WO2016011852A1 - 膀胱肿瘤相关抗原检测试剂盒 - Google Patents

膀胱肿瘤相关抗原检测试剂盒 Download PDF

Info

Publication number
WO2016011852A1
WO2016011852A1 PCT/CN2015/081159 CN2015081159W WO2016011852A1 WO 2016011852 A1 WO2016011852 A1 WO 2016011852A1 CN 2015081159 W CN2015081159 W CN 2015081159W WO 2016011852 A1 WO2016011852 A1 WO 2016011852A1
Authority
WO
WIPO (PCT)
Prior art keywords
associated antigen
bladder tumor
monoclonal antibody
hybridoma cell
produced
Prior art date
Application number
PCT/CN2015/081159
Other languages
English (en)
French (fr)
Inventor
于晖
李雨心
陈勤慧
王旭
刘洋
Original Assignee
北京普恩光德生物科技开发有限公司
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 北京普恩光德生物科技开发有限公司 filed Critical 北京普恩光德生物科技开发有限公司
Publication of WO2016011852A1 publication Critical patent/WO2016011852A1/zh

Links

Images

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
    • 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
    • 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
    • C07K16/3038—Kidney, bladder
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention belongs to the field of molecular immunology, and in particular relates to a kit for detecting the content of bladder tumor-associated antigen in a sample.
  • Bladder cancer is the most common urinary tumor in China, and about 90% of bladder cancer is bladder urothelial carcinoma.
  • the recurrence rate of bladder cancer is high, 60%-70% of patients may relapse, and 11% of patients with recurrence may progress to invasive tumors.
  • the diagnosis and follow-up of bladder cancer mainly depends on cystoscopy and urine cytology. Patients have poor compliance and are expensive. They are not easy to be used as routine microscopy. Although patients have high specificity and non-invasive, they have low superficial tumors. Low sensitivity and prone to false negative results. Therefore, the search for bladder tumor markers with high sensitivity and specificity has received increasing attention as an early diagnosis, detection and prognosis evaluation of bladder cancer.
  • Bladder tumors are the most common tumors in the urinary system, including bladder transitional cell carcinoma (BTCC).
  • BTCC bladder transitional cell carcinoma
  • the biological behavior of BTCC is complex and variable, which is characterized by recurrence, multiple, infiltration and metastasis, so these factors are easy to affect the diagnosis and treatment of bladder cancer.
  • the diagnosis of bladder cancer mainly depends on urine exfoliative cytology and cystoscopy. Cystoscopy is an invasive examination. Patients are more painful. In some cases, there is a possibility of causing urinary tract infection, so it is not suitable for bladder cancer. Group screening. Other examination methods, such as urine exfoliative cytology, although the specificity is high, but the sensitivity is poor, and it is easily interfered by factors such as urinary tract infection.
  • a new clinical early diagnosis and detection method should be non-invasive, with good sensitivity and specificity, and easy to operate, low cost of use. Therefore, finding a non-invasive method for detecting sensitivity, high specificity, and facilitating group screening is a hot spot in clinical research of bladder cancer in recent years.
  • a kit for detecting a bladder tumor associated antigen in a sample comprising:
  • the first bladder tumor associated antigen monoclonal antibody in a multiwell plate coated with a first bladder tumor associated antigen monoclonal antibody is produced by hybridoma cells of accession number CGMCC No. 9317 or 9318.
  • the monoclonal antibody in the enzyme working solution is produced by another hybridoma cell of the present invention; and vice versa, when porous
  • the monoclonal antibody on the plate is produced by a hybridoma cell having the accession number CGMCC No. 9318
  • the monoclonal antibody in the enzyme working solution is produced by a hybridoma cell having the accession number CGMCC No. 9317.
  • the sample diluent comprises sodium chloride, goat serum, Tween-20, and a preservative.
  • the sample diluent comprises 30 g/L sodium chloride, 25 ml/L goat serum, 5 ml/L Tween-20, and 0.5 ml/L Proclin-300.
  • the sample diluent can be formulated as a concentrated type or not. The skilled person will appreciate that other concentration factors are also included within the scope of the invention.
  • the enzyme working fluid comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, horseradish peroxidase-labeled second bladder tumor associated antigen monoclonal antibody, and a preservative.
  • the second bladder tumor associated antigen monoclonal antibody is produced by hybridoma cells with accession number CGMCC No. 9318 or 9317.
  • the enzyme working solution comprises 5.8 g/L disodium hydrogen phosphate, 0.593 g/L sodium dihydrogen phosphate, 8.0 g/L sodium chloride, 200 ml/L calf serum, 0.5 ml/L Proclin-300. And a 0.4 mg/L horseradish peroxidase-labeled second bladder tumor associated antigen monoclonal antibody.
  • the chemiluminescent liquid comprises chemiluminescent liquid A and chemiluminescent liquid B.
  • the chemiluminescent solution A contains luminol and a luminescence enhancer.
  • the chemiluminescent solution B contains a peroxide and a luminescence enhancer.
  • reagents of the present invention may be formulated in a concentrated form or may not be. In some embodiments, some reagents are formulated in a concentrated form, which can reduce transport, storage product. The skilled person will appreciate that other concentration factors other than the specific examples are also included in the scope of the present invention.
  • the sample is from a mammal, preferably from a human.
  • the sample is selected from the group consisting of whole blood, plasma, and serum.
  • the sample is human urine.
  • the kit of the invention may also include a calibrator as desired.
  • Calibrators are used for the drawing of standard curves.
  • the calibrator comprises a known concentration of bladder tumor associated antigen, and the concentration of bladder tumor associated antigen is in the range of 10 ng/ml to 4000 ng/ml.
  • the respective concentrations of bladder tumor associated antigen in the calibrator are 400, 200, 50, 10, 5, 0 ng/ml. The skilled person understands that other concentrations are equally applicable, for example, only three concentration points are sufficient to establish a standard curve.
  • kits of the invention may also include controls as needed.
  • the control is diluted with a high value urine sample of known bladder tumor associated antigen concentration.
  • the high value control is in the range of 160 ng/ml to 240 ng/ml; the low quality control is in the range of 16 ng/ml to 24 ng/ml.
  • a bladder tumor associated antigen monoclonal antibody is produced, which is produced by a hybridoma cell having the accession number CGMCC No. 9317.
  • a bladder tumor associated antigen monoclonal antibody is produced which is produced by a hybridoma cell having the accession number CGMCC No. 9318.
  • a bladder tumor associated antigen monoclonal antibody selected from one or both of the following in the preparation of a detection reagent: bladder tumor associated with a hybridoma cell deposited under the accession number CGMCC No. 9317 An antigen monoclonal antibody, and a monoclonal antibody against bladder tumor-associated antigen produced by a hybridoma cell having the accession number CGMCC No. 9318.
  • the two monoclonal antibodies produced by the hybridoma cells of accession numbers CGMCC No. 9317 and 9318 are used in combination to prepare a detection reagent.
  • the detection reagent is selected from the group consisting of an ELISA detection reagent, an immunoturbidimetric detection reagent, a magnetic particle detection reagent, a chemiluminescence detection reagent, an immunofluorescence detection reagent, and a radioimmunoassay reagent; preferably a chemiluminescence detection reagent; more preferably, the detection reagent is detection Detection reagent for bladder tumor associated antigen.
  • a hybridoma cell having the accession number CGMCC No. 9317. According to further embodiments, a hybridoma cell is provided having the accession number CGMCC No. 9318.
  • Figure 1 Calibration curve drawn using the kit of the invention.
  • mice Female Balb/c mice weighing 6 weeks and weighing about 20g were selected for initial immunization.
  • Factor H pure product purchased from Fitzgerald, catalog number 30C-CP2038U
  • 20-50 ⁇ g plus Freund's complete adjuvant subcutaneous injection
  • the second and third immunization doses were the same as above, and the Freund's incomplete adjuvant was injected intraperitoneally.
  • the booster was boosted 3 days before the fusion, and the dose was 20-50 ⁇ g. After 3 days, the spleen fusion was performed.
  • Preparation of feeder cell layer Take an unimmunized Balb/c mouse, 6 weeks old, sacrificed by neck, soaked in 75% alcohol for 5 min, cut the skin with sterile scissors, expose the peritoneum, and inject 6 ml with a sterile syringe.
  • Pre-cooled culture solution (do not pierce the intestine), rinse repeatedly, aspirate the rinse solution, place the rinse solution in a 10ml centrifuge tube, 1200rpm/separate for 6min, mix with 20% (v/v) fetal bovine serum (FCS)
  • FCS fetal bovine serum
  • Preparation of immune spleen cells Take well-immunized Balb/c mice, sacrifice the neck, take the spleen aseptically, wash once with 10 ml of incomplete culture solution, spleen and crush, pass through 200 mesh cell sieve, transfer the spleen cells to 10 ml centrifugation. In the tube, centrifuge at 800 rpm for 10 min, the cells were washed twice with 10 ml of the culture solution, the cells were counted, and 1 ⁇ 10 8 spleen lymphocyte suspension was taken for use.
  • Cell fusion Mix myeloma cells and spleen cells in a ratio of 1:10, wash once with a serum-free incomplete medium in a 50 ml centrifuge tube, centrifuge, 1200 rpm, 8 min; discard the supernatant and blot with a pipette. Residual liquid to avoid affecting the concentration of polyethylene glycol (PEG). Gently hit the bottom of the tube to loosen the cell pellet slightly.
  • PEG polyethylene glycol
  • the culture supernatant can be detected by indirect CLIA method, and the positive clones are screened.
  • the dried solution was immersed in 100 ⁇ l of the cell culture supernatant, the positive control was selected from the immune sera of the mouse, the negative control was selected as the SP2/0 culture supernatant, and the blank was the washing solution, which was allowed to stand at 37 ° C for 2 h.
  • the positive clones were screened, and the hybridomas were cloned by limiting dilution method.
  • the feeder cells were prepared 1 day before the cloning, and the hybridoma cells to be cloned were gently dried from the culture wells with incomplete medium and counted. Adjust the cells to 5 cells/ml. A cell culture plate of the prepared feeder cell layer was taken, and 100 ⁇ l of the diluted cells were added to each well. Incubate in a 37 ° C, 5% CO 2 incubator. The liquid was changed on the 7th day, and the liquid was changed once every 3 days. Cell clone formation was observed in 9 days, and antibody titer was detected by chemical luminescence assay. The strongest positive clone was cloned again until the cell positive rate reached 100%, and the fixed strain was established; And send it to the depository for preservation.
  • the hybridoma cell line (classified name BTAA hybridoma cell line) was deposited on June 16, 2014 at the General Microbiology Center of China Microbial Culture Collection Management Committee (No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the preservation number. They are CGMCC No.9317 and CGMCC No.9318, respectively.
  • Blocking The blocking solution (5.8 g of disodium hydrogen phosphate, 0.593 g of sodium dihydrogen phosphate, 8.0 g of sodium chloride, 100 ml of goat serum, Proclin-300 0.5 ml, and purified water to 1000 ml) was loaded on a multiwell plate. Dry at room temperature for 2 hours, dry overnight.
  • HRP horseradish peroxidase
  • the working concentration of the enzyme-labeled antibody was selected by the guide method, and 2 ⁇ l of the enzyme-labeled antibody (1 mg/ml) was added to 5 ml of the enzyme diluent, that is, a ratio of 1:2500.
  • the horseradish peroxidase-labeled monoclonal antibody prepared in the step 2.1 was dissolved in the enzyme buffer at a ratio of 1:2500.
  • the enzyme buffer formulation was: 5.8 g/L disodium hydrogen phosphate, 0.593 g/L sodium dihydrogen phosphate, 8.0 g/L sodium chloride, 200 ml/L calf serum, 0.5 ml/L Proclin-300).
  • the 1000 ml sample dilution included 30.0 g sodium chloride, 25.0 ml goat serum, 5.0 ml Tween-20, and 0.5 ml Proclin-300.
  • Chemiluminescence solution A and chemiluminescence solution B are purchased reagents (purchased from Roche), in which chemiluminescence solution A contains luminol and luminescence enhancer, and chemiluminescence solution B contains peroxide and luminescence. Enhancer. Or according to the chemiluminescence technology, self-formulation.
  • Calibrators are traceable to Fitzgerald reference materials or other reference materials as needed.
  • Human urine samples with high clinical value of bladder tumor-associated antigens are collected (the urine can be any commercially available urine sample, or collected from a clinical facility), and the high-value human urine is diluted with the sample diluent to a desired concentration range.
  • High value control 160 ng/ml to 240 ng/ml; low value control: 16 ng/ml to 24 ng/ml.
  • Each of the above reagents is assembled into a kit, and a tool such as an instruction manual or a sealing film may be incorporated into the kit as needed.
  • Test Example 1 Method for using bladder tumor-associated antigen chemiluminescence kit of the present invention
  • Sample incubation Take a sufficient number of coated plates, fix them on the frame, set the calibration hole, the control hole and the sample hole to be tested, record the position of each hole; add 45 ⁇ l sample to the sample hole to be tested. Dilutate, add 50 ⁇ l of calibrator to the well of the calibrator in sequence, add 50 ⁇ l of the control to the well of the control, and add 5 ⁇ l of the sample to be tested in the well of the sample (equivalent to a 10-fold dilution of the sample). Add 50 ⁇ l of enzyme working solution to each well (it is recommended to complete this step within 20 min), mix well by shaking, cover the sealing membrane, and incubate at 37 °C for 30 min.
  • Calibrators and controls are not required to be diluted and can be used directly.
  • Washing machine wash plate add 350 ⁇ l of washing solution to each well, each washing interval is 5-10 seconds, repeat washing 5 times, and pat dry.
  • Luminescence Add 50 ⁇ l of chemiluminescence solution A and 50 ⁇ l of chemiluminescence solution B to each well, mix well and read.
  • the recovery rate should be between 85% and 115%;
  • Blank detection limit should not be higher than 1ng/ml
  • Measurement system linearity linear correlation coefficient r ⁇ 0.9900 in the range of 10 ng / ml to 4000 ng / ml.
  • the bladder tumor-associated antigen-negative sample N1 and the positive sample P1 urine specimen were separately detected.
  • Human factor I concentration (25 ng/ml, 12.5 ng/ml, 0 ng/ml),
  • Human factor D concentration (10 ng/ml, 5 ng/ml, 0 ng/ml),
  • Bovine factor H concentration (100ng/ml, 50ng/ml, 0ng/ml)
  • Rat factor H concentration (100 ng/ml, 50 ng/ml, 0 ng/ml)
  • Cross-reacting enzymes usually have high homology, sequence similarity or structurally similar epitopes with the substance to be tested. It is conceivable that if these cross-reactants are present in the sample, the antibodies may recognize and bind to these cross-reactants, resulting in false positives. Therefore, the cross-reactivity of diagnostic kits is an important indicator. This is very important for the reliability of clinical outcomes.
  • the components of the kit can be placed at 37 ° C for 6 days.
  • R-H and R-L represent: high-value accuracy reference products and low-value accuracy reference products.
  • the double antibody sandwich method is used to determine the level of bladder tumor-associated antigen in urine, that is, the coated antibody on the enzyme-labeled porous plate and the HRP-labeled antibody and the bladder of the sample to be tested.
  • the tumor-associated antigen antigen forms a sandwich complex structure of "coated antibody-antigen-HRP-labeled antibody". The high sensitivity and specificity of the detection are achieved.
  • the invention can quickly and accurately determine the content of bladder tumor-associated antigen in urine, and provides reliable clinical reference value for early diagnosis and early treatment of bladder tumor.
  • the invention has the advantages of simple use, rapid detection, sensitivity, stability, and simple operation. Applicable to the majority of small and medium hospitals.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种膀胱肿瘤相关抗原检测试剂盒。试剂盒包含两株特异性膀胱肿瘤相关抗原（BTAA）单克隆抗体。多孔板上包被的单克隆抗体与辣根过氧化物酶（HRP）标记的单克隆抗体以及被测样本的膀胱肿瘤相关抗原形成"包被抗体-抗原-HRP标记抗体"的夹心复合物结构。HRP进而与底物反应产生信号，用以进行膀胱肿瘤相关抗原定量分析。通过所述试剂盒，能够迅速、灵敏地定量检测样本中的膀胱肿瘤相关抗原含量。

Description

膀胱肿瘤相关抗原检测试剂盒 技术领域
本发明属于分子免疫学领域，具体涉及检测样本中膀胱肿瘤相关抗原含量的试剂盒。
背景技术
膀胱癌是我国最常见的泌尿系肿瘤，大约90％的膀胱癌为膀胱尿路上皮癌。膀胱癌的复发率很高，60％-70％的患者可能复发，11％复发的患者可进展为浸润性肿瘤。目前对于膀胱癌诊断及随访主要依赖于膀胱镜检查和尿细胞学检查，患者依从性差，价格比较昂贵，不易作为常规镜检；患者虽然特异度高且无创，但对低度表浅性的肿瘤敏感性低，容易产生假阴性的结果。因此，寻找敏感度及特异度高的膀胱肿瘤标志物作为膀胱癌的早期诊断、检测以及预后评估受到越来越多的关注。
膀胱肿瘤是泌尿系统最常见的肿瘤，其中以膀胱移行细胞癌(bladder transitional cell carcinoma，BTCC最多见)。BTCC生物学行为复杂多变，表现为易复发、多发、浸润和转移，所以这些因素容易影响到对膀胱癌的诊断和治疗。目前，膀胱癌的诊断主要依靠尿脱落细胞学和膀胱镜检查，膀胱镜检查属于侵入性检查，患者较痛苦，某些情况下尚有导致尿路感染的可能性，因而不适用于膀胱癌的群体性筛查。其他检查方法，如尿脱落细胞学检查虽然特异度高，但是灵敏度较差，容易受到尿路感染等因素的干扰。一般来讲，一种新的临床早期诊断和检测方法应该具有非侵入性的特点，同时具有较好的灵敏度和特异度，并且兼顾操作简便，使用成本低。因此，寻找灵敏度高、特异度强，并且便于群体性筛查的无创性检查方法是近年来膀胱癌临床研究的热点。
建立一种简便快速、特异度强并且不具有侵入性的检查方法有利于膀胱癌的早期诊断，对善预后以及检测复发都具有重要意义。
发明内容
根据一些实施方式，提供了一种检测样本中膀胱肿瘤相关抗原的试剂盒，其包含：
包被有第一膀胱肿瘤相关抗原单克隆抗体的多孔板、
样品稀释液、
酶工作液、
化学发光液。
在一些实施方案中，包被有第一膀胱肿瘤相关抗原单克隆抗体的多孔板中第一膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9317或9318的杂交瘤细胞所产生。
当多孔板上的单克隆抗体由保藏号为CGMCC No.9317的杂交瘤细胞所产生时，则酶工作液中的单克隆抗体由本发明的另一株杂交瘤细胞产生；反之亦然，当多孔板上的单克隆抗体由保藏号为CGMCC No.9318的杂交瘤细胞所产生时，则酶工作液中的单克隆抗体由保藏号为CGMCC No.9317的杂交瘤细胞产生。
在一些实施方案中，样品稀释液包含氯化钠、山羊血清、吐温-20、和防腐剂。在具体实施方案中，样品稀释液包含30g/L氯化钠、25ml/L山羊血清、5ml/L吐温-20和0.5ml/L Proclin-300。样本稀释液可以配制成浓缩型，也可以不是。技术人员可以理解，其它浓缩倍数也包括在本发明范围内。
在一些实施方案中，酶工作液包含磷酸氢二钠、磷酸二氢钠、氯化钠、小牛血清、辣根过氧化物酶标记的第二膀胱肿瘤相关抗原单克隆抗体和防腐剂。在一些实施方案中，第二膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9318或9317的杂交瘤细胞所产生。在具体实施方案中，酶工作液包含5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300和0.4mg/L辣根过氧化物酶标记的第二膀胱肿瘤相关抗原单克隆抗体。
在一些实施方案中，化学发光液包含化学发光液A和化学发光液B。所述化学发光液A含有鲁米诺和发光增强剂。化学发光液B含有过氧化物和发光增强剂。
应当理解，本发明的各试剂可以配制成浓缩型，也可以不是。在一些实施方案中，一些试剂配制成浓缩型，这可以减少运输、储存体 积。技术人员可以理解，除了具体实例以外的其它浓缩倍数也包括在本发明范围内。
在一些实施方案中，样本来自哺乳动物，优选来自人类。样本选自全血、血浆、和血清。在具体实施方案中，样本为人尿液。
在一些实施方案中，本发明的试剂盒还可以根据需要包括校准品。校准品用于标准曲线的绘制。在具体实施方案中，校准品包含已知浓度的膀胱肿瘤相关抗原纯品，膀胱肿瘤相关抗原的浓度在10ng/ml至4000ng/ml范围内。在具体实施方案中，校准品中膀胱肿瘤相关抗原的分别浓度为400、200、50、10、5、0ng/ml。技术人员理解，其它浓度同样适用，例如只需三个浓度点就足以建立一条标准曲线。
在一些实施方案中，本发明的试剂盒还可以根据需要包括质控品。在具体实施方案中，质控品为已知膀胱肿瘤相关抗原浓度的高值尿液样本稀释而成。在具体实施方案中，高值质控的浓度在160ng/ml至240ng/ml范围内；低值质控的浓度在16ng/ml至24ng/ml范围内。
根据一些实施方案，提供了一种膀胱肿瘤相关抗原单克隆抗体，其由保藏号为CGMCC No.9317的杂交瘤细胞所产生。
根据另一些实施方案，提供了一种膀胱肿瘤相关抗原单克隆抗体，其由保藏号为CGMCC No.9318的杂交瘤细胞所产生。
根据另一些实施方案，提供了选自如下一种或两种的膀胱肿瘤相关抗原单克隆抗体在制备检测试剂中的用途：由保藏号为CGMCC No.9317的杂交瘤细胞所产生的膀胱肿瘤相关抗原单克隆抗体、和由保藏号为CGMCC No.9318的杂交瘤细胞所产生的膀胱肿瘤相关抗原单克隆抗体。在一个具体实施方案中，保藏号为CGMCC No.9317和9318的杂交瘤细胞所产生两株单克隆抗体共同用于制备检测试剂。所述检测试剂选自ELISA检测试剂、免疫比浊检测试剂、磁颗粒检测试剂、化学发光检测试剂、免疫荧光检测试剂和放射免疫检测试剂；优选化学发光检测试剂；更优选所述检测试剂为检测膀胱肿瘤相关抗原的检测试剂。
根据一些实施方案，提供一种杂交瘤细胞，其保藏号为CGMCC No.9317。根据另一些实施方案，提供一种杂交瘤细胞，其保藏号为CGMCC No.9318。
附图说明
图1：采用本发明的试剂盒绘制的校准曲线。
具体实施方式
为了使本发明易于理解，下面结合具体实施例进一步阐述本发明。以下提供了本发明实施方式中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。
实施例1.单克隆抗体的制备与鉴定
1.免疫Balb/c小鼠
选取6周龄、体重约20g的雌性Balb/c小鼠，初次免疫，取Factor H纯品(购自Fitzgerald，目录号30C-CP2038U)20-50μg加弗氏完全佐剂皮下多点注射，第14和28天分别进行第二次和第三次免疫剂量同上，加弗氏不完全佐剂腹腔内注射，融合前3天加强免疫，剂量20-50μg为宜，3天后，取脾融合。
2.细胞融合的步骤
制备饲养细胞层：取一只未免疫的Balb/c小鼠，6周龄，拉颈处死，浸泡在75％酒精内5min，用无菌剪刀剪开皮肤，暴露腹膜，用无菌注射器注入6ml预冷的培养液(严禁刺破肠管)，反复冲洗，吸出冲洗液，冲洗液放入10ml离心管，1200rpm/分离6min，用20％(v/v)胎牛血清(FCS)的培养液混悬，调整细胞数至1×105/ml，加入96孔板，100μl/孔，放入37℃CO2孵箱培养。
制备免疫脾细胞：取免疫好的Balb/c小鼠，拉颈处死，无菌取脾脏，用10ml不完全培养液洗一次，脾脏研碎，过200目细胞筛，将脾细胞转移至10ml离心管中，800rpm离心10min，细胞用10ml培养液洗2次，细胞计数，取1×108脾淋巴细胞悬液备用。
制备骨髓瘤细胞SP2/0：取对数生长骨髓瘤细胞离心，用无血清培养液洗2次，计数，取得5×107细胞备用。
细胞融合：将骨髓瘤细胞与脾细胞按1：10的比例混合在一起，在50ml离心管中用无血清不完全培养液洗1次，离心，1200rpm，8min；弃上清，用吸管吸净残留液体，以免影响聚乙二醇(PEG)浓度。轻轻弹击离心管底，使细胞沉淀略松动。
加入37℃预温的1ml 45％(g/100ml)PEG(分子量4000)溶液，边加边轻微摇动。37℃水浴作用90s。加37℃预温的不完全培养液以终止PEG作用，每隔2min分别加入1ml、2ml、3ml、4ml、5ml和6ml。离心，800rpm,6min。充上清，用含20％(v/v)小牛血清HAT选择培养液重悬。将上述细胞，加到已有饲养细胞层的96孔板内，每孔加100μl。将培养板置37℃、5％CO2培养箱中培养。
3.杂交瘤细胞的筛选
脾细胞和骨髓瘤细胞融合后5天，形成多种细胞的混合体，补加HAT培养基100μl，第10天换HT培养基培养。杂交瘤细胞布满孔底1/5面积时，即可采用间接CLIA法检测培养上清，筛选阳性克隆。以Factor H纯品(Fitzgerald)包被酶标板(5μg/ml)100μl/孔,4℃过夜，将酶标板孔中的液体倒尽，加入PBST，重复洗涤三次；加入封闭液200μl/孔进行封闭，置于37℃，1小时。甩干封闭液，加入100μl细胞培养上清，阳性对照选小鼠的免疫血清，阴性对照选SP2/0培养上清，空白为洗涤液，于37℃静置2h。加入HRP标记羊抗鼠二抗(1:5000)100μl/孔，于37℃静置60min。加入PBST，重复洗涤三次；加入底物反应液100μl/孔，37℃置暗处反应10分钟。加入H2SO4(2mol/L)50μl/孔，终止反应。酶标仪检测450nm吸光度值。以Factor H纯品(Fitzgerald)为抗原，免疫小鼠，成功得到2株分泌BTAA单克隆抗体的杂交瘤细胞株(编号12D4和11E9)，这二株单克隆抗体分别特异性识别并结合Factor H纯品(Fitzgerald)。
4.杂交瘤的克隆化
筛选得到的阳性克隆，采用有限稀释法对杂交瘤克隆化，克隆前1天制备饲养细胞层，将要克隆的杂交瘤细胞用不完全培养基从培养孔内轻轻吹干，计数。调整细胞为5个细胞/ml。取准备的饲养细胞层的细胞培养板，每孔加入稀释细胞100μl。孵育于37℃、5％CO2孵箱中。在第7天换液，以后每3天换液1次。9天可见细胞克隆形成，化学发 光法检测检测抗体效价。并将最强的阳性克隆再次克隆化，直至细胞阳性率达100％，即可定株；定株的细胞扩大培养。并送保藏中心保藏。
5.杂交瘤细胞的保藏
将杂交瘤细胞株(分类名BTAA杂交瘤细胞株)于2014年6月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号)，保藏编号分别为CGMCC No.9317、CGMCC No.9318。
实施例2.单克隆抗体的大量生产
已具备两株单克隆抗体杂交瘤细胞株：取1×107的细胞浓度注射于Balb/c小鼠腹腔，10天后收集腹水。通过以下步骤纯化单克隆抗体：
1.收集所得的腹水以2500rpm离心，取上清。加等体积的PBS(pH7.4)与1/2体积的饱和硫酸铵，4℃、静置30min；
2.以3000rpm，4℃，离心20min；
3.去沉淀，上清加等体积的饱和硫酸铵，静置30min；
4.以3000rpm，4℃，离心20min；
5.取沉淀，加5ml生理盐水与5ml饱和硫酸铵静置30min；
6.以3000rpm，4℃，离心20min；
7.沉淀加5ml生理盐水与5ml 0.02M的PB缓冲液(PH7.4)；
8.加8倍柱体积的PB缓冲液平衡Protein G凝胶柱(购自GE公司)；
9.将第7步中的混合液加到凝胶柱中；
10.使用10倍柱体积的PB缓冲液洗脱杂蛋白；
11.用0.2M pH2.8甘氨酸缓冲液洗脱抗体并收集；
12.使用再生液清洗柱子；
13.加平衡液平衡柱子。
实施例3.本发明膀胱肿瘤相关抗原的化学发光检测试剂盒的制备
1.制备包被有膀胱肿瘤相关抗原单抗的多孔板：
a)包被：采用0.05M、pH值为9.6的碳酸盐缓冲液与适当浓度的 实施例2制备的膀胱肿瘤相关抗原单克隆抗体(CGMCC No.9317)混合制成包被混合液，并将其加载于多孔板上，4℃包被12h；
碳酸盐缓冲液标准配方：
无水碳酸钠      1.600g
碳酸氢钠        2.940g
pH值9.60～9.80  纯化水定容至1000ml
b)洗板：稀释20倍浓缩洗液至1倍浓度，使用1倍浓度洗液洗板2次；20倍浓缩洗液标准配方：
Figure PCTCN2015081159-appb-000001
c)封闭：将封闭液(磷酸氢二钠5.8g，磷酸二氢钠0.593g，氯化钠8.0g，山羊血清100ml，Proclin-3000.5ml，纯化水定容至1000ml)加载于多孔板上，室温2小时，甩干，干燥过夜。
2.酶工作液的制备：
2.1标记步骤:
(1)称取1mg辣根过氧化物酶(HRP，购自Sigma)溶解于300μl蒸馏水中。
(2)于上液中加入0.6μl新配的0.1M NaIO4溶液，室温下避光搅拌20分钟。
(3)将上述溶液装入透析袋中，使用1mM pH 4.4的醋酸钠缓冲液透析，4℃过夜。
(4)加20μl 1M pH 9.5碳酸盐缓冲液，使HRP的pH升高到9.0至9.5，然后立即加入1mg本发明的另一株抗体(CGMCC No.9318)(在1ml 0.01M碳酸盐缓冲液中)，室温避光轻轻搅拌2小时。
(5)加0.05ml新配的4mg/ml NaBH4液，混匀，再置4℃2小时。
(6)将上述液装入透析袋中，使用0.15M pH7.4PBS透析，4℃过夜。
(7)加入等体积的甘油，-20℃保存，最终辣根过氧化物酶标抗体浓度为1mg/ml。
2.2酶标记抗体浓度确定：
采用方针法选择酶标抗体的工作浓度，取2μl酶标抗体(1mg/ml)加入到5ml酶稀释液中，即1：2500的比例。
2.3酶工作液的配制：
将2.1步骤中制备的辣根过氧化物酶标记的单克隆抗体，以1：2500的比例将酶标抗体溶于酶缓冲液中。酶缓冲液配方为：5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300)。
3.样品稀释液的制备：
1000ml样品稀释液包括30.0g氯化钠、25.0ml山羊血清、5.0ml吐温-20、0.5ml Proclin-300。
4.化学发光液：化学发光液A和化学发光液B为外购试剂(购自：罗氏)，其中化学发光液A含有鲁米诺和发光增强剂，化学发光液B含有过氧化物和发光增强剂。或者根据化学发光技术，自行配制。
5.校准品的配制：
用样品稀释液将Fitzgerald公司的Factor H纯品稀释，使得校准品中的膀胱肿瘤相关抗原浓度梯度分别为400、200、50、10、5、0ng/ml。
根据需要，校准品可溯源至Fitzgerald公司的参考物质或者其它参考物质。
6.质控品的配制：
收集膀胱肿瘤相关抗原临床检测高值的人尿液样本(尿液可以是任何市售尿液样本、或收集自临床机构)，用样品稀释液稀释高值人尿液至期望的浓度范围内。高值质控：160ng/ml至240ng/ml；低值质控：16ng/ml至24ng/ml。2-8℃保存备用。
将上述各试剂组装成试剂盒，根据需要还可以在试剂盒中并入使用说明书、封板膜等工具。
测试例
测试例1.本发明膀胱肿瘤相关抗原化学发光试剂盒的使用方法
1.1.实验前准备
1.1.自4℃冰箱中取出试剂盒，均应平衡到室温(18-25℃)。
1.2.20倍浓缩洗涤液用纯化水或蒸馏水稀释20倍后使用。
2.试验方法
2.1.加样温育：取足够数量的包被板，固定于框架上，分别设置校准品孔、质控品孔和待测样品孔，记录各孔位置；在待测样品孔中加入45μl样品稀释液，按顺序在校准品孔中加入校准品50μl，在质控品孔加入质控品50μl，在样品孔中分别加入5μl的待测样品(等同于样本进行了10倍稀释)。每孔加入酶工作液50μl(建议在20min内完成此步操作)，震荡混匀，加盖封板膜，37℃温育30min。
注：校准品和质控品无需稀释，可直接使用。
2.2.洗板：
手工洗板：每孔加入350μl洗液，静置5-10秒后弃尽，重复冲洗5次后，拍干；
洗板机洗板：每孔加入350μl洗液，每次洗涤间隔5-10秒，重复冲洗5次后，拍干。
2.3.发光：每孔加入50μl化学发光液A和50μl化学发光液B，充分混匀后读数。
2.4.测定：立即置化学发光仪下测定RLU值。
2.5.计算：根据校准品的浓度及对应的RLU值，以校准品S0孔调零，使用双对数线性拟合方式(log(X)-log(Y))计算结果，结果乘以稀释倍数(10倍)，即为样品最终浓度。
线性方程为log(RLU)＝4.513+1.065log(浓度)；r＝1.00。
Figure PCTCN2015081159-appb-000002
测试例2.本发明试剂盒的方法学指标
1.准确度：回收率应在85％至115％之间；
2.空白检出限：应不高于1ng/ml；
3.测量系统的线性：在10ng/ml至4000ng/ml范围内线性相关系数r≥0.9900。
4.重复性：批内变异系数CV批内应不超过10％；
5.批间差：批间变异系数CV批间应不超过15％；
表1.准确性、最低检出限、系统线性、重复性检测结果
Figure PCTCN2015081159-appb-000003
6.分析特异性：
分别检测膀胱肿瘤相关抗原阴性样本N1、阳性样本P1尿液标本。
在N1、P1样本中分别添加三个浓度梯度的交叉反应原，检测交叉反应原对膀胱肿瘤相关抗原抗体检测的交叉反应情况：
人因子I浓度(25ng/ml、12.5ng/ml、0ng/ml)、
人因子D浓度(10ng/ml、5ng/ml、0ng/ml)、
牛因子H浓度(100ng/ml、50ng/ml、0ng/ml)
鼠因子H浓度(100ng/ml、50ng/ml、0ng/ml)
结果分析：交叉反应原通常和待测物质具有较高的同源性、序列相似或者具有结构相似的表位。可以想象，如果样本中存在这些交叉反应原时，抗体可能会识别并结合这些交叉反应原，导致假阳性。因此，诊断试剂盒的交叉反应性是一项重要的指标。这对于临床结果的可靠性非常重要。
从下表4可见，当人因子I不大于25ng/ml、人因子D不大于10ng/ml、牛因子H和鼠因子H不大于100ng/ml时，对膀胱肿瘤相关抗原的检测结果没有统计学显著影响。展示出本发明试剂盒优良的抗交叉反应性。
7.稳定性：试剂盒各组分37℃可放置6天。
表2.37度存放稳定性实验结果
Figure PCTCN2015081159-appb-000004
表3.4℃保存条件下稳定性实验结果
Figure PCTCN2015081159-appb-000005
R-H、R-L分别代表：高值准确度参考品、低值准确度参考品。
8.干扰物质分析：
8.1尿红蛋白，实验结果见表5。
结论：根据结果，当轻度溶尿标本尿红蛋白≤200mg/ml时，用本试剂盒检测对结果基本无影响，当严重溶尿时检测结果可能导致假阳性，故不宜用严重溶尿标本。
8.2白蛋白，实验结果见表6。
结论：当白蛋白浓度在1000mg/dl内，对膀胱肿瘤相关抗原抗体检测结果没有显著性影响。
8.3胆红素，实验结果见表7。
结果分析：当胆红素浓度不大于20mg/dl时对膀胱肿瘤相关抗原抗体检测结果无统计学显著影响。
8.4尿酸，实验结果见表8。
结果分析：当尿酸浓度不大于100mg/dl时对膀胱肿瘤相关抗原抗体检测结果无统计学显著影响。
8.5抗坏血酸，实验结果见表9。
结果分析：当抗坏血酸浓度不大于2g/dl时对膀胱肿瘤相关抗原抗体检测结果无统计学显著影响。
8.6人IgG，实验结果见表10。
结果分析：当人IgG浓度不大于10g/dl时，对膀胱肿瘤相关抗原抗体检测结果无统计学显著影响。
小结：
1)利用本发明的二株特异性抗体制备试剂盒，采用双抗体夹心法测定尿液中膀胱肿瘤相关抗原水平，即酶标多孔板上包被的抗体与HRP标记抗体和被测样本的膀胱肿瘤相关抗原抗原形成“包被抗体-抗原-HRP标记抗体”的夹心复合物结构。实现了检测的高灵敏度和特异性。
2)本发明一方面可以快速、准确地测定尿液中膀胱肿瘤相关抗原的含量，对于膀胱肿瘤的早期诊断、早期治疗提供可靠的临床参考价值。
3)另一方面由于使用特异性的单克隆抗体作为包被固相，大大提高了膀胱肿瘤相关抗原的检测灵敏度和特异性，抗干扰能力强。
4)本发明具有使用简便、检测快速、灵敏、稳定、操作简便等优点。适用于广大中小医院使用。
Figure PCTCN2015081159-appb-000006
Figure PCTCN2015081159-appb-000007
Figure PCTCN2015081159-appb-000008
Figure PCTCN2015081159-appb-000009

Claims (9)

  1. 一种检测样本中膀胱肿瘤相关抗原的试剂盒，其包含：
    包被有第一膀胱肿瘤相关抗原单克隆抗体的多孔板、
    样品稀释液、
    酶工作液、
    化学发光液；
    其中：
    样品稀释液包含：氯化钠、山羊血清、吐温-20、和防腐剂；
    酶工作液包含：磷酸氢二钠、磷酸二氢钠、氯化钠、小牛血清、辣根过氧化物酶标记的第二膀胱肿瘤相关抗原单克隆抗体和防腐剂；
    化学发光液包含：化学发光液A和化学发光液B；所述化学发光液A含有鲁米诺和发光增强剂，化学发光液B含有过氧化物和发光增强剂；
    当所述第一膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9317的杂交瘤细胞所产生时，第二膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9318的杂交瘤细胞所产生，或者当所述第一膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9318的杂交瘤细胞所产生时，第二膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9317的杂交瘤细胞所产生；
    样本来自哺乳动物，优选人类；样本优选为人尿液。
  2. 根据权利要求1所述的检测样本中膀胱肿瘤相关抗原的试剂盒，还包括校准品，
    校准品包含已知浓度的膀胱肿瘤相关抗原纯品，膀胱肿瘤相关抗原的浓度在10ng/ml至4000ng/ml范围内。
  3. 根据权利要求1所述的检测样本中膀胱肿瘤相关抗原的试剂盒，还包括质控品，
    质控品为已知膀胱肿瘤相关抗原浓度的高值尿液样本稀释而成。
  4. 一种检测样本中膀胱肿瘤相关抗原的CLIA试剂盒，其包含：
    包被有第一膀胱肿瘤相关抗原单克隆抗体的多孔板、
    样品稀释液、
    酶工作液、
    化学发光液；
    其中：
    样品稀释液包含：30g/L氯化钠、25ml/L山羊血清、5ml/L吐温-20和0.5ml/L Proclin-300；
    酶工作液包含：5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300、和0.4mg/L辣根过氧化物酶标记的第二膀胱肿瘤相关抗原单克隆抗体；
    化学发光液由化学发光液A和化学发光液B组成；所述化学发光液A含有鲁米诺和发光增强剂，化学发光液B含有过氧化物和发光增强剂；
    当所述第一膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9317的杂交瘤细胞所产生时，第二膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9318的杂交瘤细胞所产生，或者当所述第一膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9318的杂交瘤细胞所产生时，第二膀胱肿瘤相关抗原单克隆抗体由保藏号为CGMCC No.9317的杂交瘤细胞所产生。
  5. 一种膀胱肿瘤相关抗原单克隆抗体，其由保藏号为CGMCC No.9317的杂交瘤细胞所产生。
  6. 一种膀胱肿瘤相关抗原单克隆抗体，其由保藏号为CGMCC No.9318的杂交瘤细胞所产生。
  7. 选自如下一种或两种的膀胱肿瘤相关抗原单克隆抗体在制备检测试剂中的用途：
    由保藏号为CGMCC No.9317的杂交瘤细胞所产生的膀胱肿瘤相关抗原单克隆抗体、和
    由保藏号为CGMCC No.9318的杂交瘤细胞所产生的膀胱肿瘤相关抗原单克隆抗体；
    所述检测试剂选自：ELISA检测试剂、免疫比浊检测试剂、磁颗粒检测试剂、化学发光检测试剂、免疫荧光检测试剂和放射免疫检测试剂，优选化学发光检测试剂；
    所述检测试剂优选为检测膀胱肿瘤相关抗原的检测试剂。
  8. 一种杂交瘤细胞，其保藏号为CGMCC No.9317的杂交瘤细胞。
  9. 一种杂交瘤细胞，其保藏号为CGMCC No.9318的杂交瘤细胞。
PCT/CN2015/081159 2014-07-25 2015-06-10 膀胱肿瘤相关抗原检测试剂盒 WO2016011852A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410359816.3A CN104142401B (zh) 2014-07-25 2014-07-25 膀胱肿瘤相关抗原检测试剂盒
CN201410359816.3 2014-07-25

Publications (1)

Publication Number Publication Date
WO2016011852A1 true WO2016011852A1 (zh) 2016-01-28

Family

ID=51851634

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/081159 WO2016011852A1 (zh) 2014-07-25 2015-06-10 膀胱肿瘤相关抗原检测试剂盒

Country Status (2)

Country Link
CN (1) CN104142401B (zh)
WO (1) WO2016011852A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109100512A (zh) * 2018-08-02 2018-12-28 宁波奥丞生物科技有限公司 一种检测egfr的化学发光试剂盒
CN115261401A (zh) * 2022-08-25 2022-11-01 杭州博岳生物技术有限公司 一种利用酵母细胞表面展示技术开发膀胱肿瘤抗原单克隆抗体的方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104142401B (zh) * 2014-07-25 2016-04-20 北京普恩光德生物科技开发有限公司 膀胱肿瘤相关抗原检测试剂盒
CN105017419B (zh) * 2015-07-06 2019-01-04 北京普恩光德生物科技开发有限公司 抗人nmp-22的单克隆抗体及其检测试剂盒
CN106370864A (zh) * 2016-08-31 2017-02-01 镇江华测金太医学检验所有限公司 一种中性粒细胞明胶酶相关脂质运载蛋白的化学发光盒
CN106353508A (zh) * 2016-08-31 2017-01-25 镇江华测金太医学检验所有限公司 一种膀胱肿瘤相关抗原的化学发光酶联免疫检测试剂盒
CN117089529B (zh) * 2023-10-16 2024-03-15 深圳普门科技股份有限公司 杂交瘤细胞及其制备方法、单克隆抗体及试剂盒

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101368961A (zh) * 2008-04-02 2009-02-18 北京科美东雅生物技术有限公司 尿膀胱癌抗原化学发光免疫分析定量测定试剂盒及其制备方法
CN103018461A (zh) * 2012-12-13 2013-04-03 中国科学院生物物理研究所 一种早期诊断膀胱癌的试剂盒及其制备方法
CN103033625A (zh) * 2012-12-19 2013-04-10 中国科学院生物物理研究所 一种人膀胱癌细胞化学发光检测试剂盒及其制备方法
US20140072987A1 (en) * 2012-09-12 2014-03-13 Randox Laboratories Ltd. Methods of detecing bladder cancer
CN104142401A (zh) * 2014-07-25 2014-11-12 北京普恩光德生物科技开发有限公司 膀胱肿瘤相关抗原检测试剂盒

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1440462A (zh) * 2000-04-28 2003-09-03 麦康公司 鉴定和生产抗原肽的方法并且将其用作疫苗
AU2007332473B2 (en) * 2006-12-14 2012-09-27 Forerunner Pharma Research Co., Ltd. Anti-Claudin-3 monoclonal antibody, and treatment and diagnosis of cancer using the same
CN102372773B (zh) * 2010-08-11 2013-06-05 中国科学院生物物理研究所 人膀胱癌肿瘤标志物及其抗体和应用
CN102649975A (zh) * 2011-02-23 2012-08-29 张康 补体因子h基因型、载脂蛋白上氧化磷酸胆碱和血浆补体因子h定量测定试剂盒及测定方法
CN103941017B (zh) * 2014-03-18 2016-01-20 北京普恩光德生物科技开发有限公司 C反应蛋白检测试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101368961A (zh) * 2008-04-02 2009-02-18 北京科美东雅生物技术有限公司 尿膀胱癌抗原化学发光免疫分析定量测定试剂盒及其制备方法
US20140072987A1 (en) * 2012-09-12 2014-03-13 Randox Laboratories Ltd. Methods of detecing bladder cancer
CN103018461A (zh) * 2012-12-13 2013-04-03 中国科学院生物物理研究所 一种早期诊断膀胱癌的试剂盒及其制备方法
CN103033625A (zh) * 2012-12-19 2013-04-10 中国科学院生物物理研究所 一种人膀胱癌细胞化学发光检测试剂盒及其制备方法
CN104142401A (zh) * 2014-07-25 2014-11-12 北京普恩光德生物科技开发有限公司 膀胱肿瘤相关抗原检测试剂盒

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
REN, LIXIN ET AL.: "Urine Markers of Bladder Tumours", UROLOGY AND NEPHROLOGY FOREIGN MEDICAL SCIENCES, vol. 22, no. 4, 31 December 2002 (2002-12-31), pages 202 - 205 *
ROBERT, K. ET AL.: "Complement Factor H or a Related Protein Is a Marker for Transitional Cell Cancer of the Bladder", CLINICAL CANCER RESEARCH, vol. 4, no. 10, 31 October 1998 (1998-10-31), pages 2511 - 2520, XP055036036 *
ZHANG, BING ET AL.: "Research Status of Bladder Tumour Antigen", UROLOGY AND NEPHROLOGY FOREIGN MEDICAL SCIENCES, vol. 23, no. 2, 31 December 2003 (2003-12-31), pages 135 - 137 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109100512A (zh) * 2018-08-02 2018-12-28 宁波奥丞生物科技有限公司 一种检测egfr的化学发光试剂盒
CN115261401A (zh) * 2022-08-25 2022-11-01 杭州博岳生物技术有限公司 一种利用酵母细胞表面展示技术开发膀胱肿瘤抗原单克隆抗体的方法
CN115261401B (zh) * 2022-08-25 2023-10-20 杭州博岳生物技术有限公司 一种利用酵母细胞表面展示技术开发膀胱肿瘤抗原单克隆抗体的方法

Also Published As

Publication number Publication date
CN104142401B (zh) 2016-04-20
CN104142401A (zh) 2014-11-12

Similar Documents

Publication Publication Date Title
WO2016011852A1 (zh) 膀胱肿瘤相关抗原检测试剂盒
WO2017107974A1 (zh) 血清psmd4蛋白的检测试剂盒及其检测方法与应用
CN109580959B (zh) 一种检测肝素结合性表皮生长因子的elisa试剂盒
CN112679605B (zh) 针对新型冠状病毒核衣壳蛋白的抗体或其抗原结合片段及其应用
WO2015165357A1 (zh) 白介素6检测试剂盒
CN101210927B (zh) 一种诊断前列腺癌的试剂盒
CN110865192A (zh) 一种基于胶乳增强免疫比浊法测定gp73的试剂盒及其制备使用方法
TW201802472A (zh) 抗人類血紅素單株抗體或抗體套組、抗人類血紅素單株抗體固定化不可溶性載體粒子、及使用其等之測定試劑或測定方法
CN113150133B (zh) 针对SARS-CoV-2的单克隆抗体或其抗原结合片段
EP2863224A1 (en) Kit for diagnosing malignant melanoma
CN104730249A (zh) 一种检测人附睾分泌蛋白4的酶联免疫试剂盒
JP6900515B2 (ja) 脂肪性肝炎の検出に用いられる標的マーカーｇｐ７３及び検出方法
CN111273015B (zh) 一种检测短裸甲藻毒素的酶联免疫试剂盒及其制备和应用
Maiolini et al. Study of an enzyme immunoassay kit for carcinoembryonic antigen.
CN107746430B (zh) 一种gp73 c端抗原的制备及其应用
CN116217720A (zh) 一种抗HbA1c的纳米抗体及其制备方法和应用
CN111273041B (zh) 一种检测鬼笔环肽的酶联免疫试剂盒及其制备和应用
CN111443202B (zh) 一种检测抗凝血杀鼠剂的酶联免疫试剂盒及其制备和应用
CN110687285B (zh) 诊断试剂盒及mak16在制备系统性红斑狼疮早期诊断试剂中的应用
CN114994306A (zh) 一种蛋白质pknox1在制备诊断酒精性心肌病的试剂中的应用及诊断试剂盒
CN111217910B (zh) 单克隆抗体对及其在检测髓过氧化物酶蛋白中的应用
CN111458522A (zh) 一种用于检测血浆白细胞介素6天然抗体的检测试剂、试剂盒及其应用
CN111944049A (zh) Slc12a9抗体的制备方法及其应用
JP5156677B2 (ja) 免疫学的測定試薬および免疫学的測定方法
CN104830805B (zh) 抗人翻译控制肿瘤蛋白单克隆抗体杂交瘤及其单克隆抗体与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15824525

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 12.06.2017)

122 Ep: pct application non-entry in european phase

Ref document number: 15824525

Country of ref document: EP

Kind code of ref document: A1