WO2003037934A1 - Technique de dosage d'activite de proteine kinase a au moyen d'anticorps anti-phosphokemptide dans le diagnostic du cancer - Google Patents

Technique de dosage d'activite de proteine kinase a au moyen d'anticorps anti-phosphokemptide dans le diagnostic du cancer Download PDF

Info

Publication number
WO2003037934A1
WO2003037934A1 PCT/KR2002/002020 KR0202020W WO03037934A1 WO 2003037934 A1 WO2003037934 A1 WO 2003037934A1 KR 0202020 W KR0202020 W KR 0202020W WO 03037934 A1 WO03037934 A1 WO 03037934A1
Authority
WO
WIPO (PCT)
Prior art keywords
phosphokemptide
monoclonal antibody
antibody
pka
cancer
Prior art date
Application number
PCT/KR2002/002020
Other languages
English (en)
Inventor
Uh-Hyun Kim
Kwang-Hyun Park
Mi-Sun Chung
Original Assignee
A.B.I Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by A.B.I Co., Ltd filed Critical A.B.I Co., Ltd
Priority claimed from KR1020020066311A external-priority patent/KR20030036010A/ko
Publication of WO2003037934A1 publication Critical patent/WO2003037934A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • the present invention generally, relates to a monoclonal antibody which binds to phosphokemptide and a method of measuring protein kinase A (PKA) activity using the same. More particularly, the present invention relates to a monoclonal antibody capable of specifically recognizing phosphokemptide produced in an enzymatic reaction of PKA, and a method of effectively detecting the activity of serum PKA using the anti-phosphokemptide monoclonal antibody. Since PKA expression is up-regulated in sera of cancer patients, the anti- phosphokemptide monoclonal antibody is very useful for diagnosis of cancer.
  • PKA protein kinase A
  • PKA Protein kinase A
  • enzymatic activity can be evaluated by reacting an enzyme and its substrates in a reaction solution under a suitable pH and temperature, and, after termination of the enzymatic reaction, measuring the reduction of the added substrates or accumulation of products using a biochemical method.
  • PKA uses
  • Kemptide is a heptapeptide having an amino acid sequence of Leu-Arg-Arg-Ala- Ser-Leu-Gly, and has a net positive charge, where the serine residue is phosphorylated by PKA. Thanks to its positively charged property, kemptide can be easily collected using anion filter paper, and is thus used as a substrate for biologically or medically valuable enzymes.
  • the conventional method for measuring PKA activity comprises reacting p-labelled ATP ([ p]ATP), kemptide and an enzyme using the two compounds as substrates in a buffered reaction solution; collecting produced 32 p-labelled phosphokemptide using the anion filter paper, phosphocellulose; and measuring radioactivity.
  • p-labelled ATP [ p]ATP
  • kemptide a compound that is a compound that is a compound that is a compound that isophosphate
  • an enzyme using the two compounds as substrates in a buffered reaction solution
  • collecting produced 32 p-labelled phosphokemptide using the anion filter paper, phosphocellulose and measuring radioactivity.
  • Owing to the use of the radioactive isotope such a method is problematic in terms of causing environmental pollution, requiring too much time, requiring high-cost machineries, and consisting of complex procedures making analysis by non-experts difficult. Therefore, there is a need for development of methods of easily and rapidly detecting PKA.
  • Enzyme linked Immunosorbent Assay using antigen-antibody interaction is a method of qualitatively and quantitatively detecting biomaterials by measuring binding of an antigen and an antibody and strength of the binding using enzymes or a marker such as radioactive isotopes or fluorescent materials, where the antibody specifically recognizes the antigen that is the intended analyte.
  • the analysis system facilitates study of molecular mechanisms involved in PKA activity increased in sera of cancer patients, and leads to effective diagnosis of cancer.
  • Fig. 1 is a graph showing binding specificity of an anti-phosphokemptide monoclonal antibody of the present invention to phosphokemptide, which is obtained by performing an ELISA, including binding biotinylated phosphokemptide to a plate coated with avidin and performing antigen-antibody reaction using an anti-phosphokemptide antibody;
  • Fig. 2 shows a standard curve of protein kinase A (PKA) concentration, which is obtained by performing an ELISA using an anti-phosphokemptide monoclonal antibody of the present invention; and
  • PKA protein kinase A
  • Fig. 3 is a graph showing PKA levels in sera of normal humans and cancer patients, which is obtained by ELISA using an anti-phosphokemptide antibody.
  • a mouse hybridoma cell line producing a monoclonal antibody specifically recognizing phosphokemptide, wherein the monoclonal antibody is of the IgM class.
  • a method of preparing a hybridoma cell line producing a monoclonal antibody specifically recognizing phosphokemptide in accordance with a further aspect of the present invention, there is provided a method of measuring PKA activity by detecting phosphokemptide produced by PKA in blood using a monoclonal antibody to phosphokemptide.
  • phosphokemptide was first synthesized.
  • hybridoma cells producing a monoclonal antibody to phosphokemptide were prepared by immunizing mice with phosphokemptide, fusing splenocytes and myeloma cells from the immunized mice, and selecting cell clones secreting a monoclonal antibody specifically recognizing phosphokemptide from the fused cells by ELISA.
  • Subclasses of monoclonal antibodies produced in the selected cell clones were determined by an immunodiffusion test. One of them was found to produce a monoclonal antibody of IgM class, and this mouse hybridoma cell line was designated "ABI M002".
  • the hybridoma cell line "ABI M002” was injected into mice, and ascitic fluid containing high concentrations of hybridoma cells was collected from mice having swelled abdominal cavity.
  • the anti-phosphokemptide monoclonal antibody may serve as a diagnostic and prognostic bio-marker for cancer.
  • a heptapeptide having an amino acid sequence of Leu-Arg-Arg-Ala- phosphoSer-Leu-Gly was synthesized using an amino acid synthesizer.
  • EXAMPLE 3 Immunization of mice Immunized mice to be used for development of hybridoma cell lines producing a monoclonal antibody to phosphokemptide were prepared as follows. The phosphokemptide prepared in Example 1, present as a complex with bovine serum albumin (BSA), was mixed with an equal volume of complete Freund's adjuvant, and the resulting emulsion was subcutaneously injected into Balb/c mice
  • mice (6-8 weeks old). After 4 weeks, an amount of the phosphokemptide-BSA complex equal to that used in the first injection was mixed with incomplete Freund's adjuvant, and the resulting emulsion was intraperitoneally injected into the mice. 4 to 5 days after the second injection, a small amount of blood was collected from tails of the mice, and antibody titer was determined. 3 to 4 days before performing cell fusion, 10 ⁇ g of the phosphokemptide-BSA complex dissolved in a 0.85 % physiological saline solution was intravenously and intraperitoneally injected into mice.
  • mice immunized with phospohkemptide prepared in Example 1 From the mice immunized with phospohkemptide prepared in Example 1, splenocytes and myeloma cells were obtained.
  • An immortal SP2/0 Agl4 myeloma cell used as a parent cell for cell fusion, was maintained in RPMI medium containing 10% FBS at a density of 5 ⁇ l0 5 cells/mL, and collected by two rounds of centrifugation using Han's buffered saline solution (HBSS).
  • HBSS Han's buffered saline solution
  • spleens present in the left side of the body of each mouse were collected and finely ground.
  • the ground spleens were suspended in HBSS, collected by centrifugation in a 15 mL centrifuge tube, and this suspension and centrifugation was repeated to sufficiently wash splenocytes.
  • the resuspended cells were transferred into a 96-well microtiter plate, and cultured in a humidified CO 2 incubator (Forma Scientific Inc.) at 37°C.
  • EXAMPLE 5 Selection of hybridoma cell lines producing an anti- phosphokemptide monoclonal antibody
  • the fused cells prepared in Example 4 were screened for production of a monoclonal antibody to phosphokemptide through ELISA using phosphokemptide as an antigen, as follows. First, a 50 ⁇ L solution containing 20 ⁇ g of biotinylated phosphokemptide was placed into each well of a 96-well ELISA plate coated with avidin and allowed to attach to the bottom of the well, and unbound phosphokemptide was removed by washing each well with phosphate- buffered saline containing Tween 20 (PBST).
  • PBST phosphate- buffered saline containing Tween 20
  • the cell culture supernatant of each hybridoma cell line was added to the plate, and antigen-antibody reaction was performed for 2 hrs.
  • the unbound antibody was removed by washing the plate with PBST.
  • Horseradish peroxidase-conjugated goat anti-mouse IgG was added to the plate and the plate was incubated at room temperature for 1 hr.
  • OPD o-phenylenediamine dihydrochloride
  • Optical density was measured at 492 nm using an ELISA reader (Flow. Lab Inc.).
  • hybridoma cells secreting a monoclonal antibody having high binding specificity to phosphokemptide were selected, through several repeated tests.
  • the ELISA-positive hybridomas were cloned by limiting dilutions to obtain a cell line that would be stable and continue producing anti- phosphokemptide monoclonal antibody.
  • the resultant six stable colonies were isolated and frozen for storage, while the hybridoma culture supernatants were evaluated for antibody titer by ELISA. Through an immunodiffusion test, one of the colonies was found to produce an anti-phosphokemptide monoclonal antibody belonging to IgM class.
  • the finally selected hybridoma cell line was deposited in Korean Cell Line
  • PBS phosphate-buffered saline
  • EXAMPLE 8 Quantitative analysis of PKA by ELISA using anti- phosphokemptide monoclonal antibody
  • Optical density was measured at 492 nm using an ELISA reader (Flow. Lab Inc.). As shown in Fig. 2, a standard curve in which absorbance is increased with amount of PKA was obtained. Using the standard curve, ELISA values, that is, absorbance at 492 nm, obtained by using sera of various cancer patients were converted to PKA levels. As shown in Fig. 3, cancer patients were found to have higher PKA levels than the normal humans.
  • the anti-phosphokemptide monoclonal antibody of the present invention can be used in detecting PKA activity. Since
  • the anti-phosphokemptide monoclonal antibody can be applied for diagnosis of cancer, where the diagnosis is simply and quickly performed.
  • KCLRF Korean Cell Line Research Foundation

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un anticorps monoclonal de phosphokemptide et une lignée de cellule hybridome produisant cet anticorps monoclonal anti-phosphokemptide. Cette invention concerne aussi une technique de détection d'activité de protéine kinase A au moyen de cet anticorps monoclonal anti-phosphokemptide. L'anticorps monoclonal anti-phosphokemptide de cette invention convient pour le diagnostic du cancer, le diagnostic au moyen de cet anticorps monoclonal étant réalisé simplement et rapidement par détection de la protéine kinase A dans des sérums de patients atteints de cancer.
PCT/KR2002/002020 2001-10-30 2002-10-30 Technique de dosage d'activite de proteine kinase a au moyen d'anticorps anti-phosphokemptide dans le diagnostic du cancer WO2003037934A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20010067131 2001-10-30
KR2001/67131 2001-10-30
KR2002/66311 2002-10-30
KR1020020066311A KR20030036010A (ko) 2001-10-30 2002-10-30 암 진단을 위한 항-인산화 캠타이드 단일클론 항체를이용한 혈중 프로테인키나제의 효소활성측정법

Publications (1)

Publication Number Publication Date
WO2003037934A1 true WO2003037934A1 (fr) 2003-05-08

Family

ID=26639435

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2002/002020 WO2003037934A1 (fr) 2001-10-30 2002-10-30 Technique de dosage d'activite de proteine kinase a au moyen d'anticorps anti-phosphokemptide dans le diagnostic du cancer

Country Status (1)

Country Link
WO (1) WO2003037934A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670628A (en) * 1989-03-23 1997-09-23 British Technology Group Ltd. Radio-labelling of proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670628A (en) * 1989-03-23 1997-09-23 British Technology Group Ltd. Radio-labelling of proteins

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FOXWELL B.M.: "Conjugation of monoclonal antibodies to a synthetic peptide substrate for protein kinase: a method for labelling antibodies with 32P.", BR. J. CANCER, vol. 57, no. 5, 1988, pages 489 - 493 *
LEUNG S. ET AL: "BACTERIAL EXPRESSION OF A KEMPTIDE FUSION PROTEIN FACILITATES 32P LABELING OF A HUMANIZED, ANTI-CARCINOEMBRYONIC ANTIGEN (HMN-14) ANTIBODY FRAGMENT.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 55, no. 23, 1 December 1995 (1995-12-01), BALTIMORE, MD, US, pages 5968S - 5972S, XP002038654 *
PATRICK M.R. ET AL: "In Vitro Characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody.", CANCER IMMUNOL. IMUNOTHER, vol. 46, no. 4, 1998, pages 229 - 237 *

Similar Documents

Publication Publication Date Title
JPH0367678B2 (fr)
JPH04503600A (ja) 抗インターロイキン―１α及び―１βのモノクローナル抗体、その製法、ならびに前記抗体類の、インターロイキン―１α及び―１βの検出と治療への応用
KR101338517B1 (ko) 인간 간-카르복실에스터라제 １을 특이적으로 인식하는 단일클론 항체, 상기 항체를 생산하는 하이브리도마 세포주 및 이의 용도
US5856182A (en) Monoclonal antibodies specific for the PSA-ACT complex
EP1098198A1 (fr) Procédé pour la détermination qualitative et quantitative des anticorps humains de la classe IgG
US6828114B1 (en) Monoclonal antibody against apolipoprotein A-I
KR100287382B1 (ko) 지럴레논 독소의 항체를 생산하는 방법
KR20080112246A (ko) 햅텐 화합물 및 항체
JPH07238099A (ja) モノクローナル抗体及びこれを用いる免疫学的測定法
US6764827B1 (en) Anti-human medullasin monoclonal antibody process for producing the same and immunoassay using the same
JPH10226700A (ja) Ｍｉａの検出のためのイムノアッセイ
WO2003037934A1 (fr) Technique de dosage d'activite de proteine kinase a au moyen d'anticorps anti-phosphokemptide dans le diagnostic du cancer
JPS63307900A (ja) 抗ＬＣＡ結合性ヒトα−フェトプロテインモノクロ−ナル抗体
JP4533995B2 (ja) 抗ａｄａｍｔｓ１３モノクローナル抗体
US4952507A (en) Production and diagnostic use of antibodies against pancreatic alpha-amaylase
CN117264071B (zh) 一种抗rankl单克隆抗体或其衍生物的结合剂及其应用
US20110281284A1 (en) Novel liver cancer marker
JPH1175839A (ja) モノクローナル抗体、細胞株及びｎ１，ｎ１２−ジアセチルスペルミンの測定法
KR20030036010A (ko) 암 진단을 위한 항-인산화 캠타이드 단일클론 항체를이용한 혈중 프로테인키나제의 효소활성측정법
US8349569B2 (en) Anti-fibronectin fragment monoclonal antibody
WO1986002362A1 (fr) Anticorps monoclonaux et leur utilisation
KR100246126B1 (ko) N-아세틸글루코사민 전이효소ⅲ에 대한 단일클론 항체, 이를생산하는 융합세포주 및 그들의 제조방법
WO1986002363A1 (fr) Anticorps monoclonaux et leur utilisation
WO1986003498A1 (fr) Anticorps monoclonaux et leur utilisation
JP3849001B2 (ja) 抗−２′−デオキシシチジン抗体並びにその製造及び使用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP