD antibodies in diabetic patients.
  • FIG. 6 is a diagram showing the correlation between the ELISA method and the ICT-EIA-2 method for GAD antibodies in diabetic patients.
  • FIG. 7 is a table summarizing the positive rates of insulin autoantibodies and GAD antibodies by the ICT-EIA-2 method and ELISA kit.
  • FIG. 8 is a diagram showing an operation procedure for simultaneous detection of the ICT-EIA-2 method for insulin autoantibodies and GAD antibodies. That is, in the simultaneous detection method of the present invention, the total signal of each autoantibody can be detected in one measurement by using a detection system in which each label of insulin autoantibodies and GAD antibodies is combined. it can.
  • FIG. 9 is a diagram showing comparison of detection sensitivity of insulin autoantibodies by ICT-EIA-2 method, ICT-EIA-1 method and ELISA kit.
  • Serum of type 1 diabetic patients diluted with insulin-removed serum of healthy subjects was measured using ICT-EIA method and ELISA kit, and the detection sensitivity was compared.
  • ⁇ represents ICT-EIA-2 method
  • ⁇ represents ICT-EIA-1 method
  • ⁇ represents measurement by ELISA kit.
  • the upper figure shows all signals, and the lower figure shows specific signals.
  • the “immunoconjugate transferase immunoassay method (ICT-EIA method)” of the present invention relates to an improved method for improving the sensitivity of the non-competitive method (sandwich) enzyme immunoassay method (EIA) (Non-patent Document 1). reference).
  • EIA enzyme immunoassay method
  • FIG. 1 in the ICT-EIA method, the nonspecific adsorption (background) of the antibody to be used could be lowered, so that it is possible to measure below amol level (zmol) of many macromolecular bioactive substances.
  • the “GAD antibody” of the present invention is an autoantibody against glutamic acid decarboxylase (GAD).
  • GAD is an enzyme that synthesizes ⁇ -aminobutyric acid (GABA) from L-glutamic acid and is highly expressed in the brain and islet ⁇ cells.
  • GABA ⁇ -aminobutyric acid
  • the role of GABA in islet ⁇ cells is thought to be responsible for the regulation of insulin synthesis and secretion.
  • GAD antibodies have appeared several years before the onset of type 1 diabetes, and the predictive rate of GAD antibodies is as high as 60% or more (Kawasaki E, Gill RG, Eisenbarth GS: Type 1 diabetics melitus.
  • the “enzyme” of the present invention is not particularly limited as long as it is an enzyme that causes the degradation product of the substrate to emit fluorescence because concentration measurement is performed by fluorescence.
  • ⁇ -D-galactosidase examples thereof include horse radish peroxidase and alkaline phosphatase.
  • the “insulin autoantibody” of the present invention is an antibody against insulin and is an antibody specific for pancreatic ⁇ cells. Insulin autoantibodies are useful as markers for predicting the onset of type 1 diabetes, and if insulin autoantibodies are positive, it is considered that they may shift to insulin-dependent diabetes in the future.
  • the positive rate of insulin autoantibodies varies depending on the age of onset, and shows about 70% in child-onset type 1 diabetes.
  • the “method for evaluating the progress of diabetes” according to the present invention is a method for accurately evaluating the progress of a diabetic disease in a subject by measuring the concentration of GAD antibody or insulin autoantibody in serum. is there.
  • This evaluation method makes it possible to accurately perform interventional therapy such as administration of therapeutic agents and lifestyle guidance for suppressing and improving the progression of diabetic disease in the subject.
  • interventional therapy such as administration of therapeutic agents and lifestyle guidance for suppressing and improving the progression of diabetic disease in the subject.
  • the concentration of GAD antibody in the serum is 0.1 U / ml or more, it is diagnosed that it is early in diabetes, intervention therapy is started, and the progression of diabetes is suppressed / improved. Do.
  • the “intervention therapy” of the present invention refers to improvement of lifestyle (meal / physical activity center) based on a protocol or insulin therapy.
  • lifestyle meal / physical activity center
  • dietary and exercise regimens are incorporated into lifestyle habits to control the necessary and consumed calories, and guidance is given to reduce mental stress, eliminating and reducing factors related to the onset of diabetes. Do things.
  • insulin treatment as early as possible is included as intervention therapy.
  • early intervention therapy can be started to suppress or avoid a decrease in insulin secretion ability and improve symptoms.
  • Example 1 ICT-EIA method for GAD antibody (1) Reagents a) General reagent : Bovine serum albumin (BSA) was purchased from Nacalai Tesque (Kyoto), and streptavidin was purchased from Wako Pure Chemical Industries (Osaka). Other general reagents were purchased from Nacalai Tesque and Wako Pure Chemical Industries. b) antibody : Rabbit anti-2,4-dinitrophenyl group (DNP) -bovine serum albumin (BSA) serum was purchased from Shibayagi (Gunma).
  • BSA Bovine serum albumin
  • DNP rabbitt anti-2,4-dinitrophenyl group
  • BSA bovine serum albumin
  • b) antigen Recombinant human GAD65 was purchased from RSR Limited (Cardiff, UK).
  • Buffer 0.1M NaCl, 0.1% BSA, 1 mM MgCl 2 And 0.01 M sodium phosphate buffer (pH 7.0) containing 0.1% NaN3, buffer A, 0.4 M NaCl, 0.1% BSA, 1 mM MgCl 2 0.01M sodium phosphate buffer (pH 7.0) containing 0.1% NaN3 and buffer solution B, 0.1M NaCl, 0.01% BSA, 1 mM MgCl 2 And 0.01M sodium phosphate buffer (pH 7.0) containing 0.1% NaN3 as buffer C, and 0.01M sodium phosphate buffer (pH 7.0) containing 0.1M NaCl as buffer.
  • ELISA kit The GAD antibody ELISA kit was purchased from RSR Limited (Cardiff, UK). (2) Labeled antigen, etc. a) DNP labeled GAD, ⁇ -D-galactosidase labeled GAD : 1) Preparation of thiol group-introduced GAD : 10 ⁇ l of 13 mM SATA dissolved in DMF was added to 0.4 mg / 0.48 ml of GAD solution dissolved in 0.1 M Tris-HCl, pH 8.5, and incubated at 30 ° C. for 30 minutes.
  • reaction solution was subjected to centrifugal desalting using a Sephadex G-50 (5 ml) column equilibrated with 0.1 M sodium phosphate buffer (pH 7.0) (reference), excess reagent was removed, and DNP-labeled antigen A conjugate (for capture) was obtained.
  • ⁇ -D-galactosidase labeled GAD The maleimide group-introduced ⁇ -D-galactosidase and the thiol group-introduced GAD were mixed at 3.4 ⁇ M and 8.5 ⁇ M, respectively, and incubated at 4 ° C. for 17 hours. After incubation, 10 ⁇ l of 100 mM ME dissolved in buffer E is added to the reaction solution. After incubation at 30 ° C. for 5 minutes, 20 ⁇ l of 100 mM NEM is added and incubated at 30 ° C. for 5 minutes to block unreacted thiol groups and maleimide groups. did.
  • DNP biotinylated label GAD 1) Preparation of thiol group-introduced GAD : 10 ⁇ l of 13 mM SATA dissolved in DMF was added to 0.4 mg / 0.48 ml of GAD solution dissolved in 0.1 M Tris-HCl, pH 8.5, and incubated at 30 ° C. for 30 minutes. After the incubation, 10 ⁇ l of 4M hydroxylamine was added to the reaction solution and incubated at 30 ° C. for 5 minutes.
  • Biotin-Lys 1.0mg 0.1M sodium phosphate buffer solution (pH 7.0) 0.9ml, 0.2mM EMCS 0.1ml dissolved in DMF is added, incubated at 30 ° C for 30 minutes, maleimide Group-introduced biotin-Lys was prepared.
  • DNP biotinylated labeled GAD A mixture of maleimide group-introduced DNP and maleimide group-introduced biotin was prepared by combining maleimide group-introduced DNP and maleimide group-introduced biotin at the same ratio.
  • the mixed solution and GAD introduced with a thiol group were mixed to 78 ⁇ M and 19 ⁇ M, respectively, and incubated at 4 ° C. for 17 hours. After incubation, 10 ⁇ l of 100 mM ME dissolved in buffer E is added to the reaction solution. After incubation at 30 ° C. for 5 minutes, 20 ⁇ l of 100 mM NEM is added and incubated at 30 ° C.
  • the reaction solution is a dialysis cellulose tube (Sanko Junyaku Co., Ltd., Tokyo), dialyzed against a buffer solution (0.1 M sodium phosphate buffer, pH 7.0) to remove excess reagent, and DNP biotin. A labeled antigen conjugate (for capture) was obtained. 1.3 molecules of DNP and 2.9 molecules of biotin were introduced per GAD molecule.
  • b) Blood sample Serum samples were collected on an early hungry after fasting for 12 hours (NIPRO, with 22G holder, Osaka), allowed to stand at room temperature for 30 minutes, and then centrifuged (table top cooling centrifuge 2800 KUBOTA, Kubota Corporation, Tokyo). And centrifuged at 3000 rpm for 10 minutes to obtain serum. Serum was stored at minus 20 ° C.
  • Dextran-charcoal adjustment Charcoal (Nacalai Tesque) 1.2 g was washed several times with distilled water and then made up to 10 ml with distilled water. To this, 0.3 g of dextran (Sigma) was added and stirred.
  • methyl cellulose (Sigma-Aldric) was placed on 10 ml of Buffer A and dissolved at 4 ° C. for 16 hours without stirring.
  • a methylcellulose solution was mixed with the prepared dextran-charcoal solution in the same ratio, stirred for 15 minutes to obtain a dextran-charcoal (hereinafter referred to as DC) solution, and stored at 4 ° C.
  • DC dextran-charcoal
  • Serum treatment with DC solution After dilution by adding 150 ⁇ l of buffer A to 50 ⁇ l of serum, 13.6 ⁇ l of 0.4 M HCl was further added for acidification.
  • ICT-EIA-1 method for GAD antibody Serum (100 ⁇ l) diluted 20-fold was mixed with a mixture solution (100 ⁇ l) of DNP-labeled GAD and ⁇ -D-galactosidase-labeled GAD, 1.0 ⁇ M inactive ⁇ -D-galactosidase in buffer A, and 4 ° C. After incubation for 16 hours, an immune complex consisting of an enzyme-labeled antigen, a GAD antibody, and a supplementary labeled antigen was formed.
  • one affinity-purified anti-DNP-IgG insolubilized solid phase polystyrene bead was added to the reaction solution and reacted for 0.5 hour to supplement the immune complex on the bead.
  • the beads were washed twice with buffer C (2 ml) and then reacted with 2 mM DNP-Lys (150 ⁇ l) dissolved in buffer A for 0.5 hours to elute immune complexes from the beads.
  • the mixture was incubated at 30 ° C., 0.1 M glycine sodium buffer (pH 10.5) (2 ml) was added to stop the reaction, and measurement was performed using a fluorescence spectrophotometer (F-2500, Hitachi).
  • the excitation wavelength is 360 nm
  • the fluorescence wavelength is 450 nm
  • the fluorescence intensity is 10 -8 M 4MU was converted to 100.
  • ICT-EIA-2 method for GAD antibody To a diluted serum (100 ⁇ l) diluted 20-fold, a mixture (100 ⁇ l) of DNP-labeled GAD, biotin-labeled GAD, and 1.0 ⁇ M inactive ⁇ -D-galactosidase dissolved in buffer B was added and mixed. By incubating for a period of time, an immune complex composed of DNP-labeled antigen, autoantibody and Biocytin-labeled antigen was formed. Next, one affinity-purified anti-DNP-IgG insolubilized polystyrene bead was added to this reaction solution and reacted for 0.5 hour to supplement the immune complex on the bead.
  • the mixture was incubated at 30 ° C., 0.1 M glycine sodium buffer (pH 10.5) (2 ml) was added to stop the reaction, and measurement was performed using a fluorescence spectrophotometer (F-2500, Hitachi).
  • the excitation wavelength is 360 nm
  • the fluorescence wavelength is 450 nm
  • the fluorescence intensity is 10 -8 M 4MU was converted to 100.
  • Target people a) Target audience: Non-diabetic subjects (73 patients), hyperinsulinemia patients (9 patients), Graves' disease patients (30 patients), Hashimoto's disease (20 patients), type 2 diabetes patients (30 patients) who are taking antithyroid drugs (methimazole) ; Insulin not treated: 7 people, Insulin treatment: 15 people, Insulin treatment unknown: 8 people, Samples were collected from type 1 diabetes patients (24 people; Insulin treatment: 21 people, Insulin treatment unknown: 3 people) . b) Informed consent: The tests conducted in this study were approved by the Tokushima Bunri University Ethics Committee (approval number 4).
  • the subject was given informed consent and the test was conducted after obtaining consent.
  • c) Statistical processing The cut-off value was set to the average value of fluorescence intensity of non-diabetic subjects + 2SD. As for the correlation between the two variables, the Spearman rank correlation coefficient was calculated. For statistical analysis, SPSS 20.0.0 was used, and the statistical significance level was set to 5%.
  • (2) Examination of serum usage The amount of serum used in the ICT-EIA-1 method and the ICT-EIA-2 method was examined using type 1 diabetes patient serum. Serum of a healthy person was used for dilution of type 1 diabetes patient serum.
  • the ICT-EIA-1 method was prepared so that serum of type 1 diabetes patient was contained in 100 ⁇ l of healthy subject serum, 10 ⁇ l, 20 ⁇ l, 30 ⁇ l and 50 ⁇ l, respectively.
  • the ICT-EIA-1 method showed a proportional increase in fluorescence intensity up to 20 ⁇ l, but no significant increase in fluorescence intensity was observed after 20 ⁇ l.
  • the increase in fluorescence intensity could be confirmed up to 20 ⁇ l, the difference was only 1.3 times compared to the fluorescence intensity of 10 ⁇ l. Therefore, the maximum amount of serum used by the ICT-EIA-1 method was set to 10 ⁇ l.
  • the ICT-EIA-2 method was prepared such that 1 ⁇ l, 2 ⁇ l, 5 ⁇ l, 10 ⁇ l, 20 ⁇ l, 30 ⁇ l, 40 ⁇ l, and 50 ⁇ l of type 1 diabetes patient serum were contained in 100 ⁇ l of healthy subject serum.
  • an increase in fluorescence intensity was observed up to 5 ⁇ l, but a tendency to decrease in fluorescence intensity was observed after 5 ⁇ l. Therefore, the maximum amount of serum used by the ICT-EIA-2 method was 5 ⁇ l.
  • the 1st generation ELISA method, the 2nd generation ELISA method, and the 3rd generation ELISA method mean the following.
  • the first generation ELISA method captures a specific antibody to be measured in serum with an antigen insolubilized in a solid phase, and then reacts this specific antibody with an enzyme-labeled anti-immunoglobulin antibody (second antibody). This is a method of labeling and finally measuring the enzyme activity.
  • second antibody enzyme-labeled anti-immunoglobulin antibody
  • a large amount of non-specific antibody in serum adsorbs non-specifically to the solid phase, the non-specific antibody and the enzyme-labeled second antibody bind to each other, and the enzyme activity of this enzyme is also measured together. Therefore, the background becomes high and it becomes difficult to measure a small amount of specific antibody.
  • a second generation ELISA method has been developed to compensate for the disadvantages of the first generation.
  • the second generation ELISA method first, total immunoglobulin including specific antibodies to be measured in serum is captured by a second antibody insolubilized in a solid phase.
  • an enzyme-labeled antigen is reacted with the specific antibody in the captured total antibody, and finally the enzyme activity is measured.
  • the second antibody insolubilized in the solid phase also captures non-specific antibodies other than the specific antibody to be measured, there is a limit to the amount of the second antibody on the solid phase, and only a small amount of serum sample is required.
  • a third generation ELISA method has been developed.
  • a specific antibody to be measured in serum is first captured by an antigen insolubilized in a solid phase, then an enzyme-labeled antigen is reacted with the captured specific antibody, and finally, an enzyme
  • This is a method for measuring activity.
  • the detection sensitivity was improved as compared with the second generation, but the background was still high.
  • this method detects all subclass antibodies and antigen-binding substances other than antibodies, so it is difficult to determine whether they are autoantibodies.
  • the GAD antibody could not be detected when the first generation ELISA method and the second generation ELISA method were used.
  • the detection sensitivity by the 3rd generation ELISA method and the 3rd generation improved ELISA method was 10 U / ml and 100 U / ml, and the 3rd generation improved ELISA method was 10 times more sensitive than the 3rd generation ELISA method. It was.
  • the detection sensitivity of the 3rd generation improved ELISA method and ELISA kit was 10 U / ml, but the nonspecific fluorescence intensity of the 3rd generation improved ELISA method was high.
  • the detection sensitivity by the ICT-EIA-1 method and the ICT-EIA-2 method was both 0.3 U / ml, which was 30 times higher than the ELISA kit (see FIG. 2).
  • Example 4 Reproducibility of ICT-EIA-1 method and ICT-EIA-2 method
  • Simultaneous reproducibility of the ICT-EIA-1 method was examined using type 1 diabetic patient serum diluted 50 to 100 times.
  • the results are shown in Table 2 below.
  • Detection evaluation of GAD antibody by ICT-EIA method (1) Detection of GAD antibody by ICT-EIA-1 method Serum of non-diabetic subjects (32 people), type 2 diabetic patients (30 people), and type 1 diabetic patients (23 people) was measured using the ICT-EIA-1 method (see FIG. 3). The cut-off value was set at a fluorescence intensity of 1.8. As a result, only 1/33 non-diabetic subjects showed a fluorescence intensity exceeding the cutoff value. The number of patients with type 2 diabetes was 3/29. On the other hand, there were 11/22 patients with type 1 diabetes. A subject who exceeded the cut-off value was confirmed to be positive by the same method as in Example 2 (1).
  • the fluorescence intensity when high concentration GAD was added was subtracted from the fluorescence intensity when high concentration GAD was not added to the subject's serum, and expressed as a specific GAD antibody value (hereinafter referred to as a specific signal).
  • the cut-off value at this time was set to 0.7.
  • 0/32 persons in non-diabetic subjects and 9/30 persons in type 2 diabetic patients showed fluorescence intensity exceeding the cut-off value.
  • Example 5 Correlation between ELISA method and ICT-EIA-1 method or ICT-EIA-2 method
  • the correlation between the ELISA method and the ICT-EIA-1 method or the ICT-EIA-2 method was examined from the results of patients with type 1 diabetes and type 2 diabetes.
  • a specific signal was used, and those in which no specific signal was detected were excluded from the ICT-EIA method and the ELISA method, and the correlation was examined.
  • r 0.555, p ⁇ 0.000
  • the number of type 2 diabetic patients was 14/29. On the other hand, there were 17/22 patients with type 1 diabetes (see Table 3).
  • Example 6 Detection sensitivity of insulin autoantibodies by ICT-EIA-2 method (1) Study sample: Comparison was made using serum of insulin autoantibody positive persons. (2) Detection method: The insulin autoantibodies were measured according to the method described in the publicly known literature (Cli Biochem. 2012, 45 (13-14): 1086-91).
  • insulin is labeled with a low molecular weight DNP or biotin, and then these labeled body and antibody. It is characterized in that an immune complex is formed by reacting. Serum of an insulin autoantibody positive person (type 1 diabetic patient) diluted with an insulin-removed serum of a healthy person was measured using the above literature method and an ELISA kit, and the detection sensitivity was compared. (3) Detection sensitivity: As shown in FIG.
  • the ICT-EIA-2 method was able to detect insulin self-response 1,000 times more sensitively than the ELISA kit. Moreover, it was 10 times more sensitive than the ICT-EIA-1 method.
  • Example 7 Method for simultaneous detection of ICT-EIA-2 method against insulin autoantibodies and GAD antibodies
  • a detection system in which each label for insulin autoantibodies and GAD antibodies is combined is used.
  • the ICT-EIA-2 method used in this detection method is one of its features, but if the labeled antigen is changed, various antibodies can be detected with the same solid phase and the same operation.
  • one reagent for use such as two types of labeled antigens for insulin autoantibodies and GAD antibodies
  • the total signal of each autoantibody can be detected in one measurement.
  • the difference in fluorescence intensity between the positive and negative ones greatly opens depending on the presence or absence of each autoantibody, so early detection of these autoantibody positive patients Becomes easier. From the above, it becomes possible to more accurately predict or diagnose type 1 diabetes and SPIDDM.
  • the GAD antibody measurement method of the present invention is a method that enables detection and measurement of a GAD antibody with a sensitivity 30 times or more that of a conventional ELISA method. Therefore, by using the present invention, it has become possible to detect and diagnose the early onset of type 1 diabetes and slowly progressive type 1 diabetes (SPIDDM). And treatment or prevention of symptom progress can be aimed at by improving life such as meal and exercise and implementing insulin therapy at an early stage.
  • the method for measuring GAD antibody of the present invention makes it possible to diagnose early symptoms of type 1 diabetes and slowly progressing type 1 diabetes, and to prevent these progressions at an earlier stage. Further, by using a single reagent such as a labeled antigen for insulin autoantibodies and GAD antibodies, it is possible to more accurately predict or diagnose type 1 diabetes and SPIDDM.

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Abstract

La présente invention aborde le problème de fournir un procédé d'essai pour un anticorps GAD, en particulier un procédé d'essai pour un anticorps GAD ayant une sensibilité supérieure au procédé ELIZA classique. Pour résoudre ce problème, l'invention concerne un procédé d'essai pour un anticorps GAD dans le sang, ledit procédé d'essai étant construit par l'utilisation du procédé d'immuno-essai d'enzyme de transfert de complexe immunitaire (ICT-EIA), une sensibilité 33 fois ou plus aussi élevée que le procédé ELIZA classique pouvant être établie. Par conséquent, le diabète sucré de type 1 peut être détecté au stade d'apparition précoce de sorte qu'un traitement d'intervention approprié peut être réalisé.
PCT/JP2014/052600 2013-02-05 2014-01-29 Procédé d'essai hautement sensible pour un anticorps gad en tant que marqueur pour le diagnostic précoce du diabète sucré de type 1 Ceased WO2014123131A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106405099A (zh) * 2015-07-31 2017-02-15 希森美康株式会社 被检物质的检测方法、检测用试剂盒及检测用试剂
WO2017138497A1 (fr) * 2016-02-08 2017-08-17 シスメックス株式会社 Méthode de détection d'analyte et kit de réactifs permettant de détecter un analyte
WO2017138660A1 (fr) * 2016-02-12 2017-08-17 国立大学法人 岡山大学 Procédé d'inspection des fonctions immunitaires, procédé de tri des patients atteints du cancer, procédé de prévision des effets thérapeutiques sur le cancer, agent d'élévation de la concentration en ions calcium dans les cellules, agent d'amélioration sélectif de fonctions d'effecteur/mémoire (em) et d'effecteur (eff) dans un tissu tumoral, et procédé de surveillance de résultats d'agent thérapeutique contre le cancer
JP2020134329A (ja) * 2019-02-20 2020-08-31 シスメックス株式会社 被検物質の情報の取得方法及び被検物質の捕捉方法
JP7373286B2 (ja) 2019-02-20 2023-11-02 シスメックス株式会社 被検物質の情報の取得方法及び被検物質の捕捉方法

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