WO2021056906A1 - 杂交瘤细胞lcz8a3及其分泌的单克隆抗体和应用 - Google Patents

杂交瘤细胞lcz8a3及其分泌的单克隆抗体和应用 Download PDF

Info

Publication number
WO2021056906A1
WO2021056906A1 PCT/CN2019/129874 CN2019129874W WO2021056906A1 WO 2021056906 A1 WO2021056906 A1 WO 2021056906A1 CN 2019129874 W CN2019129874 W CN 2019129874W WO 2021056906 A1 WO2021056906 A1 WO 2021056906A1
Authority
WO
WIPO (PCT)
Prior art keywords
lcz8a3
kit
monoclonal antibody
hybridoma cell
protein
Prior art date
Application number
PCT/CN2019/129874
Other languages
English (en)
French (fr)
Inventor
钱泽
孟洁
Original Assignee
昂科生物医学技术(苏州)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 昂科生物医学技术(苏州)有限公司 filed Critical 昂科生物医学技术(苏州)有限公司
Publication of WO2021056906A1 publication Critical patent/WO2021056906A1/zh

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
    • 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
    • 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
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/342—Prostate diseases, e.g. BPH, prostatitis

Definitions

  • the invention belongs to the technical field of clinical diagnosis, and particularly relates to a hybridoma cell LCZ8A3 and a preparation method thereof, and the application of the monoclonal antibody and the monoclonal antibody secreted by the hybridoma cell LCZ8A3 in a kit for detecting human prostatitis.
  • Chronic prostatitis is a common disease in adult men. Prostatitis is divided into four types: Type I: Acute bacterial prostatitis; Type II: Chronic bacterial prostatitis; Type III: Chronic prostatitis/chronic pelvis Pain syndrome, this type is divided into two subtypes: inflammatory (III A) and non-inflammatory (III B) according to the white blood cell count in prostate massage fluid and semen; Type IV: asymptomatic inflammatory prostatitis.
  • the common clinical types of prostatitis are mainly type II, type III A and type III B.
  • CP is one of the most common diseases in urology, due to the diverse symptoms and complex etiologies of patients, there is an urgent need for a simple, non-invasive, and non-invasive CP detection method to assist and improve the detection rate of CP.
  • Prostate corpuscle is a subcellular structure secreted by human prostate epithelial cells, with an average diameter of 150nm (50-500nm). It is secreted or leaked into the lumen by prostatic epithelial cells through exocytosis and permeation. It can be located in the cell capsule Intravesicular, extracellular acinar ducts, or prostate fluid and semen, etc., can also be found in the culture fluid of prostate cell lines.
  • the main components of the prostate corpuscle include sphingomyelin, which contains phosphocholine and ceramide. The ratio of cholesterol to phospholipids is 2:1, while the ratio on a typical mammalian cell membrane is 1:1.
  • prostate bodies are rich in cholesterol, sphingomyelin, calcium, guanosine diphosphate, and many transmembrane proteins (CD13, CD46, CD55, CD59).
  • CD59 is a membrane inhibitor of reactive cell lysis .
  • Utleg et al. used high performance liquid chromatography and electrospray ionization mass spectrometry to identify 139 kinds of proteins in the prostatic corpuscle, which are divided into six categories: enzymes (35%), transport structural proteins (19%), guanosine triphosphate GTP binding protein (14%), chaperone protein (6%), signal transduction protein (17%), derivative protein (9%).
  • Poliakov et al. used trypsin digestion and liquid chromatography/mass spectrometry analysis to identify 440 human prostatic body proteins, which have antibacterial, antioxidant, and multiple immunomodulatory functions.
  • the antibacterial properties of the prostatic body may be due to the presence of the antimicrobial protein belonging to the antimicrobial peptide family-human cationic microbial protein 18 (hcap-18) on its membrane, which can release the antimicrobial peptide LL37.
  • Reactive oxygen nuclide ROS
  • ROS Reactive oxygen nuclide
  • Prostate bodies are different from other antioxidants that directly eliminate ROS, but transfer cholesterol and sphingomyelin from the prostate bodies to the cell membrane, inhibiting the NADPH oxidase activity of polymorphonuclear leukocytes, thereby reducing the ROS reaction. Since the prostatic corpuscle can be separated from blood and urine, it can be used as a good class of prostate disease markers.
  • the main technical problem solved by the present invention is to provide a hybridoma cell LCZ8A3 and a preparation method thereof, a monoclonal antibody secreted by the hybridoma cell and a kit for detecting human prostatitis.
  • the present invention adopts the following technical solutions:
  • a hybridoma cell LCZ8A3 characterized in that: the hybridoma cell LCZ8A3 is deposited in the Chinese Type Culture Collection, and the deposit number is CCTCC No: C2019144.
  • the present invention also provides a monoclonal antibody, which is secreted and produced by the hybridoma cell LCZ8A3.
  • the present invention also provides a kit, which includes the hybridoma cell or the monoclonal antibody.
  • the kit is a chemiluminescence kit.
  • the invention also provides the use of the hybridoma cell or the monoclonal antibody in the preparation of a kit.
  • the kit is a kit based on immunoassay.
  • the kit is a chemiluminescence kit.
  • the kit is used to detect human prostatitis.
  • the present invention also provides the use of the hybridoma cell or the monoclonal antibody in preparing a kit for the diagnosis of human prostatitis.
  • the hybridoma cells can stably produce monoclonal antibodies against human prostatic cytosolic protein, with stable chromosomes and high antibody titer, and can be used in research on the application of human prostatic cytostatic protein;
  • the monoclonal antibody against human prostatic corpuscles protein can be used in the preparation of human prostatitis diagnostic kits.
  • the test samples include human urine, blood, semen, prostate massage fluid and other human samples for use in human prostate.
  • the diagnosis of inflammation greatly makes up for the insufficiency of the existing clinical diagnosis technology.
  • the detection method of the present invention is not limited to chemiluminescence immunological technology, but also includes colloidal gold immunochromatographic technology, fluorescence immunochromatographic technology and other similar immunological technologies.
  • Example 1 The preparation of hybridoma cell LCZ8A3 includes the following steps:
  • mouse anti-human prostate vesicle protein hybridoma cells includes the following steps:
  • the ELISA detection method is specifically: adding the coating buffer to the 96-well plate to dilute the prostatic corpuscle protein antigen to 4ug/ml, adding 50u1 to each reaction well, 1 hour, and washing 3 times with the lotion , Add 100u1/well blocking solution and seal at 37°C for 2h, wash 3 times, and pat dry. Add the serum to be tested, dilute 1:800 in the first well, and dilute in a 1:2 gradient, incubate at 37°C for 30 minutes, wash the plate 4 times, and pat dry. Take horseradish enzyme-labeled goat anti-mouse IgG diluted 1:2500, add 100u1/well to the reaction well, incubate at 37°C for 30min, wash 4 times, and pat dry.
  • the indirect ELISA method was used to detect whether all 8 positive cells could specifically recognize the prostatic vesicle protein: a positive cell named A can specifically bind to the prostatic vesicle protein, indicating that A can specifically recognize the prostate corpuscle protein.
  • a hybridoma cell A that can stably secrete a monoclonal antibody against human prostate vesicle protein was obtained.
  • the hybridoma cell A was classified and named as hybridoma cell LCZ8A3, which was deposited in China on August 28, 2019.
  • the Type Culture Collection, the address is Wuhan University, China, and the deposit number is CCTCC N0: C2019144.
  • Example 2 The preparation and purification of mouse anti-human prostatic corpuscle ascites monoclonal antibody, including the following steps:
  • mice choose healthy female BALB/C mice aged 8-12 weeks, weighing about 20g, and inject sterilized liquid paraffin, 0.5ml per mouse; 7 days later, inject 1 ⁇ 10 6 mice into each mouse.
  • the 8 monoclonal hybridoma cells LCZ8A3-1, LCZ8A3-2, LCZ8A3-3, LCZ8A3-4, LCZ8A3-5, LCZ8A3-6, LCZ8A3-7 and LCZ8A3-8 of Example 1 were injected into each of the monoclonal hybridoma cells Two mice
  • UV spectroscopy is used to determine the absorbance values A280 and A260 of monoclonal antibodies at 280nm and 260nm. The result is that the protein content is 18 mg/ml.
  • the kit uses chemiluminescence immunoassay.
  • the labeling buffer is a CB system, the pH is 19.5, the feed ratio (molar ratio) is 20:1 (acridine/antibody), and lysine is added when the reaction is terminated (the ratio of lysine/acridine is 140:1).
  • the labeled antibody is purified by Sephadex G-50 Sephadex column;

Landscapes

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

Abstract

杂交瘤细胞LCZ8A3及其分泌的单克隆抗体和应用，所述杂交瘤细胞LCZ8A3保藏于中国典型培养物保藏中心，保藏编号为CCTCC No：C2019144。该杂交瘤细胞能够稳定产生抗人前列腺小体外泄蛋白单克隆抗体能够应用于人前列腺小体外泄蛋白的应用的研究中。

Description

杂交瘤细胞LCZ8A3及其分泌的单克隆抗体和应用 技术领域
本发明属于临床诊断技术领域，特别是涉及一种杂交瘤细胞LCZ8A3及其制备方法，杂交瘤细胞LCZ8A3分泌的单克隆抗体和单克隆抗体在检测人前列腺炎的试剂盒中的应用。
背景技术
慢性前列腺炎(chronic prostatitis，CP)是成年男子的常见疾病，前列腺炎分为四型：Ⅰ型：急性细菌性前列腺炎；Ⅱ型：慢性细菌性前列腺炎；Ⅲ型：慢性前列腺炎/慢性骨盆疼痛综合征，此型根据前列腺按摩液、精液中白细胞计数又分为炎症性(Ⅲ A)和非炎症性(Ⅲ B)两个亚型；Ⅳ型：无症状炎症性前列腺炎。前列腺炎常见的临床类型主要是Ⅱ型、Ⅲ A型和Ⅲ B型。尽管CP是泌尿外科最多的疾病之一，但由于患者的症状多样、病因复杂，临床上急需简便易行、非侵入性的CP检测方法，来辅助并提高CP的检出率。
前列腺小体是由人类前列腺上皮细胞分泌的一种亚细胞结构，平均直径150nm(50-500nm)，由前列腺上皮细胞通过胞吐作用和胞透作用分泌或外泄至管腔，可位于细胞囊泡内、细胞外腺泡管、或前列腺液和精液等，也可在前列腺细胞系的培养液中发现。前列腺小体的主要成分包括鞘磷脂，含磷酸胆碱和神经酰胺。胆固醇和磷脂的比例是2:1，而典型的哺乳动物细胞膜上的此比例是1:1。与其他外泌体相比，前列腺小体富含胆固醇、鞘磷脂、钙、二磷酸鸟苷、和许多跨膜蛋白(CD13、CD46、CD55、CD59)，CD59是反应性细胞溶解的膜抑制剂。Utleg等人用高效液相色谱电喷离子质谱法鉴定了前列腺小体有139种蛋白质，分为六大类：酶(35％)，转运结构蛋白(19％)，三磷酸鸟苷GTP结合蛋白(14％)，伴侣蛋白(6％)，信号转导蛋白(17％)，派生蛋白(9％)。Poliakov等用胰蛋白酶消化和液相色谱/质谱分析鉴定了440种人前列腺小体蛋白，有抗菌、抗氧化和多重免疫调节的功能。
前列腺小体的抗菌特性可能是因其膜上存在属于抗菌肽家族的抗微生物蛋白-人阳离子微生物蛋白18(hcap-18)，可释放抗微生物肽LL37。而反应性氧核 素(ROS)是男性特发性不育的主因，不育病人精液中40％发现ROS增高，而精液中白细胞浸润，特别是多形核白细胞是产生ROS的主因。前列腺小体不同于其它抗氧化剂直接清除ROS，而是将胆固醇、鞘磷脂从前列腺小体转移至细胞膜，抑制多形核白细胞的NADPH氧化酶活性，从而减少ROS反应。由于前列腺小体可以从血液和尿液中分离获得，因此它可以作为一类很好的前列腺疾病标记物。
发明内容
本发明主要解决的技术问题是提供一种杂交瘤细胞LCZ8A3及其制备方法、该杂交瘤细胞分泌的单克隆抗体和检测人前列腺炎的试剂盒。
为解决上述技术问题，本发明采取了以下技术方案：
一种杂交瘤细胞LCZ8A3，其特征在于：所述杂交瘤细胞LCZ8A3保藏于中国典型培养物保藏中心，保藏编号为CCTCC No：C2019144。
本发明还提供一种单克隆抗体，所述单克隆抗体由所述的杂交瘤细胞LCZ8A3分泌产生。
本发明还提供一种试剂盒，所述试剂盒包括所述的杂交瘤细胞或所述的单克隆抗体。
进一步地说，所述试剂盒为化学发光试剂盒。
本发明还提供了所述的杂交瘤细胞或所述的单克隆抗体在制备试剂盒中的用途。
进一步地说，所述试剂盒是基于免疫检测的试剂盒。
进一步地说，所述试剂盒为化学发光试剂盒。
进一步地说，所述试剂盒用于检测人前列腺炎。
本发明还提供了所述的杂交瘤细胞或所述的单克隆抗体在制备用于人前列腺炎诊断的试剂盒中的用途。
本发明的有益效果至少具有以下几点：
1、该杂交瘤细胞能够稳定产生抗人前列腺小体外泄蛋白单克隆抗体，且细胞染色体稳定、抗体效价高，完全能够应用于人前列腺小体外泄蛋白的应用的 研究中；
2、该抗人前列腺小体外泄蛋白的单克隆抗体可应用于人前列腺炎诊断试剂盒的制备中，检测样本包括了人体尿液、血液、精液、前列腺按摩液等人体样本，用于人前列腺炎的诊断，大大弥补现有临床诊断技术的不足。
3、本发明的检测方法不仅限于化学发光免疫技术，也包括了胶体金免疫层析技术、荧光免疫层析技术等各种类似的免疫技术。
具体实施方式
下面对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易于被本领域技术人员理解，从而对本发明的保护范围做出更为清楚明确的界定。
实施例1：杂交瘤细胞LCZ8A3的制备，包括以下步骤：
1、鼠抗人前列腺小体外泄蛋白杂交瘤细胞的建立，包括以下步骤：
a.选用9只8-12周龄、体重20g左右且健康的雌性BALB/C小鼠，适用性饲养1周后，采集阴性血作为对照用；
b.初次免疫用72ug/ml前列腺小体外泄蛋白加弗氏完全佐剂等体积搅拌乳化，肌肉注射。两周后，用36ug/ml前列腺小体外泄蛋白加弗氏不完全佐剂强化免疫2次，每次间隔2周，ELISA检测血清抗体效价，免疫小鼠效价明显达到1:150000以上，准备加强免疫；
其中，ELISA检测法具体为：在96孔板中加入用包被缓冲液将前列腺小体外泄蛋白抗原稀释至4ug/ml，在每个反应孔中加50u1，1小时，用洗液洗涤3次，加100u1/孔封闭液在37℃封闭2h，洗涤3次，拍干。加入待测血清，第一孔1:800稀释，往下1:2的梯度倍比稀释，37℃孵育30min，洗板4次，拍干。取辣根酶标记的羊抗鼠IgG按照1:2500倍稀释，100u1/孔加入反应孔，37℃孵育30min，洗涤4次，拍干。最后加入TMB 100u1/孔，显色15-30min。然后加入终止液，50u1/孔。以450nm单波长测定各孔OD值，并用阳性对照孔作为判断阳性。结果为血清效价为1:150000的阳性小鼠，可以用于细胞融合。
c.用44ug/ml前列腺小体外泄蛋白肌肉注射强化免疫(不含佐剂)，3天后尾 部采血，同时取脾脏。
d.将脾脏细胞与骨髓瘤细胞按细胞数10:1左右进行混合，并在聚乙二醇(PEG，分子量为1500)的促融作用下进行融合，选择培养基为含HAT的RPMI1640培养基(50ml)。11天后，通过间接ELISA方法筛选出能与前列腺小体外泄蛋白反应的阳性杂交瘤细胞，并将初筛得到的阳性杂交瘤细胞扩大培养，并进行标签蛋白杂交瘤细胞的排除，以复筛出针对前列腺小体外泄蛋白而非标签的杂交瘤细胞；
e.用有限稀释法将获得的阳性杂交瘤细胞进行克隆，经过7次筛选最终确定8株能稳定分泌单克隆抗体的杂交瘤细胞A-1，A-2，A-3，A-4，A-5，A-6，A-7和A-8。
间接ELISA方法检测所有阳性8株细胞是否能够特异识别前列腺小体外泄蛋白：一株命名为A的阳性细胞可以与前列腺小体外泄蛋白特异结合，说明A可以特异识别前列腺小体外泄蛋白。经筛选得到能稳定分泌抗人前列腺小体外泄蛋白的单克隆抗体的杂交瘤细胞A，该杂交瘤细胞A的分类命名为杂交瘤细胞LCZ8A3，已于2019年8月28日保藏于保藏于中国典型培养物保藏中心，地址为中国武汉大学，保藏号为CCTCC N0:C2019144。
2、鼠抗人前列腺小体外泄蛋白单克隆抗体的制备及单抗亚型的鉴定
取1000u1杂交瘤细胞LCZ8A3(8株杂交瘤细胞LCZ8A3-1，LCZ8A3-2，LCZ8A3-3，LCZ8A3-4，LCZ8A3-5，LCZ8A3-6，LCZ8A3-7和LCZ8A3-8)的培养上清，采用sigma分型试剂盒鉴定单克隆抗体的亚型，结果显示：单克隆抗体的重链为IgGl型，轻链为Kappa型。
实施例2：鼠抗人前列腺小体外泄蛋白的腹水单克隆抗体制备及纯化，包括以下步骤：
a.选择8-12周龄、体重20g左右且健康的雌性BALB/C小鼠，腹腔注射灭菌液体石蜡，0.5m1/只；7天后，给每只小鼠腹腔注射1×10 6个实施例1的8株单克隆杂交瘤细胞LCZ8A3-1，LCZ8A3-2，LCZ8A3-3，LCZ8A3-4，LCZ8A3-5，LCZ8A3-6，LCZ8A3-7和LCZ8A3-8，每株单克隆杂交瘤细胞注射两只小鼠；
b.7天后观察小鼠腹水生产情况，如腹部明显膨大，即可抽取腹水。腹水采 集后13000rpm离心l0min，除去油脂和沉淀，收集上清液，即为腹水单克隆抗体。采用硫酸按沉淀和透析法纯化腹水单克隆抗体。
对腹水单克隆抗体进行ELISA效价测定、分子质量鉴定和浓度测定：
1)ELISA效价的测定：采用间接ELISA测定纯化的腹水单克隆抗体，1ug/ml的抗原100u1/孔包被96孔板做ELISA，结果显示纯化后效价为l:260000。
2)抗体的分子质量鉴定:采用SDS-PAGE进行测定单克隆抗体，显示单克隆抗体的重链约为46KD，轻链约为25KD。
3)单克隆抗体浓度测定:紫外分光法测定单克隆抗体在280nm和260nm处的吸光度值A280和A260。结果为蛋白质含量为18mg/ml。
实施例3：病原的临床诊断
1.试剂盒制备
试剂盒采用化学发光免疫法。
1)将兔源抗人前列腺小体外泄蛋白检测抗体(EliOnco美国)包被在已经活化的磁珠上，取100ul已活化的Mag COOH磁珠，加入100ug兔源抗人前列腺小体外泄蛋白检测抗体，25℃垂直混合120分钟，磁分离清洗3次，除去游离抗体，再加300ul 1％BSA封闭液，25℃垂直混合60分钟，磁分离清洗3次，重新悬浮于1ml PBST溶液中；
2)将吖啶酯标记在抗人前列腺小体外泄蛋白单克隆抗体LCZ8A3上。标记缓冲液为CB体系，pH为19.5，投料比(摩尔比)为20：1(吖啶/抗体)，终止反应时加入赖氨酸(赖氨酸/吖啶比例为140:1)。标记抗体纯化采用sephadex G-50葡聚糖凝胶柱分离；
3)在各反应孔中，加入50ul上述偶联磁珠，再各孔对应加入待测标准品或者尿液50ul。37℃恒温箱孵育30分钟，磁分离洗板3次。加入吖啶酯标记的抗人前列腺小体外泄蛋白单克隆抗体LCZ8A3，37℃恒温箱孵育30分钟，磁分离洗板3次后，用化学发光检测仪进行测定。仪器参数：化学发光底物A液和B液各加液50ul，整合时间3S。根据不同浓度的标准品化学发光值绘出标准曲线，将病人的数值与阳性标准孔数值比较，从而得出被测试者尿样本的实际前列腺小体外泄蛋白浓度。
2.结果检测
用上述试剂盒检测前列炎癌症和正常人尿样本。根据临床样本检测真阳性a，假阳性b，假阴性c，真阴性d，求出检测灵敏度、特异度和总符合率。
表1患者和正常人群中前列腺小体外泄蛋白含量(ng/ml)比较表
研究对象分组 病例数 平均值 标准偏差 中位数
患者组 200 4.63 1.251 2.41
正常对照组 200 0.48 0.215 0.38
*Note：Z＝P＜0.05
由表1可知，前列腺炎患者尿液中前列腺小体外泄蛋白含量与正常人相比较明显升高，统计学有显著性差异。
对上述200例样本进行临床金标准和本试剂盒的两种检测。计算前列腺小体外泄蛋白检测试剂盒临床检测的灵敏度、特异性、总符合率、阳性预期值、阴性预期值等统计学结果，结果如表2。
表2临床金标准和本试剂盒前列腺小体蛋白检测临床数据归纳表
Figure PCTCN2019129874-appb-000001
灵敏度＝a/(a+c)×100％＝187/200×100％＝93.5％；
特异度＝d/(b+d)×100％＝189/200×100％＝94.5％；
总符合率＝(a+d)/(a+b+c+d)＝(187+189)/400×100％＝94.0％。
由表2可知，该细胞分泌的抗人前列腺小体外泄蛋白抗体对前列腺小体外泄蛋白检测的灵敏度和特异度达到93％以上。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (9)

  1. 一种杂交瘤细胞LCZ8A3，其特征在于：所述杂交瘤细胞LCZ8A3保藏于中国典型培养物保藏中心，保藏编号为CCTCC No：C2019144。
  2. 一种单克隆抗体，其特征在于：其由权利要求1所述的杂交瘤细胞LCZ8A3分泌产生。
  3. 一种试剂盒，其特征在于：所述试剂盒包括权利要求1所述的杂交瘤细胞或权利要求2所述的单克隆抗体。
  4. 根据权利要求3所述的试剂盒，其特征在于：所述试剂盒为化学发光试剂盒。
  5. 权利要求1所述的杂交瘤细胞或权利要求2所述的单克隆抗体在制备试剂盒中的用途。
  6. 根据权利要求5所述的用途，其特征在于：所述试剂盒是基于免疫检测的试剂盒。
  7. 根据权利要求6所述的用途，其特征在于：所述试剂盒为化学发光试剂盒。
  8. 根据权利要求5所述的用途，其特征在于：所述试剂盒用于检测人前列腺炎。
  9. 权利要求1所述的杂交瘤细胞或权利要求2所述的单克隆抗体在制备用于人前列腺炎诊断的试剂盒中的用途。
PCT/CN2019/129874 2019-09-29 2019-12-30 杂交瘤细胞lcz8a3及其分泌的单克隆抗体和应用 WO2021056906A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910932461.5A CN112574302B (zh) 2019-09-29 2019-09-29 杂交瘤细胞lcz8a3及其分泌的单克隆抗体和应用
CN201910932461.5 2019-09-29

Publications (1)

Publication Number Publication Date
WO2021056906A1 true WO2021056906A1 (zh) 2021-04-01

Family

ID=75111029

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/129874 WO2021056906A1 (zh) 2019-09-29 2019-12-30 杂交瘤细胞lcz8a3及其分泌的单克隆抗体和应用

Country Status (2)

Country Link
CN (1) CN112574302B (zh)
WO (1) WO2021056906A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115184616A (zh) * 2022-06-23 2022-10-14 昂科生物医学技术(苏州)有限公司 前列腺小体外泄蛋白抗原及其抗体在制备良性前列腺增生诊断试剂盒中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104357404A (zh) * 2014-11-10 2015-02-18 昂科生物医学技术(苏州)有限公司 一种杂交瘤细胞及其分泌的单克隆抗体与应用
CN104897900A (zh) * 2014-03-03 2015-09-09 昂科生物医学技术(苏州)有限公司 一种前列腺小体外泄蛋白抗原及其抗体与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104897900A (zh) * 2014-03-03 2015-09-09 昂科生物医学技术(苏州)有限公司 一种前列腺小体外泄蛋白抗原及其抗体与应用
CN104357404A (zh) * 2014-11-10 2015-02-18 昂科生物医学技术(苏州)有限公司 一种杂交瘤细胞及其分泌的单克隆抗体与应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GU CHENG-YUAN, HUANG YONG-QIANG, HAN CHENG-TAO, ZHU YAO, BO DAI, MENG JIE, QIN XIAO-JIAN, YE DING-WEI: "Clinical significance of urine prostatic exosomal protein in the diagnosis of prostate cancer", AMERICAN JOURNAL OF CANCER RESEARCH, vol. 9, no. 5, 1 May 2019 (2019-05-01), pages 1074 - 1078, XP055795744 *
SHAO, XUEFENG ET AL.: "Detection and diagnostic evaluation of urine prostatic exosomal protein in patients with NIH-Ⅲb prostatitis", JOURNAL OF MODERN UROLOGY, vol. 21, no. 11, 15 November 2016 (2016-11-15), pages 861 - 863, XP055795741 *
YANG, ZHICHAO ET AL.: "Clinical application of prostatic exosomal protein( PSEP) in the diagnosis of chronic prostatitis", JOURNAL OF SOUTHEAST UNIVERSITY (MEDICAL SCIENCE EDITION), vol. 36, no. 5, 25 October 2017 (2017-10-25), pages 800 - 803, XP055795738 *
YIN LIANLI, TANG YINGHUA, PAN AIPING, YANG LAN, ZHU XU, LIU YONGGANG: "The application of IL-10 and TNF-α in expressed prostatic secretions and prostatic exosomal protein in urine in the diagnosis of patients with chronic prostatitis", MEDICINE, WILLIAMS AND WILKINS, BALTIMORE., US, vol. 98, no. 33, 1 August 2019 (2019-08-01), US, pages e16848, XP055795732, ISSN: 0025-7974, DOI: 10.1097/MD.0000000000016848 *

Also Published As

Publication number Publication date
CN112574302B (zh) 2022-05-06
CN112574302A (zh) 2021-03-30

Similar Documents

Publication Publication Date Title
WO2010038974A2 (ko) 이뮤노글로블린 에이 신증과 티지비엠 신증의 진단용 조성물 및 키트
US11016107B2 (en) Method for diagnosing or monitoring kidney function or diagnosing kidney dysfunction
US8399207B2 (en) Monoclonal antibodies against osteopontin
EP2233503A1 (en) Anti-ofloxacin monoclonal antibody and immunoassay method of ofloxacin using the same
WO2012091465A2 (ko) 혈청 아밀로이드 ａ에 대한 단일클론항체 및 이를 생산하는 하이브리도마 세포
WO2021129247A1 (zh) 柯萨奇病毒a6型实心病毒的单克隆抗体及其应用
WO2019000267A1 (zh) Gm杂交瘤细胞、单克隆抗体、试剂盒及其制备方法与应用
CN104357404B (zh) 一种杂交瘤细胞及其分泌的单克隆抗体与应用
WO2022174570A1 (zh) 一种氨基末端脑尿钠肽检测方法和试剂盒
WO2021056906A1 (zh) 杂交瘤细胞lcz8a3及其分泌的单克隆抗体和应用
WO2020184409A1 (ja) 生体試料中のβ－Ｄ－グルカンの免疫学的分析方法、及びβ－Ｄ－グルカン分析用キット
EP3342861B1 (en) Specifically purified anti-presepsin antibody
JP6743322B2 (ja) 生体試料中の（１→３）−β−Ｄ−グルカンの免疫学的分析方法、（１→３）−β−Ｄ−グルカン分析用キット、及び（１→３）−β−Ｄ−グルカンの免疫学的分析方法に使用するための生体試料用アルカリ前処理液
WO2023088444A1 (zh) 一种抗hiv-1 p24的抗体及其制备方法和用途
KR20120132227A (ko) 다중 타입 구제역 바이러스 검출용 단일클론 항체 및 이를 이용한 구제역 바이러스 검출방법
JP3154724B2 (ja) 下痢性貝毒に特異的なモノクローナル抗体、ハイブリドーマ及び下痢性貝毒の検出方法
WO2022207014A2 (zh) 抗真菌(1，3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用
CN112574955B (zh) 杂交瘤细胞lcz9h4及其分泌的单克隆抗体和应用
WO2023245801A1 (zh) 一种抗念珠菌甘露聚糖单克隆抗体及其应用
WO2024103504A1 (zh) 一种检测炎症相关疾病的标志物和方法
CN110079506B (zh) 一种人血清纤维胶凝蛋白ficolin-2的单克隆抗体及其应用
EP2006682B1 (en) Diagnosis of acute enteritis by determination of intestinal fatty acid-binding protein in the blood
KR100254414B1 (ko) 항-결핵균 항체 및 나균 항원에 대한 항혈청을 포함하는 결핵균 항원 검출용 진단키트
WO2009132484A1 (zh) Vero细胞裂解蛋白、其制备方法以及包含该蛋白的vero细胞hcp检测试剂盒
JP6558568B2 (ja) 細菌由来成分の免疫学的検出方法

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: 19947033

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19947033

Country of ref document: EP

Kind code of ref document: A1