WO2016127278A1 - Application d'une protéine du composant 3 (c3) du complément de l'urine - Google Patents

Application d'une protéine du composant 3 (c3) du complément de l'urine Download PDF

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Publication number
WO2016127278A1
WO2016127278A1 PCT/CN2015/000626 CN2015000626W WO2016127278A1 WO 2016127278 A1 WO2016127278 A1 WO 2016127278A1 CN 2015000626 W CN2015000626 W CN 2015000626W WO 2016127278 A1 WO2016127278 A1 WO 2016127278A1
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urine
protein
complement
complement component
detection
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PCT/CN2015/000626
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English (en)
Chinese (zh)
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张曼
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张曼
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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 urine complement C3 protein, in particular to the normal expression of urine complement C3 protein as an internal reference of a urine test method, and abnormal expression as a biomarker for disease diagnosis.
  • specimens with invasive, lipemia or hemolysis in blood specimens are not detectable for certain items, and collection equipment is often a weapon. This requires not only well-trained and experienced blood collectors, but also blood products themselves that increase occupational exposure. risk. On the other hand, at least 8 hours on an empty stomach increases the risk of hypoglycemia in patients such as pregnant women, the elderly and children. For patients who need frequent and even life-long blood collection, such as diabetes, blood diseases, cancer chemotherapy, etc., it also brings great pain to patients.
  • Urine is the ultrafiltrate of blood. It is the terminal metabolite produced by glomerular filtration, renal tubular and collecting tube reabsorption, excretion and secretion. It is a window reflecting the changes of the body. Its composition is changed by certain diseases.
  • the specific manifestation of the state, and the collection of urine specimens is simple, non-invasive, multi-volume, repeatable multiple times, convenient to store, high patient compliance, and does not require the help of medical personnel.
  • urine protein is relatively simple and easy to analyze, and has great advantages in the diagnosis of diseases by body fluids.
  • composition, quantity and trait changes of urine not only carry various information about the occurrence, development and prognosis of urinary system diseases, but also reflect the overall metabolic state of the body, for studying organ function, evaluating body state, metabolism, and dynamic monitoring. Various information is provided on aspects such as disease progression and judgment of efficacy.
  • urine also contains a greater number of polypeptide fragments. Proteins and peptides with a molecular weight of less than 10 kDa can be freely filtered through the glomerulus. The polypeptide fragments in the urine are likely to be products of proteolysis. Analysis of urine may be more suitable for diagnosing systemic diseases and revealing the pathophysiological characteristics of the disease. It is therefore an ideal material for diagnosing and judging disease progression.
  • Complement C3 is the core protein in the complement system and is mainly produced by hepatocytes and macrophages.
  • the gene encoding complement C3 is located on chromosome 19 and consists of 41 exons.
  • the full length of the cDNA coding sequence is about 5052 bp, which in turn encodes the leader peptide, beta chain and alpha chain.
  • the complement C3 mature protein contains 1663 amino acids with a relative molecular weight of 180 kDa.
  • C3 has the highest serum content; in terms of function, C3 is also at the center, which is the intersection of several activation pathways and the basis of C3b-dependent positive feedback loop; meanwhile, C3 is activated and lysed. After that, the series of fragments and their binding proteins produced are complex and diverse, and play an important role in immune defense, immune regulation and immunopathology.
  • the non-complement function of C3 associated with cell proliferation and differentiation has also been reported, such as the proliferation and establishment of bud base and lens vesicles, involved in the proliferation and growth of B cells in vitro, which may be attributed to the complement component in mitogenic events.
  • Role, C3 may also play a role in early dedifferentiation and even in later differentiation. With the deepening of research, C3 is thought to play a role in tumor formation, signal transduction, and apoptosis. At the same time, it can protect against oxidative stress.
  • the concentration of markers related to urine diseases depends not only on the excretion rate of the marker but also on the urine flow.
  • 24h urine is the gold standard for assessing urine markers, but the collection of such specimens is cumbersome and the patient's compliance is poor.
  • the effects of creatinine, urinary permeation or urine specific gravity on the calculation of urine volume are used in the study.
  • the present invention first screens for high abundance protein polypeptides that are stably present in urine.
  • the urine protein polypeptide can be stabilized at a certain level when the body is in a normal physiological state, and an increase or decrease in its level is associated with certain diseases.
  • a urine complement C3 protein Complement Component 3
  • amino acid sequence of the urine complement C3 protein is as shown in SEQ ID NO: 1:
  • the preparation is a urine complement C3 protein detection kit.
  • the kit is an antibody antigen reaction.
  • the antigen-antibody reaction is coated or labeled with a urine complement C3 protein 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. After mixing 1 ⁇ l of the sample with 10 ⁇ l of the substrate (0.3% ⁇ -cyano-4-hydroxycinnamic acid, HCCA), 1 ⁇ l of the spot was taken on an Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target plate, and the sample was ionized. Mass spectrometry analysis was performed to acquire data in the range of 1000-10000 Da, and a mass spectrometric peptide map composed of protein peaks of different mass-to-charge ratios was obtained.
  • Anchorchip Autoflex MALDI TOF, Bruker-Dalton
  • the present invention confirmed by studies that the complement C3 protein can stably appear in the urine of a person who is normally examined. Therefore, it is proposed to detect the use of urine complement C3 protein for urine related examination.
  • the invention exerts the advantage of obtaining non-invasive urine specimens, and uses the random urine specimen to detect the complement C3 protein 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 1726.8 expressed in 30 normal medical specimens.
  • Figure 3 is a mass spectrum of the complement C3 protein.
  • 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 complement C3 in all urine samples. It can be seen from the figure that m/z 1726.8 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 the International Protein Index (IPI human v3.45fasta 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 complement C3 protein was searched in the database, and the mass spectrum of the complement C3 protein is shown in Fig. 3.
  • the antibody and antigen concentrations were determined according to Pierce's BCA Protein Concentration Kit instructions, and then the rabbit anti-human complement C3 protein 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.
  • Murine anti-human complement C3 monoclonal antibody (Abcam) 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 complement C3 protein 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 complement C3 monoclonal antibody, a labeled secondary antibody, and the like.
  • Complement C3 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-complement C3 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 complement C3 was detected.
  • the results showed that the reagent could detect the concentration of 0.01 ng/ml complement C3 protein, indicating a higher detection sensitivity.
  • the above experiments show that the kit of the present invention detects the content of complement C3 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 antigen (centrifuged urine sample) was diluted 1:3, added to the previously coated enzyme plate, incubated at 37 ° C for 2 hours, washed away unbound The antigen, blotting the residual liquid.
  • the mouse anti-human complement C3 protein monoclonal antibody was added and incubated at 37 ° C for 1 hour, the unbound antibody was washed away, and the residual liquid was blotted.
  • the labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, washed 4 times, and the residual liquid was blotted dry. Add the chromogenic substrate, leave it at room temperature for 10 minutes, stop the reaction by adding a stop solution, and read the microplate reader to calculate the content of complement C3 protein in the sample.

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  • Life Sciences & Earth Sciences (AREA)
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  • Engineering & Computer Science (AREA)
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  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention concerne une application d'une protéine du composant 3 (C3) du complément de l'urine pour une détection non effractive de l'urine. Les segments de la protéine du composant 3 (C3) du complément peuvent être exprimés de manière stable dans l'urine d'une personne normale. L'avantage d'acquérir un échantillon d'urine est obtenu de manière non invasive, la protéine du composant 3 (C3) du complément de l'urine est détectée en utilisant l'échantillon d'urine, le problème goulot d'étranglement existant pour l'analyse d'urine en permanence, à savoir comment éliminer les influences du volume d'urine dans la détection de protéine/polypeptide dans l'urine, est résolu, les défauts se rapportant à l'utilisation de la créatinine comme référence interne pour la détection associée de l'urine sont résolus, et un procédé de détection plus simple et plus commode est obtenu pour le diagnostic de maladies.
PCT/CN2015/000626 2015-02-10 2015-09-01 Application d'une protéine du composant 3 (c3) du complément de l'urine WO2016127278A1 (fr)

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CN201510067728.0 2015-02-10
CN201510067728.0A CN105988006A (zh) 2015-02-10 2015-02-10 尿液补体c3蛋白的应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114280309A (zh) * 2021-11-29 2022-04-05 西安交通大学 一种原发性抑郁症的血清多肽诊断标志物c3的应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112048017B (zh) * 2019-09-03 2021-04-20 南京市妇幼保健院 一种卵母细胞体外成熟培养方法
CN113092769A (zh) * 2019-12-23 2021-07-09 首都医科大学附属北京世纪坛医院 尿液补体c4-a及其多肽片段在过敏性疾病中的应用

Citations (5)

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CN1423130A (zh) * 2002-04-26 2003-06-11 帕弗瑞生物技术(北京)有限公司 流式测定固定补体抗体的hla交叉配型方法和试剂盒
CN1444044A (zh) * 2002-06-18 2003-09-24 帕弗瑞生物技术(北京)有限公司 以酶联免疫测定为基础的hla补体依赖性细胞毒性抗体检测方法和试剂盒
CN101014861A (zh) * 2004-08-13 2007-08-08 因迪维姆德有限公司 C3a及其衍生物作为结肠直肠腺瘤/或结肠直肠癌的生物标志的用途；使用同样生物标志的诊断方法和测试系统
CN101238373A (zh) * 2005-08-19 2008-08-06 因迪维姆德有限公司 内质蛋白片段及其衍生物作为结肠直肠腺瘤和/或癌的生物标记的用途；用于检测的方法和测试系统
US20140038203A1 (en) * 2012-07-09 2014-02-06 Musc Foundation For Research Development Methods for detecting or predicting kidney disease

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1423130A (zh) * 2002-04-26 2003-06-11 帕弗瑞生物技术(北京)有限公司 流式测定固定补体抗体的hla交叉配型方法和试剂盒
CN1444044A (zh) * 2002-06-18 2003-09-24 帕弗瑞生物技术(北京)有限公司 以酶联免疫测定为基础的hla补体依赖性细胞毒性抗体检测方法和试剂盒
CN101014861A (zh) * 2004-08-13 2007-08-08 因迪维姆德有限公司 C3a及其衍生物作为结肠直肠腺瘤/或结肠直肠癌的生物标志的用途；使用同样生物标志的诊断方法和测试系统
CN101238373A (zh) * 2005-08-19 2008-08-06 因迪维姆德有限公司 内质蛋白片段及其衍生物作为结肠直肠腺瘤和/或癌的生物标记的用途；用于检测的方法和测试系统
US20140038203A1 (en) * 2012-07-09 2014-02-06 Musc Foundation For Research Development Methods for detecting or predicting kidney disease

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114280309A (zh) * 2021-11-29 2022-04-05 西安交通大学 一种原发性抑郁症的血清多肽诊断标志物c3的应用

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