WO2014208911A1 - Dosage immunologique pour mesurer la concentration en gliadorphine dans l'urine - Google Patents

Dosage immunologique pour mesurer la concentration en gliadorphine dans l'urine Download PDF

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Publication number
WO2014208911A1
WO2014208911A1 PCT/KR2014/005159 KR2014005159W WO2014208911A1 WO 2014208911 A1 WO2014208911 A1 WO 2014208911A1 KR 2014005159 W KR2014005159 W KR 2014005159W WO 2014208911 A1 WO2014208911 A1 WO 2014208911A1
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WO
WIPO (PCT)
Prior art keywords
gliadolpine
gliadolphin
immunoassay
solution
urine
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PCT/KR2014/005159
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English (en)
Korean (ko)
Inventor
김종배
정승필
김영기
김인보
정주성
이아영
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(주)바이오닉스
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Application filed by (주)바이오닉스 filed Critical (주)바이오닉스
Publication of WO2014208911A1 publication Critical patent/WO2014208911A1/fr

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids

Definitions

  • the present invention relates to an immunoassay using an antigen-antibody reaction capable of measuring the concentration of gliadolphin in urine.
  • Gluten is an insoluble protein in grains such as barley and wheat. A small amount of water is added to the flour and kneaded to form a mass, and when it is kneaded in a large amount of water, the starch is suspended in water to remove it. What remains is gluten. When gluten is mixed with 50-70% ethyl alcohol, there are soluble and insoluble ingredients. The soluble component is called gliadin, and the insoluble ingredient is called glutenin. Gliadophin is one of the peptides that are produced when the gliadin protein is digested. It is also called an opioid peptide or opiate peptide. Opioid peptides exhibit analgesic activity by specifically binding to morphine receptors.
  • gluten When eaten as food, it is broken down into peptide chains in the stomach, followed by digestion with smaller molecules of amino acids, such as gliadolpine, which are then absorbed into the blood through the intestines, circulate into the brain, and schizophrenia. It is known to cause symptoms such as autism.
  • Immunoassay is a technique that analyzes trace components such as hormones based on a competitive reaction principle with an antibody that reacts specifically with a target substance to be measured.
  • the method used to measure gliadolphin is an enzyme linked immunosorbent assay (ELISA) using enzyme and substrate reaction.
  • ELISA enzyme linked immunosorbent assay
  • an object of the present invention to provide an assay that can measure gliadolphin excreted in the urine.
  • Another object of the present invention is to provide a method for diagnosing related diseases through the gliadolpine assay.
  • the above object of the present invention comprises the steps of preparing an immunogen gliadolphin-KLH antigen for antibody production; Mixing the immunogen gliadolpine-KLH antigen prepared in the step with a complete immunopotentiator and injecting the same into an object to produce a polyclonal antibody; Selecting only those with good titer and good specificity of the antibody after fusion and production of B cells and myeloma cells to the measurement target injected with gliadolphin-KLH in this step; Achieved through the step of applying the DELFIA method for quantifying gliadolphin.
  • the immunoassay according to the present invention provides a method for quantifying urinary gliadolphin as a new method with higher sensitivity and utility than the immunoassay that has been used so far. It is a very useful invention in the pharmaceutical industry because it can be used as a method for diagnosing various diseases by measuring gliadolphin.
  • 1 is a method for generating gliadolpine monoclonal antibody.
  • 2 is a graph of gliadolpine monoclonal antibody titers.
  • Figure 3 is a diagram showing the principle of the ELISA for gliadolpine measurement.
  • 5 is a diagram illustrating the principle of DELFIA for measuring gliadolphin.
  • Figure 7 is a graph comparing the results of applying the existing kit and ELISA method.
  • the urine of the subject to be measured is collected, centrifuged at 4 ° C, and only the supernatant is collected and stored at -20 ° C until analysis.
  • Gliadolpine peptides were coupled with Camino-terminal one by one Fmoc SPPS (Solid Phase Peptide Synthesis) method with 7 amino acids of Tyr-Pro-Gln-Pro-Gln-Pro-Phe. C-terminal
  • the first amino acid was used as attached to the resine. All amino acid raw materials used in the synthesis were protected by N-terminal with Fmoc and all residues were removed from trifluoroacetic acid (TFA) using N, N- containing 20% piperidine. Fmoc was removed using dimethylmethanamide (DMF) solution.
  • TFA EDT: Thioanisole: TIS: H 2 O at a ratio of 90: 2.5: 2.5: 2.5: 2.5, respectively.
  • the keyhole limpet hemocyanin (KLH) was combined as a carrier to use the gliadolpine peptide, which is an incomplete antigen, as an immunogen.
  • 2 mg of the synthesized gliadolphin peptide was dissolved in 200 uL distilled water or dimethyl sulfoxide (DMSO). 1 uL of the solution was added to a mixed solution of 10 uL of ellmans reagent and 89 uL of distilled water to identify yellow free thiol.
  • the solution was dissolved in 800 uL of KLH 2 mg and the peptide solution was stirred for 2 hours.
  • Bovine serum albumin (BSA) was combined to facilitate marker binding to small size gliadolphin peptides.
  • the solution dissolved in 800 uL and the peptide solution were mixed and reacted for 2 hours. 5 uL was added to the reaction solution in a mixed solution of 10 uL of Elmans reagent and 85 uL of distilled water, and the reaction was confirmed.
  • the sample dissolved in DMSO only the supernatant was desalted using a Hi-trap column (GE healthcare), mixed with a precipitate, and combined with gliadolphin and BSA protein.
  • an immunogen prepared by binding KLH to a gliadolphin peptide was mixed 1: 1 with an adjuvant and injected with 100 ug of antigen per animal to experimental Balb / c mice. After two weeks, the same antigen was mixed with an adjuvant in the mouse injected with the antigen, followed by a second adjuvant injection. After 1 week, the animals were sacrificed to separate B cells from the spleen and then myeloma tumor cells. , Fusion with 'SP / 20' and selection medium; After culturing in HGPRT broth, hybridoma cells were obtained, and clones that secrete antibodies were selected to produce monoclonal clones by limiting dilution.
  • the clones were named 4H2, 5E1, 8C1, 9H1, 10D2, respectively.
  • clones with high titers of antibodies were selected, and among them, hybridoma 10D2 having the best properties was selected.
  • hybridoma cells were injected into experimental Balb / c mice injected with 200 uL of Freund's adjuvant to obtain ascitic fluid. Protein-G or Protein-A affinity column (Pharmacia, USA) After purification using and used as an antibody.
  • Eu-labeling kit Perkin Elmer, USA was used to bind europium (Europium, Eu) to the gliadolphin-BSA conjugate and was used for DELFIA measurement.
  • Europium, Eu europium
  • gliadolpine-BSA was prepared at a concentration of 4 mg / mL, and 250 uL of gliadolphin-BSA was added to a 100 mmol / L Na 2 CO 3 solution at pH 9.3. And reacted at 4 ° C. overnight.
  • the peak at 280 nm was confirmed by passing through a Sephadex G-50 column using TSA buffer containing 0.9% NaCl and 0.05% sodium azide in 50 mmol / L Tris-HCl solution at pH 7.8 as elution buffer.
  • the separated europium-coupled gliadolphin-BSA-Eu conjugate was used by measuring its concentration.
  • Table 1 shows Table 1 as a standard curve graph.
  • the ELISA method applied to the existing kit and the present invention showed a result similarity of about 96%, and the result value was also about 96% similarity when comparing the existing kit and the DELFIA method applied to the present invention. Showed. Finally, the ELISA method and the DELFIA method of the present invention shown in Figure 9 showed about 98% similarity. Through this, it showed high reliability by showing similar results with existing kits.
  • the immunoassay method according to the present invention is not only used as a substitute for the existing method but also produced and utilized as a business kit, so it is a very useful invention in the biopharmaceutical industry because of the excellent effect expected from the business.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention, qui concerne un nouveau dosage immunologique inconnu précédemment pour mesurer la concentration en gliadorphine, à l'aide d'urine, présente un excellent effet dans la réalisation d'un dosage immunologique.
PCT/KR2014/005159 2013-06-28 2014-06-12 Dosage immunologique pour mesurer la concentration en gliadorphine dans l'urine WO2014208911A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20130076096A KR20150003059A (ko) 2013-06-28 2013-06-28 뇨 중 글리아돌핀(Gliadorphin) 농도 측정을 위한 면역분석법
KR10-2013-0076096 2013-06-28

Publications (1)

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WO2014208911A1 true WO2014208911A1 (fr) 2014-12-31

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050170333A1 (en) * 2004-02-03 2005-08-04 Aristo Vojdani Identification of etiology of autism
US6984493B1 (en) * 2000-05-08 2006-01-10 Toray Industries, Inc. Method for examining the involvement of opioid peptides in prurtis
US20090305434A1 (en) * 2006-10-03 2009-12-10 Serguei Fetissov Method of diagnosing neurophsychiatric diseases, eating disorders or metabolic diseases
WO2012100070A2 (fr) * 2011-01-20 2012-07-26 Immunosciences Lab, Inc. Méthodes et appareil pour la détection de la sensibilité au gluten, et sa différenciation de la maladie cœliaque

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6984493B1 (en) * 2000-05-08 2006-01-10 Toray Industries, Inc. Method for examining the involvement of opioid peptides in prurtis
US20050170333A1 (en) * 2004-02-03 2005-08-04 Aristo Vojdani Identification of etiology of autism
US20090305434A1 (en) * 2006-10-03 2009-12-10 Serguei Fetissov Method of diagnosing neurophsychiatric diseases, eating disorders or metabolic diseases
WO2012100070A2 (fr) * 2011-01-20 2012-07-26 Immunosciences Lab, Inc. Méthodes et appareil pour la détection de la sensibilité au gluten, et sa différenciation de la maladie cœliaque

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VOJDANI: "The characterization of the repertoire of wheat antigens and peptides involved in the humoral immune responses in patients with gluten sensitivity and crohn's disease", ISRN ALLERGY, vol. 2011, 2011, pages 1 - 12, XP055114214, DOI: doi:10.1053/j.gastro.2009.03.059 *

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