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WO2013067750A1 - Trousse pour spectrométrie de masse et immunologie à anticorps polyclonal d'un marqueur du cancer du foie - Google Patents

Trousse pour spectrométrie de masse et immunologie à anticorps polyclonal d'un marqueur du cancer du foie Download PDF

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Publication number
WO2013067750A1
WO2013067750A1 PCT/CN2012/000222 CN2012000222W WO2013067750A1 WO 2013067750 A1 WO2013067750 A1 WO 2013067750A1 CN 2012000222 W CN2012000222 W CN 2012000222W WO 2013067750 A1 WO2013067750 A1 WO 2013067750A1
Authority
WO
WIPO (PCT)
Prior art keywords
polyclonal antibody
polypeptide
solid phase
phase carrier
protein
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2012/000222
Other languages
English (en)
Chinese (zh)
Inventor
魏开华
肖汉族
原剑
孙云波
付海媛
周晓明
杨保安
侯利平
黄亚娟
郑俊杰
甄蓓
张拓
王东茂
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing C & N International Sci-Tech Co Ltd
HUNAN JINJIAN PHARMACEUTICAL CO Ltd
Original Assignee
Beijing C & N International Sci-Tech Co Ltd
HUNAN JINJIAN PHARMACEUTICAL 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 Beijing C & N International Sci-Tech Co Ltd, HUNAN JINJIAN PHARMACEUTICAL CO Ltd filed Critical Beijing C & N International Sci-Tech Co Ltd
Publication of WO2013067750A1 publication Critical patent/WO2013067750A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • C07K16/303—Liver or Pancreas
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a multi-anti-immuno-mass spectrometry kit and a detection method thereof. Background technique
  • Primary liver cancer is one of the most common cancers in the world.
  • the five-year survival rate of early diagnosis of malignant tumors can reach 70% -95%, while the five-year survival rate of advanced tumors is only 20% -30%.
  • improving the early diagnosis rate of malignant tumors achieving early detection and early treatment of malignant tumors is an effective means to improve the overall survival rate of patients with malignant tumors, improve the quality of life, and reduce medical expenses.
  • Current diagnostic tools commonly used in clinical practice include physical examination, imaging examination, serum tumor marker detection, and pathological examination. Due to the small volume of the early stage of the cancer, the liver is hidden in the deep upper part of the abdomen, and the ribs are used as a barrier.
  • liver cancer It is difficult to find early by using B-ultrasound and CT scanning.
  • the liver has a strong compensatory function and often has no clinical symptoms in the early stage. It also brings difficulties to the early diagnosis of liver cancer.
  • pathological examination is a diagnostic gold standard, it is difficult to obtain materials, and most of them are invasive examinations, which are not suitable for population screening.
  • the non-invasive and reproducible serum tumor markers have theoretically become an ideal means for population screening for malignant tumors.
  • the mass spectrometry technology has the characteristics of high sensitivity and high throughput. Can provide a huge amount of information. However, many factors in the experiment may affect the results of mass spectrometry. Such as the collection, processing, preservation of the sample, the conditions of the test and even the temperature and humidity at the time of detection may have more effects. In addition, from previous studies, different mass spectrometry systems, different sample processing methods, and different bioinformatics methods have been used to treat different diseases with different results.
  • the biological mass spectrometer that has emerged in recent years has been successfully applied to the diagnosis of several cancers, such as breast cancer, ovarian cancer, prostate cancer, etc., mainly using surface-enhanced laser desorption ionization.
  • the sensitivity, specificity, and positive predictive measures of ovarian cancer were 100%, 95%, and 94%, respectively, which were significantly better than traditional CA125 tests, especially for the diagnosis of early stage ovarian cancer (stage I). This technique is expected to be used for early or early warning of ovarian cancer.
  • This technology can effectively enrich and analyze low molecular weight proteins/peptides in samples such as blood, and it also has the advantages that SELDI does not have, such as the sample after enrichment is easy to elute for identification.
  • this system has the disadvantages of being expensive and disadvantageously popularized with SELDI-TOF-MS. Therefore, the search for serum biomarker enrichment detection technology with low cost, strong enrichment ability and high repetitiveness has become one of the important research hotspots. Summary of the invention
  • the present invention relates to a multi-antibody immuno-mass spectrometry kit for liver cancer markers.
  • the polyclonal antibody anti-immunoassay detection kit for liver cancer markers comprises a serum polypeptide polyclonal antibody and a buffer, wherein the polyclonal antibody is immobilized on a solid phase carrier, and the solid phase carrier is coated egg A ( Protein A) or protein G (protein G) agarose particles.
  • the polyclonal antibody is an anti-synthetic peptide 6 antibody, and the full length sequence of the synthetic polypeptide 6 is: NLGHG HKHDR DHGHG HQo
  • the buffer is preferably 0.01 mol/L, pH 7.4 in PBS buffer.
  • Agarose / Protein G Agarose is a matrix for immunoprecipitation and is more cost effective than Protein A or Protein A magnetic G magnetic beads.
  • the multi-antibody immuno-mass spectrometry kit provided by the present invention preferably further comprises an eluent selected from the group consisting of a 0.1 mol/l glycine-HC1 solution having a pH of 2.7 and a mixture of 70% ACN containing 0.1% TFA. Mixture of 50% ACN with 0.1% TFA or 5°/. Acetic acid.
  • the preferred anti-synthetic peptide 6 antibodies of the invention are prepared as follows:
  • the invention also provides a preparation method of the multi-antibody immuno-mass spectrometry kit, which comprises the steps of: mixing the purified polyclonal antibody with the solid phase carrier suspension to obtain a concentration of 0.075 g/L- 0.6 g ⁇ L, incubate at 4 ° C for 5 minutes - 4 hours, place 1-5 min precipitation, filter, use 0.01mol / L, pH 7.4 PBS buffer 100-200 ⁇
  • the solid phase carrier was washed 2 to 5 times to obtain the polypeptide immunoassay kit.
  • the invention also provides a method of using the kit in a serum epitope antigen, comprising the steps of:
  • step 2) taking 10-40 ⁇ 1 serum sample, 10-50 ⁇ PBS, mixing with the solid phase carrier after step 1), rotating and mixing at 4 ° C for 8-24h;
  • the invention also provides a method for detecting a polypeptide marker by immuno Mass Spectrometry, and the polypeptide standard and the serum polypeptide marker antigen separated by the kit of the invention are detected by MALDI-TOF-MS, and the peak value and the theoretical peak value are less than 0.3 Da, This peak is the marker peak.
  • the detection conditions of MALDI-TOF-MS are positive ion detection mode, the ion source acceleration voltage is 20kV, N 2 laser, laser wavelength 337 nm, energy 2500, ion delay extraction time 390 ns, mass spectrometry signal single scan cumulative 2000 times, Using the peptide II standard kit ion peak calibration, the mass scan range is 1000-10000 Da.
  • the invention selects a plurality of polypeptide markers of liver cancer, and correspondingly prepares a plurality of polyclonal antibodies.
  • the invention selects a plurality of polypeptide markers of liver cancer, and correspondingly prepares a plurality of polyclonal antibodies.
  • detection and analysis and data statistics it is unexpectedly found that the specificity and sensitivity of one of the polyclonal antibodies are much higher than other ones, indicating This marker has a strong ability to diagnose liver diseases.
  • an immunomass detection kit and a detection method containing the polyclonal antibody are provided.
  • the immuno-mass spectrometry of the present invention uses a polyclonal antibody to enhance the enrichment specificity, and at the same time, the preparation process is simplified, further reducing the cost of the immuno-mass spectrometry kit.
  • the invention combines conventional immunoassay technology with the most advanced mass spectrometry technology to make full use of the two technologies. High specificity, high throughput, high accuracy, high sensitivity, and low false positive are the latest frontiers of diagnostic technology.
  • FIG. 1 is a schematic diagram of a multi-antibody immuno-mass spectrometric detection method of the present invention.
  • Figure 2 is a mass spectrum of a synthetic peptide 6 standard sample in an embodiment of the present invention.
  • Figure 3 is a mass spectrum of a liver cancer serum sample in an embodiment of the present invention.
  • Figure 4 shows the detection specificity and sensitivity of the kit of the present invention. detailed description
  • the synthetic polypeptide 6 conjugated to the carrier protein KLH was used as an immunogen to immunize the white rabbit; its full length sequence is: NLGHGHKHDRDHGHGHQ (SEQ ID No. 1).
  • Protein A agarose particles (Protein A Agarose, Santa Cruz), place in a 0.2 mLEppendorf tube, add 24 g of the polyclonal antibody prepared in Example 1, rotate at 4 ° C (rotation speed 5 r / min), mix 2 Hour, place for 3 minutes, remove the supernatant, use lOO LPBS The resulting solid phase carrier was washed 5 times with a buffer (0.01 mol/l, pH 7.4).
  • Fig. 1 The flow of the polypeptide immunoassay detection method of the present invention is shown in Fig. 1. Specifically: 1) Take 20 L of Protein A agarose particles (Protein A Agarose, Santa Cruz), placed in a 0.2 mLEppendorf tube, add 7.5 g of the polyclonal antibody prepared in Example 1, and rotate at 4 ° C (rotation speed 5 r /min), mixed for 0.5 hours, placed for 2 min, the supernatant was removed, and Protein G Agarose was washed 3 times with 100 mL of PBS buffer (0.01 mol/l, pH 7.4);
  • the detection condition is positive ion detection mode
  • the ion source acceleration voltage is 20kV
  • the N 2 laser the laser wavelength is 337 nm
  • the energy is 2500
  • the ion ion extraction time (PIE) is 390 ns
  • the mass spectrometry signal is accumulated 2,000 times in a single scan.
  • the mass scan range is 1000 ⁇ 10000Da.
  • Fig. 1 The flow of the polypeptide immunoassay detection method of the present invention is shown in Fig. 1. Specifically:
  • the detection condition is positive ion detection mode
  • ion source acceleration voltage is 20kV
  • N 2 laser laser wavelength 337 nm
  • energy 2500 ion ion extraction time (PIE) 390 ns
  • Fig. 1 The flow of the polypeptide immunoassay detection method of the present invention is shown in Fig. 1. Specifically:
  • FIG. 6 Another 30 L test 100 cases of liver cancer serum and 100 normal serum were detected by MALDI-TOF-MS.
  • the mass spectrum of liver cancer serum is shown in Figure 3.
  • Figure 3 is similar to Figure 2, with fewer peaks, indicating a higher specificity of the target peak.
  • Figure 4 shows the sensitivity of the method of the invention of 83.0% and specificity of 93.3%.
  • the detection condition is positive ion detection mode
  • ion source acceleration voltage is 20kV
  • N 2 laser laser wavelength 337 nm
  • energy 2500 ion ion extraction time (PIE) 390 ns
  • Synthetic peptide 6 83.0% 93.3% Synthetic peptide 7 50.9% 67.7% Synthetic peptide 8 41.3% 78.7%
  • the immunoassay detection kit and detection method of the polyclonal antibody of the invention combines the conventional immunoassay technology with the most advanced mass spectrometry test technology, and fully utilizes the two technologies with high specificity, high throughput, high accuracy and high sensitivity.
  • the advantages of sex, low false positive, etc. have realized the detection of liver cancer markers, and have important economic value and application prospects.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

L'invention concerne une trousse pour spectrométrie de masse et immunologie à anticorps polyclonal d'un marqueur du cancer de foie, et un procédé de détection. La trousse comprend un anticorps polyclonal polypeptidique du sérum et une solution tampon, l'anticorps polyclonal étant fixé sur un vecteur à phase solide, le vecteur à phase solide étant des particules d'agarose revêtues par une protéine A ou protéine G, l'anticorps polyclonal polypeptidique du sérum étant un anticorps polyclonal dirigé contre le polypeptide 6 synthétique, et la séquence de longueur totale du polypeptide 6 synthétique étant NLGHG HKHDR DHGHG HQ.
PCT/CN2012/000222 2011-11-09 2012-02-21 Trousse pour spectrométrie de masse et immunologie à anticorps polyclonal d'un marqueur du cancer du foie Ceased WO2013067750A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 201110353728 CN102507936B (zh) 2011-11-09 2011-11-09 一种肝癌标志物多抗免疫质谱试剂盒
CN201110353728.9 2011-11-09

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Cited By (1)

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CN110687284A (zh) * 2019-08-26 2020-01-14 中国医学科学院肿瘤医院 检测血清中six2自身抗体的试剂的应用

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CN112067807B (zh) * 2019-06-10 2022-07-26 中国医学科学院肿瘤医院 肝癌患者血清中筛选预后相关蛋白的方法及其应用
CN113567680A (zh) * 2021-06-30 2021-10-29 广州医科大学 一种痰上清抗中性粒细胞胞浆抗体的检测方法
CN114295706B (zh) * 2021-09-28 2024-11-01 岛津企业管理（中国）有限公司 一种基于统计学的非靶向非小细胞肺癌的病理分型方法
CN119798401B (zh) * 2024-12-30 2025-10-10 常州因普迈生物科技有限公司 基于pdzk1ip1蛋白的多肽、抗体及其应用

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CN102507936A (zh) 2012-06-20

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