WO2016127276A1 - Application d'une alpha-foetoprotéine de l'urine - Google Patents

Application d'une alpha-foetoprotéine de l'urine Download PDF

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WO2016127276A1
WO2016127276A1 PCT/CN2015/000624 CN2015000624W WO2016127276A1 WO 2016127276 A1 WO2016127276 A1 WO 2016127276A1 CN 2015000624 W CN2015000624 W CN 2015000624W WO 2016127276 A1 WO2016127276 A1 WO 2016127276A1
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fetoprotein
urine
alpha
antibody
application
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PCT/CN2015/000624
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English (en)
Chinese (zh)
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张曼
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张曼
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Publication of WO2016127276A1 publication Critical patent/WO2016127276A1/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

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  • the invention relates to a new use of urinary alpha fetoprotein, in particular to the expression of alpha fetoprotein in urine and its application in the detection of urine content.
  • Alpha-fetoprotein is the main component of embryonic plasma protein, and its gene encoding human albumin and vitamin D binding protein belongs to the albumin gene family, which are located on chromosome 4. It is produced by yolk sac and fetal liver. It is mainly composed of fetal liver synthesis after 12 weeks of gestation. The serum concentration during fetal period is very high, and it decreases after birth. From February to March, alpha-fetoprotein is basically replaced by albumin. More difficult to check out.
  • Alpha-fetoprotein has many important physiological functions, the most basic of which is the transport function. Alpha-fetoprotein can bind estrogen, fatty acid, bilirubin, Cu2+ and Ni2+. One of the most important functions is to transport fatty acids. Both fetal and embryonic alpha-fetoprotein can bind 2 to 3 fatty acid molecules. Part is unsaturated fatty acids. In addition, alpha-fetoprotein is involved in cell proliferation, regulation of metabolism, and interaction between macrophages and T lymphocytes. The role of alpha-fetoprotein in the immune response is immunosuppression, mainly manifested by inhibition of the maternal immune response to embryonic development and tumor patients' immune response to tumors.
  • Alpha-fetoprotein can also be synthesized in hepatocellular carcinoma, embryonic tumors, and some extrahepatic tumors. Therefore, alpha-fetoprotein is associated with primary liver cancer, gastric cancer, lung cancer, pancreatic cancer, cholangiocarcinoma, and testicular tumor. It can also be elevated in patients with partial hepatitis (15% to 58%) and cirrhosis (11% to 47%).
  • alpha-fetoprotein Due to the special source of alpha-fetoprotein, it is difficult to detect it in the serum of normal adults. This only indicates that the serum concentration is too low, exceeding the minimum detection limit of the general test, so that it cannot be detected.
  • our research has confirmed that a part of alpha-fetoprotein is found in the urine of normal people. Therefore, normal people can still synthesize a certain amount of alpha-fetoprotein, which plays a certain role in normal physiological metabolism, and can also be used in urine. A certain amount of a fragment of alpha-fetoprotein is stably present.
  • Urine is the ultrafiltrate of blood, which is the terminal metabolite produced by the body.
  • the composition change is a specific manifestation of certain disease states, and the collection of urine specimens is non-invasive, multi-volume, patient compliance, and not
  • the advantage of requiring the help of medical personnel, in summary, the present invention aims to apply the advantages of urine specimens, and provides a simple kit for detecting alpha-fetoprotein fragments in urine.
  • amino acid sequence of the urine alpha-fetoprotein is as shown in SEQ ID NO: 1:
  • the preparation is a urine alpha-fetoprotein detection kit.
  • the kit is an antibody antigen reaction.
  • the antigen-antibody reaction is coated or labeled with a urine alpha-fetoprotein or polypeptide and an antibody thereof in a solid phase or liquid phase carrier.
  • the inventors first collected random urine specimens of normal physical examination, took the supernatant after centrifugation, and purified and separated urine specimens using weak cation exchange magnetic beads. 1 ⁇ l of sample with 10 ⁇ l of substrate (0.3% ⁇ -cyano-4- After mixing hydroxycinnamic acid and HCCA), 1 ⁇ l was spotted on an Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target plate, and the sample was ionized and subjected to mass spectrometry. Data in the range of 1000-10000 Da was collected to obtain different mass loads. A mass spectrometric peptide map composed of protein peaks.
  • the present invention confirmed by studies that alpha fetoprotein can stably appear in the urine of a person who is normally examined. Therefore, it is proposed to detect the application of urine protein alpha fetoprotein in urine related examination.
  • the invention exerts the advantage of non-invasiveness of urine specimens, and uses random urine specimens to detect alpha-fetoprotein or polypeptide.
  • Figure 1 is the average value of all points in the normal physical examination specimens between 1000 and 10000.
  • Figure 2 is a scatter plot of the point of mass-to-charge ratio of 1893.6 expressed in 30 normal medical specimens.
  • Figure 3 is a mass spectrum of alpha-fetoprotein.
  • the urine sample was taken out from the -80 ° C refrigerator, recombined at 4 ° C, centrifuged (3000 rpm, 10 min), and the supernatant was taken for use.
  • the weak cation magnetic beads (MB-WCX) were equilibrated at room temperature and the magnetic bead suspension was manually mixed.
  • the magnetic beads are separated from the suspended liquid. Use a sample gun to remove the suspended clear liquid. The tip should avoid contact with the magnetic beads and avoid picking up the magnetic beads.
  • FIG. 1 which shows the average value of all the mass-to-charge ratio points between 1000-10000 Da in 30 urine samples; the peak area as a quantitative standard, and FIG. 2 shows the expression of alpha fetoprotein in all urine samples. It can be seen from the figure that m/z 1893.6 has a peak area greater than 600 in all specimens.
  • the magnetic bead eluate in the sample tube was evaporated to dryness, dissolved in 20 ul of mobile phase A (5% acetonitrile, 0.1% formic acid in water), and transferred to a sample bottle.
  • the injection volume was 18 ul, firstly desalted into the trap column at a rate of 15 ⁇ l/min, and the capture time was 3 min. Then enter the analytical column at a flow rate of 400 nl / min for gradient elution, the elution gradient is 5% B-50% B-80% B-80% B-50% B-5% B (mobile phase B: 95% acetonitrile) , 0.1% aqueous solution of formic acid, see Table 1).
  • the analysis time was 60 min, the column temperature was 35 ° C, and all eluted components were analyzed by mass spectrometry.
  • Nano ion source spray voltage 1.8kV; mass spectrometry mode for data dependence and dynamic exclusion, scan range 400-2000m/z; first-order scan (MS) using Obitrap, resolution set to 100000; CID and secondary scan using LTQ; A single isotope of the strongest 10 ions was selected as the parent ion in the MS spectrum for MS/MS (single charge exclusion, not as parent ion).
  • the mass spectrometry scan time was 60 min. Sequest TM search was performed using the data analysis software Bioworks Browser 3.3.1 SP1.
  • the search database is International Protein Index (IPI human v3.45 fasta with 71983entries).
  • the mother ion error was set to 100 ppm
  • the fragment ion error was set to 1 Da
  • the digestion method was non-enzymatically cut
  • the variable modification was methionine oxidation.
  • the search result parameters are set to deltacn ⁇ 0.10, two charges Xcorr 2.6, three charges Xcorr 3.1, and three charges above Xcorr 3.5.
  • the protein alpha-fetoprotein is retrieved in the database, and the mass spectrum of the alpha-fetoprotein is shown in Figure 3.
  • the antibody and antigen concentrations were determined according to Pierce's BCA Protein Concentration Kit instructions, and then the rabbit anti-human alpha-fetoprotein polyclonal antibody (Abcam) was diluted to a concentration using a standard checkerboard assay. 10.0 ng/ml, 1.0 ng/ml, and 0.1 ng/ml were coated on solid phase ELISA plates and liquid phase magnetic beads, respectively, each concentration consisting of three wales, overnight at 4 ° C, and washed 3 times. A strong positive antigen solution was added to one of the transverse coated wells, a weak positive antigen solution was added to the other row, and a negative control was added to the third row. Incubate for 2 hours at 37 ° C and wash 3 times.
  • a murine anti-human alpha fetoprotein monoclonal antibody (Abeam) was added, incubated at 37 ° C for 1 hour, and washed 3 times.
  • the labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, washed 4 times, the substrate was added, and the mixture was allowed to stand at room temperature for 20 minutes in the dark, and the stop solution was added for reading. Choose the optimal concentration of coated antibody.
  • the rabbit anti-human alpha-fetoprotein polyclonal antibody was diluted with a coating buffer, added to a solid phase microplate and liquid phase magnetic beads, and gently shaken overnight at 4 °C.
  • the uncoated liquid was poured out, washed 3 times, and a blocking solution was added to prevent non-specific binding sites, incubated at 37 ° C for 1 hour, and washed 3 times. Store at 4 ° C for later use.
  • the kit is divided into a mouse anti-human alpha fetoprotein monoclonal antibody, a labeled secondary antibody, and the like.
  • the alpha-fetoprotein recombinant protein (OriGene, Germany) was diluted with PBS to 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 10 ng/ml, 2 ng/ml, 0.5 ng/ml, 0.05 ng/ml, 0.01. Ng/ml, 0 ng/ml, 100 ul per well was added to the above coated ELISA plate and the liquid magnetic beads, incubated at 37 ° C for 2 hours, and washed 3 times.
  • the mouse anti-alphafetoprotein monoclonal antibody was diluted 1:2000, 100 ul was added per well, incubated at 37 ° C for 1 hour, and washed 3 times.
  • the labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, and washed 3 times.
  • the substrate was added to room temperature for 15 minutes, and the stop solution was added for reading.
  • the lowest amount of alpha-fetoprotein was detected.
  • the results showed that the reagent could detect the concentration of 0.01 ng/ml alpha-fetoprotein, indicating a higher detection sensitivity.
  • the above experiments show that the kit of the present invention detects the content of alpha-fetoprotein in the urine sample, and has high sensitivity.
  • the minimum detection limit of the sample is 0.01 ng/ml, and the recovery rate is 90% ⁇ 13%.
  • the kit requires fewer instruments and requires only a microplate reader, an oscillator, a centrifuge, a pipette, etc., and the cost is low.
  • the inventors examined the content of alpha-fetoprotein in 100 normal urine urine samples with an accuracy of more than 97%, and had good specificity.

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

Abstract

La présente invention concerne une application d'une alpha-foetoprotéine (AFP) de l'urine, en particulier une expression de l'alpha-foetoprotéine de l'urine dans l'urine et une application associée à la détection du contenu de l'urine. Une recherche vérifie qu'une alpha-foetoprotéine dans l'urine présente une expression d'abondance relativement élevée, et, par conséquent, des informations sur le diagnostic de maladies sont obtenues en détectant la quantité d'expression de l'alpha-foetoprotéine dans l'urine. Les avantages d'acquisition d'un échantillon d'urine de manière non effractive et de sauvegarde de manière pratique sont obtenus, et l'alpha-foetoprotéine est détectée en utilisant l'échantillon d'urine.
PCT/CN2015/000624 2015-02-10 2015-09-01 Application d'une alpha-foetoprotéine de l'urine WO2016127276A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510067727.6A CN105988005A (zh) 2015-02-10 2015-02-10 尿液甲胎蛋白的应用
CN201510067727.6 2015-02-10

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WO2016127276A1 true WO2016127276A1 (fr) 2016-08-18

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4892500B2 (ja) * 2008-02-12 2012-03-07 富士フイルム株式会社 非特異吸着抑制剤を用いたイムノクロマトグラフ方法
US20140155279A1 (en) * 2012-11-14 2014-06-05 JBS Science Inc. Detection of a panel of urine DNA markers for HCC screening and disease management

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4892500B2 (ja) * 2008-02-12 2012-03-07 富士フイルム株式会社 非特異吸着抑制剤を用いたイムノクロマトグラフ方法
US20140155279A1 (en) * 2012-11-14 2014-06-05 JBS Science Inc. Detection of a panel of urine DNA markers for HCC screening and disease management

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank 23 September 2014 (2014-09-23), "Alpha-fetoprotein Precursor [Homo sapiens", Database accession no. NP_001125.1 *
HE, GENGHUA ET AL.: "Detection of Alpha-Fetoprotein in the Urine of a Patient with Primary Liver Cancer", CANCER RESEARCH ON PREVENTION AND TREATMENT, no. 5, 31 December 1980 (1980-12-31), pages 1 - 3 *
JING, CHENGBAO ET AL.: "Detecting AFP in Urine by using Solid Phase Immunity Hemagglutination Technology", SHANXI JOURNAL OF MEDICAL LABORATORY SCIENCES, vol. 8, no. 2, 31 May 1993 (1993-05-31), pages 89 *
JING, CHENGBAO;: "Discussion about Applying Urine to Detect AFP", CHINESE JOURNAL OF CLINICAL LABORATORY SCIENCE, vol. 12, 31 December 1994 (1994-12-31), pages 63 *
JING, CHENGBAO;: "Discussion about Applying Urine to Detect AFP", CHINESE JOURNAL OF CLINICAL LABORATORY SCIENCE, vol. 12, no. 31, December 1994 (1994-12-01), pages 63 *

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