WO2022207014A2 - 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 - Google Patents
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 Download PDFInfo
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- WO2022207014A2 WO2022207014A2 PCT/CN2022/107379 CN2022107379W WO2022207014A2 WO 2022207014 A2 WO2022207014 A2 WO 2022207014A2 CN 2022107379 W CN2022107379 W CN 2022107379W WO 2022207014 A2 WO2022207014 A2 WO 2022207014A2
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- glucan
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- 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
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
- G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the purpose of the present application is to obtain a monoclonal antibody that only binds strongly to (1,3)- ⁇ -D glucan, and has little or no binding to other substances that interfere with detection.
- the specific identification of the antifungal (1,3)- ⁇ -D glucan monoclonal antibody obtained after recombinant expression by genetic engineering technology was carried out again.
- the specificity of the antibody can be measured by whether it has cross-reactivity with the analog of (1,3)- ⁇ -D glucan and the magnitude of the cross-reaction.
- the specific identification method is as follows:
- the purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody was diluted with 0.1mol/L PBS buffer to the concentrations of 0.001 ⁇ g/mL, 0.01 ⁇ g/mL, 0.1 ⁇ g/mL, respectively. mL, 1 ⁇ g/mL, and 10 ⁇ g/mL of purified antibody dilutions.
- a purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody was used as capture antibody and/or detection antibody to prepare a fungal (1,3)- ⁇ -glucan that can be used in clinic.
- D-glucan magnetic particle chemiluminescence detection kit the main components of the kit include: biotin-labeled purified antifungal (1,3)- ⁇ -D-glucan monoclonal antibody, chemiluminescent group label
- the purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody [this example uses acridine sulfonamide-labeled purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody
- the cloned antibody can also be labeled with at least one of acridine sulfonamide, acridine ester, ruthenium complex, luminol, (diaramane)-1,2-dioxane or alkaline phosphatase], Strept
- the resulting dialysis Proclin300 with a final concentration of 0.1% and trehalose 1% were added to the product to obtain biotin-labeled purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody. 8°C for use (4°C in this example).
- the purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody labeled with biotin and streptavidin magnetic particles can be mixed 1:1.
- Preparation method of high-value calibrator (200pg/mL): take (1,3)- ⁇ -D glucan and add ultrapure water to make an aqueous solution with a concentration of 4ng/mL, take 5mL of which, add 20mL mannitol solution, 6mL Polyvinylpyrrolidone (PVP), 20 mL of newborn bovine serum, then add ultrapure water to 100 mL, mix well, and freeze-dried at low temperature to obtain a high-value calibrator (200 pg/mL).
- PVP Polyvinylpyrrolidone
- Preparation method of quality control substance (100pg/mL): take (1,3)- ⁇ -D glucan and add ultrapure water to make an aqueous solution with a concentration of 2ng/mL, take 5mL of which, add 20mL mannitol solution, 6mL poly Vinylpyrrolidone (PVP), 20 mL of newborn bovine serum, then add ultrapure water to 100 mL, mix well, and freeze-dried at low temperature as the quality control product (100 pg/mL).
- PVP poly Vinylpyrrolidone
- the prepared fungal (1,3)- ⁇ -D glucan magnetic particle chemiluminescence detection kit can use the magnetic particle chemiluminescence method to realize automatic detection on an automatic chemiluminescence analyzer.
- the specific steps are: adding to the reaction cup 50 ⁇ L streptavidin magnetic particles and 50 ⁇ L biotin-labeled purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody, mixed well, incubated at 37°C for 15 min, and then separated under an applied magnetic field Magnetic particles, aspirate the liquid in the reaction cup, add 300 ⁇ L of PBST washing buffer to the reaction cup for washing, aspirate the supernatant, wash three times repeatedly, then add 100 ⁇ L of the sample to be tested in the reaction cup, mix well, and incubate at 37°C 15min; separate the magnetic particles under an external magnetic field again, suck off the liquid in the reaction cup, add 300 ⁇ L of PBST washing buffer to the reaction cup for washing, aspirate the supernatant
- the magnetic particles were separated under an external magnetic field, the liquid in the cuvette was sucked off, 300 ⁇ L of PBST washing buffer was added to the cuvette for washing, the supernatant was aspirated, and the washing was repeated 3 times.
- the fungal (1,3)- ⁇ -D glucan magnetic particle chemiluminescence detection kit of the present application is to detect the fungal (1,3)- ⁇ -D glucan in the sample by the sandwich method.
- the carboxylated magnetic particles couple conjugated capture antibody (biotin-labeled purified antifungal (1,3)- ⁇ -D glucan monoclonal antibody), (1,3)- ⁇ -D glucan and chemiluminescent group-modified
- the detection antibody (the purified antifungal (1,3)- ⁇ -D-glucan monoclonal antibody labeled with a chemiluminescent group) formed a capture antibody-antigen-detection antibody immune complex, and magnetic separation and washing removed the non-magnetic After the substance bound by the particles, add excitation solution (chemiluminescence excitation solution A and chemiluminescence excitation solution B in this example) to emit light, and analyze the fungal (1,3)- ⁇ -D in the sample by detecting the chemiluminescence intensity The content
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Abstract
本申请提供一种抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用,属于生物和医疗检测技术领域,所述抗体包括轻链可变区决定簇互补区,其氨基酸序列包括:SEQ ID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2和SEQ ID NO:3所示的VL-CDR3;所述抗体还包括重链可变区决定簇互补区,其氨基酸序列包括:SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2和SEQ ID NO:6所示的VH-CDR3。该抗体与真菌(1,3)-β-D葡聚糖特异性结合,抗体亲和力高,不与干扰物质产生交叉反应。
Description
本专利申请要求于2021年09月07日提交的中国专利申请No.CN202111045511.1的优先权。在先申请的公开内容通过整体引用并入本申请。
本申请属于医药生物检测技术领域,尤其涉及一种抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用。
侵袭性真菌感染(Invasive fungal infection,IFI)是指真菌侵入人体组织、血液或器官,并在其中生长繁殖引起组织损害、器官功能障碍、炎症反应性病理改变的病理生理过程。近年来,由于免疫抑制剂、广谱抗生素、糖皮质激素、抗肿瘤药物等的广泛使用及外科手术中导管、置管的应用,侵袭性真菌感染的发病率和死亡率逐渐增多。又由于IFI的临床症状和体征缺乏特异性,导致目前缺少有效的早期诊断手段。
目前,检测真菌感染的主要方法有:组织活检法、血培养法、传统鲎试剂检测法、新一代鲎试剂检测法、分子生物学检测法等。其中,①组织活检属于有创性检查,临床标本很难获取。②血培养方法技术要求高,耗时长,培养阳性率低(有些真菌如曲霉菌几乎培养不出阳性结果),不能做到早期诊断。③传统鲎试剂检测法(即G试验)依赖海洋生物鲎的血细胞,但鲎试剂特异性较差,不同批次间试剂活性差异大,检测易受内毒素(脂多糖)和某些药物干扰,假阳性率高,且鲎是濒危物种,属于国家二级保护动物,采血来源受限。④新一代G试验是利用基因工程技术重组鲎血G因子(即人工鲎试剂)来代替采集野生或人工养殖的鲎血而制成的传统鲎试剂,解决了鲎血来源和内毒素(脂多糖)干扰问题,但仍存在(1,3)-β-D葡聚糖类似物(半乳甘露聚糖、甘露聚糖、肽聚糖、纤维素)和某些药物(多粘菌素、香菇多糖等)干扰引起的检测假阳性的问题。⑤分子生物学检测是基于聚合酶链式反应(Polymerase Chain Reaction, PCR)法检测真菌DNA,由于真菌细胞壁厚,很难使之破碎释放出DNA,因此PCR法检测真菌感染阳性率低,目前尚处于研发阶段。
以上各种方法的局限性导致目前临床上侵袭性真菌感染的早期诊断非常困难。因此,临床上十分需要一种灵敏度高、特异性好、干扰小且操作简单,适于真菌感染早期诊断的检测方法。
针对上述问题,本申请的目的在于提供一种抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用,以克服现有真菌感染检测方法的弊端。
为实现上述目的,本申请采用的技术方案为:
本申请的第一方面提供一种抗真菌(1,3)-β-D葡聚糖单克隆抗体,包括轻链可变区决定簇互补区和重链可变区决定簇互补区;
所述轻链可变区决定簇互补区的氨基酸序列包括:SEQ ID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2及SEQ ID NO:3所示的VL-CDR3;
所述重链可变区决定簇互补区的氨基酸序列包括:SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2及SEQ ID NO:6所示的VH-CDR3。
进一步的,所述抗真菌(1,3)-β-D葡聚糖单克隆抗体的轻链可变区决定簇互补区的氨基酸序列为SEQ ID NO:7;重链可变区决定簇互补区的氨基酸序列为SEQ ID NO:8。
本申请的第二方面提供一种编码上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因,所述轻链可变区决定簇互补区的核苷酸序列为SEQ ID NO:9,所述重链可变区决定簇互补区的核苷酸序列为SEQ ID NO:10。
本申请的第二方面提供一种编码上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因,所述轻链可变区决定簇互补区的核苷酸序列如SEQ
ID NO:11-13所示,所述重链可变区决定簇互补区的核苷酸序列如SEQ
ID NO:14-16所示。
本申请的第三方面提供一种包含上述抗真菌(1,3)-β-D葡聚糖单克隆抗体基因的重组载体;
所述重组载体为含有抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区决定簇互补区的基因的真核重组表达质粒pCMVp NEO BAN-Anti-glucan。
本申请的第四方面提供一种包含上述抗真菌(1,3)-β-D葡聚糖单克隆抗体基因的重组表达细胞;
所述重组表达细胞为抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞。
所述重组载体及将重组载体导入细胞后所得的所述重组表达细胞都属于本申请保护范围,所述重组载体是指所有包含上述抗真菌(1,3)-β-D葡聚糖单克隆抗体基因的重组载体(也可以称为重组质粒),所述重组表达细胞是指所有包含上述抗真菌(1,3)-β-D葡聚糖单克隆抗体基因的重组表达细胞,并不限于本申请所指出的含有抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区决定簇互补区的基因的真核重组表达质粒pCMVp NEO BAN-Anti-glucan及抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞,应理解为,本申请要求保护的技术方案中所涉及的重组载体为将上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因插入到本领域技术人员能够购买到的任一表达载体(也可以称为表达质粒)中所获得的重组载体;重组表达细胞为将上述重组载体导入任一表达细胞后所获得的重组表达细胞。
本申请的第五方面提供一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,含有上述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
在其中一个实施例中,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中含有采用生物素标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
在其中一个实施例中,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中还含有采用化学发光基团标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
本申请的第六方面提供一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测方法,利用上述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,采用磁微粒化学发光法进行检测。
本申请提供的抗真菌(1,3)-β-D葡聚糖单克隆抗体与真菌(1,3)-β-D葡聚糖特异性结合,抗体亲和力高,不与内毒素(如脂多糖)、半乳甘露聚糖、甘露聚糖、肽聚糖、纤维素、多粘菌素、香菇多糖等物质产生交叉反应。基于该抗体特性研制的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒灵敏度高、特异性好、定量检测范围宽、检测速度快、可实现机器自动化操作,特别是可以有效避免由内毒素(如脂多糖)、(1,3)-β-D葡聚糖结构类似物(如半乳甘露聚糖、甘露聚糖、肽聚糖、纤维素)和某些药物(如多粘菌素、香菇多糖等)等引起的假阳性检测结果,为临床提供更准确的检测信息,可以满足临床对真菌感染早期诊断的要求,具有很好的临床应用价值。
图1是本申请实施例1中克隆细胞筛选情况过程图;
图2是本申请实施例2中Marker及纯化单抗IgG的电泳测定结果图;
图3是本申请实施例3中不同浓度的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的抗体效价测定结果图;
图4是本申请实施例4中纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体与其它干扰检测的物质交叉反应测定结果图。
本申请的实施方式
下面对本申请实施例中的技术方案进行清楚、完整地描述。在下面的描述中阐述了很多具体细节以便于充分理解本申请,但是本申请还可以采用其他不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本申请内涵的情况下做类似推广,因此本申请不受下面公开的具体实施例的限制。
(1,3)-β-D葡聚糖购自Sigma-Aldrich公司,CAS号:9051-97-2,货号:89862;
弗氏完全佐剂/弗氏不完全佐剂购自Sigma-Aldrich公司,货号:F5881/F5506;
酶标第二抗体为辣根过氧化物酶(Horseradish Peroxidase,HRP)标记的羊抗小鼠IgG,购自美国Jackson公司,货号:115-035-003;
TMB购自全式金公司,货号:HE111-01;
中国仓鼠卵巢细胞购自中国医学科学院基础医学研究所细胞资源中心(细胞编号:1101HAM-PUMC000116)
链霉亲和素磁微粒为Dynabeads™ M-270 链霉亲和素,购自Thermo
Fisher公司,货号65305;
Sulfo-NHS-LC-Biotin购自上海阿拉丁生化科技股份有限公司,CAS号:191671-46-2,货号:S288926;
NSP-SA-NHS购自苏州美仑生物科技有限公司,CAS号:199293-83-9,货号:MB3332。
以下实施例中所用试剂/培养基(培养液)如无特殊说明均为本领域常规试剂/培养基(培养液)。
实施例1 杂交瘤细胞的制备
一)动物免疫
单克隆抗体可以使用通常的免疫学技术,以(1,3)-β-D葡聚糖为抗原,进行动物免疫,再使用被免疫动物的细胞制备杂交瘤细胞而获得。杂交瘤细胞制备时使用的被免疫动物的种类没有特别限定,如小鼠、大鼠、仓鼠、兔等,本实施例使用BALB/c小鼠作为被免疫动物。抗原在进行动物免疫时,配合使用佐剂效果更佳,佐剂能够提高被免疫动物对抗原的免疫应答。佐剂的种类没有特别限定,例如可使用弗氏完全佐剂、弗氏不完全佐剂、百日咳疫苗、铝佐剂等,其中本实施例初次免疫时使用弗氏完全佐剂,在第二次及以后的免疫时使用弗氏不完全佐剂。
(1,3)-β-D葡聚糖与弗氏完全佐剂等体积混合且充分乳化作为初次免疫试剂。
取6~8周龄BALB/c小鼠(1号鼠、2号鼠和3号鼠),用初次免疫试剂于皮下多点注射,初次免疫试剂使用剂量相当于每只小鼠注射30μg的(1,3)-β-D葡聚糖。以后每隔2周免疫一次,从第二次免疫开始使用等体积混合且充分乳化的(1,3)-β-D葡聚糖与弗氏不完全佐剂,于腹腔注射,免疫剂量相当于每只小鼠注射30μg的(1,3)-β-D葡聚糖。第四次免疫7天后,取小鼠内眦静脉血用酶联免疫吸附实验(Enzyme Linked Immunosorbent Assay,ELISA)法测定抗体效价,具体抗体效价测定方法(ELISA法)如下:
11)包被抗原:用包被缓冲液(1.059g的Na
2CO
3、2.93g的NaHCO
3、加超纯水至1000mL,调pH值为9.6)将(1,3)-β-D葡聚糖稀释至1μg/mL加入酶标板,每孔加0.1mL,4℃过夜。次日弃去孔内的液体,同时用PBST洗涤缓冲液(0.2g的KH
2PO
4、2.9g的Na
2HPO
4∙12H
2O、8.0g的NaCl、0.2g的KCl、0.5mL的Tween-20,加超纯水至100mL,调pH值为7.4)洗涤3次,每次洗涤时每孔加PBST洗涤缓冲液300μL(以下未明确说明时,洗涤所用试剂和用量均与本次相同),拍干。
12)封闭:每孔中加入0.1mL的含3%牛血清白蛋白(BSA)的PBST洗涤缓冲液,置于37℃孵育1h,再经PBST洗涤缓冲液洗涤3次,拍干,得待测用酶标板。
13)加待测的鼠血清样本:取不同小鼠的内眦静脉血,于2000rpm离心5min后取血清,用0.1mol/L的PBS缓冲液将不同小鼠(1号鼠、2号鼠和3号鼠)的血清均分别稀释1000倍、1万倍、10万倍、50万倍和100万倍,得不同浓度的免疫小鼠血清。
分别取100μL不同浓度的免疫小鼠血清加至待测用酶标板孔内,同时以未免疫小鼠的血清作为阴性对照,以0.1mol/L的PBS缓冲液作为空白对照,置于37℃孵育1h,弃去孔中液体,再经PBST洗涤缓冲液洗涤3次,拍干。
14)加酶标第二抗体:先用稀释缓冲液(0.1g的BSA加至PBST洗涤缓冲液中,定容至100mL)将酶标第二抗体稀释至工作浓度,即稀释10000倍,得酶标第二抗体稀释液;
每孔分别加入100μL的酶标第二抗体稀释液,37℃孵育1h,PBST洗涤缓冲液洗涤,拍干。
15)显色:各孔中分别加入100μL的显色底物TMB,37℃孵育15min。
16)终止反应:各孔中分别加入100μL的终止液(浓度为1mol/L的HCl)终止反应。
17)判定结果:在酶标仪上于450nm处以空白对照孔调零后测各孔吸光度值(A450nm),当待测样品的A450nm值与阴性对照的A450nm值的比大于2.1时,判定为阳性,此时抗体的最大稀释度即为抗体效价。具体抗体效价测定结果见下表:
由表1可以看出,2号鼠的抗体效价达到1:10万以上,其抗体效价最佳,选取该小鼠进行后续试验。
二)杂交瘤细胞制备
1)骨髓瘤SP2/0细胞悬液的制备
融合前2周常规复苏小鼠骨髓瘤细胞(SP2/0细胞),并接种于培养基中(培养基为含10~20%胎牛血清的RPMI 1640培养液,补加青霉素100IU/mL、链霉素100μg/mL),置于5%的CO
2恒温培养箱中,37℃培养。融合前48~36h改用完全培养液(含20%胎牛血清的RPMI 1640培养液),置于37℃、5%CO
2恒温培养箱中进行扩大培养。
融合当天,用吸管将细胞轻轻从瓶壁上吹下,收集于50mL离心管中,于1500rpm离心6min,弃去上清,所得瘤细胞沉淀中加入30mL不完全培养液【不完全培养液是在RPMI 1640中补加2%的NaHCO
3、3%的4-羟乙基哌嗪乙磺酸(Hepes)、0.011%的丙酮酸钠,并用1mol/L的HCl调节pH为6.8~7.0制得】,再次于1500rpm离心6min,然后用PBST洗涤缓冲液洗涤一次,镜下观察细胞,并选取等大等圆、透亮、活性好的细胞作为SP2/0细胞,收集SP2/0细胞,使SP2/0细胞数达2×10
7cell,再将SP2/0细胞重悬至10mL不完全培养液,混匀,得骨髓瘤SP2/0细胞悬液。
2)免疫脾细胞悬液的制备
在细胞融合前3天,使用等体积混合且充分乳化的(1,3)-β-D葡聚糖与弗氏不完全佐剂,通过尾静脉注射对2号小鼠进行加强免疫,加强免疫的剂量相当于每只小鼠注射10μg的(1,3)-β-D葡聚糖。3天后摘除眼球放血,并分离血清供检测抗体用。将小鼠拉颈处死,浸泡于75%酒精内5min,随即放入超净台中,用无菌手术开腹,取出脾脏,放入盛有8mL不完全培养液的平皿中冲洗,并细心剥去周围结缔组织,得处理后的脾脏。
将处理后的脾脏移入另一盛有5mL不完全培养液的平皿中,置于200目不锈钢网上,用剪刀将处理后的脾脏剪成小块,然后用注射器内芯挤压研磨,并用平皿内的不完全培养液轻轻冲洗钢网,使脾细胞全部被挤压到液体中去,所得脾细胞溶液转至50mL离心管中,加不完全培养液至30mL混匀,于1500rpm离心6min,弃去上清后,再加不完全培养液至30mL混匀,再次于1500rpm离心6min,所得沉淀细胞加不完全培养液重悬至10mL,混匀,得脾细胞悬液(一般每只鼠可得1~2.5×10
8个脾细胞。以台盼兰染色用相差显微镜检查,活细胞数高于80%为合格)。
3)细胞融合
分别吸取含有1×10
8个脾细胞的脾细胞悬液和含有2×10
7个骨髓瘤细胞的骨髓瘤SP2/0细胞悬液,加至50mL离心管中,补加不完全培养液至30mL,充分混匀,再于室温下经1200r/min离心6min,弃去上清,轻轻弹击管底,使沉淀细胞松散均匀成糊状,再于37℃水浴条件下,将1mL的PEG 1650沿转动的管壁滴入,静置90s,然后将离心管从水浴中取出,以1000r/min离心6min,弃去上清液,所得沉淀中加入10mL的HAT培养液,轻轻吹吸沉淀的细胞,使其悬浮并混匀,得融合后细胞悬液。
将融合后细胞悬液加入含有HAT培养液的96孔培养板中,每孔2滴,置于37℃恒温培养箱培养7天(d),7d后更换培养液,更换培养液时吸去1/2~2/3培养液(本实施例吸去2/3培养液),加入等量HT培养液,再次培养7d后再次更换培养液,更换培养液时吸去1/2~2/3培养液(本实施例吸去2/3培养液),加入等量的完全培养液(含20%胎牛血清的RPMI 1640培养液),得杂交瘤细胞悬液。
4)杂交瘤细胞的筛选与检测
用ELISA法(具体操作步骤参见实施例1动物免疫中相应内容)检测杂交瘤细胞悬液的抗体效价,以完全培养液做阴性对照,以0.1mol/L的PBS缓冲液为空白对照,检测得杂交瘤细胞悬液的A450nm值与阴性对照的A450nm值的比值大于2.1,杂交瘤细胞悬液的抗体效价为阳性。
5)克隆化培养
将抗体效价为阳性的杂交瘤细胞悬液从96孔培养板扩大至24孔培养板培养3-5天,再将24孔培养板中待克隆化的杂交瘤细胞用加样器反复吹打均匀后,取20μL待克隆化的杂交瘤细胞悬液加入到1mL浓度为0.1mol/L的PBS缓冲液中,进行50倍稀释,得稀释后的细胞悬液。
用细胞计数板对稀释后的细胞悬液进行细胞计数,计算公式如下:细胞悬液的细胞数 /mL =(计数板上4个大格细胞总数 /4)×10000×50(稀释倍数),细胞数为3×10
6/mL。
根据上述细胞计数浓度,取含有230个活细胞的细胞悬液悬浮于4.6mL完全培养液(含20%胎牛血清的RPMI 1640培养液)中,所得活细胞液接种于96孔培养板,每孔0.1mL活细胞液,共36孔(记为第一组)。余下的1mL活细胞液中,再加入4mL完全培养液(含20%胎牛血清的RPMI 1640培养液),共得5mL活细胞液,然后再将活细胞液接种至其余36孔,每孔0.1mL活细胞液(记为第二组)。剩余的1.4mL活细胞液,再补加1.4mL完全培养液(含20%胎牛血清的RPMI 1640培养液),接种剩余的24孔,每孔0.1mL活细胞液(记为第三组)。这样共以三种不同的细胞浓度进行克隆化,第一组36孔,平均每孔5个细胞,第二组36孔,平均每孔1个细胞,第三组24孔,平均每孔0.5个细胞。
将96孔培养板置于37℃、5%CO
2恒温培养箱培养5d,5d后在倒置显微镜下观察细胞克隆生长情况,更换培养液并采用ELISA法检测抗体分泌情况,取呈阳性的单克隆孔再次依据上述克隆方法进行克隆,选择两次克隆均为阳性的单克隆孔转入24孔培养板,再转入细胞培养瓶中扩大培养并冻存,具体克隆细胞筛选过程见图1。
6)杂交瘤细胞的冻存
观察在细胞培养瓶中扩大培养的细胞状态,将处于对数生长期的杂交瘤细胞倒掉上清培养液,用吸管加入1mL不完全培养液,加入0.8mL胎牛血清和0.2mL二甲基亚砜,混合均匀,细胞计数,分装入冻存管,每个冻存管加入细胞数不少于1×10
6个细胞。将冻存管置于4℃预冷30min,-20℃放置2h,再移至-70℃低温冰箱中过夜,最后投入液氮中保存。
实施例2 抗真菌(1,3)-β-D葡聚糖单克隆抗体的制备
收集筛选呈阳性的杂交瘤细胞进行克隆,并收集其分泌的抗体,即为抗真菌(1,3)-β-D葡聚糖单克隆抗体,收集抗真菌(1,3)-β-D葡聚糖单克隆抗体,测定抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区的基因序列,其轻链可变区决定簇互补区的氨基酸序列是由SEQ ID NO:1所示的VL-CDR1,SEQ ID NO:2所示的VL-CDR2和SEQ ID NO:3所示的VL-CDR3组成,其具体氨基酸序列为SEQ
ID NO:7,核苷酸序列为SEQ ID
NO:9。重链可变区决定簇互补区的氨基酸序列是由SEQ ID NO:4所示的VH-CDR1,SEQ ID NO:5所示的VH-CDR2和SEQ ID NO:6所示的VH-CDR3组成,其具体氨基酸序列为SEQ ID NO:8,核苷酸序列为SEQ ID NO:10。
测序结果:
A、轻链可变区序列信息
1)VL-CDR1
氨基酸序列:WDIVETQSETPSYASVIPVGGTYVTDIKSITKCSDKDSE(SEQ ID NO:1)
核苷酸序列:
tgggacatagtagaaacacaaagcgaaacaccaagctacgcaagcgtaataccagtaggaggaacatacgtaacagacataaaaagcataacaaaatgcagcgacaaagacagcgaa(SEQ ID NO:11)
2)VL-CDR2
氨基酸序列:TLCDRETQECESDIKAGQKKRLSRYVPTINK(SEQ ID NO:2)
核苷酸序列:
acactatgcgacagagaaacacaagaatgcgaaagcgacataaaagcaggacaaaaaaaaagactaagcagatacgtaccaacaataaacaaa(SEQ ID NO:12)
3)VL-CDR3
氨基酸序列:DMEYPESQLGDFGGSGGFVGFVEVKVRRIGEGGVTRSLE(SEQ ID NO:3)
核苷酸序列:
gacatggaatacccagaaagccaactaggagacttcggaggaagcggaggattcgtaggattcgtagaagtaaaagtaagaagaataggagaaggaggagtaacaagaagcctagaa(SEQ ID NO:13)
轻链可变区决定簇互补区的氨基酸序列:
WDIVETQSETPSYASVIPVGGTYVTDIKSITKCSDKDSETLCDRETQECESDIKAGQKKRLSRYVPTINKDMEYPESQLGDFGGSGGFVGFVEVKVRRIGEGGVTRSLE(SEQ ID NO:7)
轻链可变区决定簇互补区的核苷酸序列:
tgggacatagtagaaacacaaagcgaaacaccaagctacgcaagcgtaataccagtaggaggaacatacgtaacagacataaaaagcataacaaaatgcagcgacaaagacagcgaaacactatgcgacagagaaacacaagaatgcgaaagcgacataaaagcaggacaaaaaaaaagactaagcagatacgtaccaacaataaacaaagacatggaatacccagaaagccaactaggagacttcggaggaagcggaggattcgtaggattcgtagaagtaaaagtaagaagaataggagaaggaggagtaacaagaagcctagaa(SEQ ID NO:9)
B、重链可变区序列信息
1)VH-CDR1
氨基酸序列:VQSQLEEVSGGRRLVQPTPGSTPKLTSAFSDRTAYSGFSWS(SEQ ID NO:4)
核苷酸序列:
gtacaaagccaactagaagaagtaagcggaggaagaagactagtacaaccaacaccaggaagcacaccaaaactaacaagcgcattcagcgacagaacagcatacagcggattcagctggagc(SEQ ID NO:14)
2)VH-CDR2
氨基酸序列:QKWVRQAPGKGLEWIGDRSTYIPLMSLLKEV(SEQ ID NO:5)
核苷酸序列:
caaaaatgggtaagacaagcaccaggaaaaggactagaatggataggagacagaagcacatacataccactaatgagcctactaaaagaagta(SEQ ID NO:15)
3)VH-CDR3
氨基酸序列:FTDSKYTPTKVDLMPTDAYYFCANYDRDAVMEWGPEITVS(SEQ ID NO:6)
核苷酸序列:
ttcacagacagcaaatacacaccaacaaaagtagacctaatgccaacagacgcatactacttctgcgcaaactacgacagagacgcagtaatggaatggggaccagaaataacagtaagc(SEQ ID NO:16)
重链可变区决定簇互补区的氨基酸序列:
VQSQLEEVSGGRRLVQPTPGSTPKLTSAFSDRTAYSGFSWSQKWVRQAPGKGLEWIGDRSTYIPLMSLLKEVFTDSKYTPTKVDLMPTDAYYFCANYDRDAVMEWGPEITVS(SEQ ID NO:8)
重链可变区决定簇互补区的核苷酸序列:
gtacaaagccaactagaagaagtaagcggaggaagaagactagtacaaccaacaccaggaagcacaccaaaactaacaagcgcattcagcgacagaacagcatacagcggattcagctggagccaaaaatgggtaagacaagcaccaggaaaaggactagaatggataggagacagaagcacatacataccactaatgagcctactaaaagaagtattcacagacagcaaatacacaccaacaaaagtagacctaatgccaacagacgcatactacttctgcgcaaactacgacagagacgcagtaatggaatggggaccagaaataacagtaagc(SEQ ID NO:10)
委托生工生物工程(上海)股份有限公司,构建含有抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区的基因的真核重组表达质粒pCMVp‑NEO‑BAN-Anti-glucan。
将真核重组表达质粒pCMVp‑NEO‑BAN-Anti-glucan导入中国仓鼠卵巢细胞(CHO细胞),得抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞。
将抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞接种于含10%胎牛血清的DMEM培养基,添加1mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)作为诱导剂,于37℃、5%CO
2条件下诱导表达48h,收集细胞培养上清(表达产物)。
饱和硫酸铵法制备单克隆抗体IgG的粗提物:取细胞培养上清5mL,依次加5mL生理盐水和10mL饱和硫酸铵水溶液,4℃放置过夜后,于3000rpm离心30min,弃上清液,剩余沉淀中加入5mL生理盐水和5mL饱和硫酸铵水溶液,4℃放置过夜,然后于3000rpm离心30min,弃上清液,得到的沉淀中再次加入5mL生理盐水和2.5mL饱和硫酸铵水溶液,4℃放置过夜,于3000rpm离心30min,弃上清液,所得沉淀溶于2mL生理盐水中,对生理盐水透析18小时,即得单克隆抗体IgG的粗提物。
Protein A凝胶亲和层析法纯化单抗IgG:取1mL的Protein A,装柱后用20mmol/L的磷酸钠缓冲液(取20mmol/L的Na
2PO
3和20mmol/L的NaH
2PO
3等体积混合,调PH=7.0)平衡,将单克隆抗体IgG的粗提物5mL在4℃反复上柱20次,以20mmol/L磷酸钠缓冲液充分淋洗柱子,至分光光度测定OD280约等于0,然后换成0.1mol/L的甘氨酸-盐酸缓冲液(pH=2.7)洗脱,每次500μL,洗脱液采用20μL浓度为1.0mol/L的Tris-HCl缓冲液(pH=9.0)中和,所得抗体洗脱液采用CBG法测定蛋白含量,收集蛋白峰,然后对其采用0.01mol/L的PBS缓冲液(pH=7.4)充分透析,得纯化单抗IgG。
采用SDS-PAGE电泳法测定Marker、单克隆抗体IgG的粗提物和纯化单抗IgG,参见图2,可以看出两条蛋白带根据蛋白分子量回归方程计算出两条染色带分子量约为54kD和25kD,与文献报道的IgG重链及轻链分子量相符。因此,纯化单抗IgG即为纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体。
实施例3 纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的效价测定
31)用包被缓冲液将(1,3)-β-D葡聚糖稀释至1μg/mL加入酶标板,每孔加0.1mL,4℃过夜。次日弃去孔内的液体,同时用PBST洗涤缓冲液洗涤3次,每次洗涤时每孔加PBST洗涤缓冲液300μL,拍干。
32)每孔中加入0.1mL的含3%BSA的PBST洗涤缓冲液,置于37℃孵育1h,再经PBST洗涤缓冲液洗涤3次,拍干,得待测用孔板。
33)将纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体用0.1mol/L的PBS缓冲液分别稀释1000倍、1万倍、10万倍、50万倍、100万倍、200万倍、500万倍和1000万倍,得不同浓度的抗体稀释液。
分别取100μL不同浓度的抗体稀释液加至待测用孔板的孔内,同时以完全培养液做阴性对照,以0.1mol/L的PBS缓冲液为空白对照,置于37℃孵育1h,弃去孔中液体,再经PBST洗涤缓冲液洗涤3次,拍干。
34)每孔分别加入100μL的酶标第二抗体稀释液,37℃孵育1h,PBST洗涤缓冲液洗涤,拍干。
35)各孔中分别加入100μL的显色底物TMB,37℃孵育15min。
36)各孔中加入100μL的终止液(浓度为1mol/L的HCl)终止反应。
37)在酶标仪上于450nm处以空白对照孔调零后测各孔吸光度值(A450nm),当待测样品的A450nm值与阴性对照的A450nm值的比大于2.1时,判定为阳性。参见图3,可以看出纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体稀释500万倍时,A450nm值仍大于对照组2.1倍以上,说明纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的效价在1:500万以上,其亲和力非常高。
实施例4 纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体特异性鉴定
本申请是为了获得仅与(1,3)-β-D葡聚糖强力结合,而与其它干扰检测的物质很少结合甚至不结合的单克隆抗体。杂交瘤细胞经过严格的筛选和单克隆化,对通过基因工程技术重组表达后,纯化所得的抗真菌(1,3)-β-D葡聚糖单克隆抗体,再一次进行了特异性鉴定。该抗体的特异性可以用其与(1,3)-β-D葡聚糖的类似物是否存在交叉反应性及交叉反应的大小来衡量。具体操鉴定方法如下:
41)用包被缓冲液分别将脂多糖、半乳甘露聚糖、甘露聚糖、肽聚糖、多粘菌素及(1,3)-β-D葡聚糖稀释成10μg/mL,分别加至包被酶标板,每孔加0.1mL,4℃过夜。次日弃孔内液体,用PBST洗涤缓冲液洗涤3次,每次洗涤时每孔加PBST洗涤缓冲液300μL,拍干。
42)每孔中加入0.1mL的含3%BSA的PBST洗涤缓冲液,置于37℃孵育1h,再经PBST洗涤缓冲液洗涤3次,拍干,得含有不同包被多糖的孔板。
43)将纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体用0.1mol/L的PBS缓冲液分别稀释至浓度为0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL及10μg/mL的纯化后抗体稀释液。
44)分别取100μL不同浓度的纯化后抗体稀释液加至含有不同包被多糖的孔板内,且含有相同包被多糖的孔内均需分别加入上述5种浓度的纯化后抗体稀释液,同时以完全培养液作为阴性对照,PBS缓冲液作为空白对照,置于37℃孵育60min,弃去孔内液体,用PBST洗涤缓冲液洗涤3次,每孔加入100μL酶标第二抗体稀释液,37℃孵育1h,PBST洗涤缓冲液洗涤,拍干。
45)各孔中分别加入100μL的显色底物TMB,37℃孵育15min。
46)各孔中加入100μL的终止液(浓度为1mol/L的HCl)终止反应。
47)用在酶标仪上于450nm处以空白对照孔调零后测各孔吸光度值(A450nm),当待测样品的A450nm值/阴性对照的A450nm值大于2.1时为阳性,即表示纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体在该浓度下与包被的多糖存在交叉反应。参见图4,可以看出,纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体在0.001-10μg/mL浓度范围内与脂多糖、半乳甘露聚糖、甘露聚糖、肽聚糖、多粘菌素均无交叉反应,且与(1,3)-β-D葡聚糖交叉反应良好,说明纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体具有非常好的特异性,且受其它物质干扰程度很小。
实施例5 真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的制备和应用
一)真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的制备
本实施例采用纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体作为捕获抗体和/或检测抗体,制得了一种可用于临床的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,该试剂盒主要组分包括:生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体、化学发光基团标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体【本实施例采用吖啶磺酰胺标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,也可用吖啶磺酰胺、吖啶酯、钌复合物、鲁米诺、(金钢烷)‑1,2‑二氧乙烷或碱性磷酸酶中的至少一种进行标记】、链霉亲和素磁微粒(Dynabeads™ M-270 链霉亲和素)、低值校准品【(1,3)-β-D葡聚糖冻干品,用超纯水复溶后浓度为50pg/mL】、高值校准品【(1,3)-β-D葡聚糖冻干品,用超纯水复溶后浓度为200pg/mL】、质控品【(1,3)-β-D葡聚糖冻干品,用超纯水复溶后浓度为100pg/mL】;化学发光激发液A液(0.1mol/L的HNO
3、1.32%的H
2O
2)、化学发光激发液B液(0.35mol/L的NaOH)和PBST洗涤缓冲液。
采用纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体偶联在固相载体上,所述固相载体为羧基化磁微粒、生物素化磁微粒中的至少一种,本实施例采用羧基化磁微粒作为固相载体。具体地,真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒如下:
51)生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的制备
取200μg的(1,3)-β-D葡聚糖抗体,用0.02mol/L的PBS缓冲液(pH=7.4)稀释至1 mg/mL,得抗体溶液。
取1mg的Sulfo-NHS-LC-Biotin(生物素抗体标记用)溶于100μL的二甲基亚砜中,取3μL加至抗体溶液中混匀,避光室温反应1h,反应结束后将反应产物装入透析袋中(排阻MW:8000),以0.02mol/L的PBS缓冲液(pH=7.4)作为透析液,2~8℃透析24h(本实施例在4℃进行透析),所得透析产物中加入终浓度为0.1%的Proclin300和1%的海藻糖,得生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,密封避光置于2~8℃备用(本实施例为4℃)。使用时生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体与链霉亲和素磁微粒1:1混合即可。
52)吖啶磺酰胺标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的制备
取200μg的(1,3)-β-D葡聚糖抗体,用0.1mol/L的PBS缓冲液(pH=7.2)稀释至1mg/mL,待化学标记的抗体溶液;
取10μL浓度为2mg/mL的NSP-SA-NHS(吖啶磺酰肼胺内盐)加至待化学标记的抗体溶液中混匀,避光室温反应1h,再加4μL浓度为10%的赖氨酸水溶液,混匀,避光置于室温下反应30min,反应结束后将反应产物装入透析袋中(排阻MW:8000),用0.02mol/L的PBS缓冲液(pH=7.2)在2~8℃透析过夜(本实施例在4℃进行透析),产物即为吖啶磺酰胺标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体。
53)校准品及质控品的制备
低值校准品(50pg/mL)的配制方法:取(1,3)-β-D葡聚糖加入超纯水配成浓度为1ng/mL的水溶液,取其中5mL加入20mL甘露醇溶液、6mL聚乙烯吡咯烷酮(PVP)、20mL新生牛血清,然后加超纯水至100mL,混匀,低温冷冻干燥后即为低值校准品(50pg/mL)。
高值校准品(200pg/mL)的配制方法:取(1,3)-β-D葡聚糖加入超纯水配成浓度为4ng/mL的水溶液,取其中5mL加入20mL甘露醇溶液、6mL聚乙烯吡咯烷酮(PVP)、20mL新生牛血清,然后加超纯水至100 mL,混匀,低温冷冻干燥后即为高值校准品(200pg/mL)。
质控品(100pg/mL)的配制方法:取(1,3)-β-D葡聚糖加入超纯水配成浓度为2ng/mL的水溶液,取其中5mL加入20mL甘露醇溶液、6mL聚乙烯吡咯烷酮(PVP)、20mL新生牛血清,然后加超纯水至100mL,混匀,低温冷冻干燥后即为质控品(100pg/mL)。
二)真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的应用
所制真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒可以采用磁微粒化学发光法,在全自动化学发光分析仪上实现自动检测,具体步骤为:反应杯中加入50μL链霉亲和素磁微粒和50μL生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,混匀,37°C孵育15min,然后在外加磁场下分离磁微粒,吸去反应杯中液体,向反应杯中加300μLPBST洗涤缓冲液进行清洗,吸去上清,反复清洗3次,再在反应杯中加入100μL待测样品,混匀,37°C孵育15min;再次在外加磁场下分离磁微粒,吸去反应杯中液体,向反应杯中加300μLPBST洗涤缓冲液进行清洗,吸去上清,反复清洗3次,然后在反应杯中加入100μL吖啶磺酰胺标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,混匀,37°C孵育15min。第三次在外加磁场下分离磁微粒,吸去反应杯中液体,向反应杯中加300μLPBST洗涤缓冲液进行清洗,吸去上清,反复清洗3次。在反应杯中加入化学发光激发液A液和化学发光激发液B液各50μL,立即测定化学发光值,即可检测出待测样品中真菌(1,3)-β-D葡聚糖的含量。
本申请的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒是利用夹心法检测样本中的真菌(1,3)-β-D葡聚糖,羧基化磁微粒偶联的捕获抗体(生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体)、(1,3)-β-D葡聚糖和化学发光基团修饰的检测抗体(化学发光基团标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体)形成了捕获抗体-抗原-检测抗体免疫复合物,磁分离洗涤去除未与磁微粒结合的物质后,加入激发液(本实施例中为化学发光激发液A液和化学发光激发液B液)发光,通过检测化学发光强度来分析样本中真菌(1,3)-β-D葡聚糖的含量,所述试剂盒具有灵敏度高、特异性好、干扰物质少、检测速度快、可实现机器自动化操作等优点,适于临床真菌感染早期诊断。
实施例6 真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的抗干扰效果
本实施例中向含有108pg/mL和413pg/mL的(1,3)-β-D葡聚糖临床样本中分别加入不同浓度的脂多糖、半乳甘露聚糖、甘露聚糖、肽聚糖、多粘菌素(具体添加浓度见表2),添加干扰物的样本再次用实施例5所制的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒按照实施例5中的应用方法进行测定,并计算干扰物质对(1,3)-β-D葡聚糖检测的干扰程度,计算公式如下:
干扰程度(%)=(添加干扰物后样本(1,3)-β-D葡聚糖实测浓度-未添加干扰物时样本(1,3)-β-D葡聚糖实际浓度)/未添加干扰物时样本(1,3)-β-D葡聚糖实际浓度×100%。
通过计算干扰物质对(1,3)-β-D葡聚糖检测的干扰程度,所得结果见下表:
通过上表可以看出,即使添加高浓度的干扰物后,采用本申请所制真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒对(1,3)-β-D葡聚糖进行检测,其受干扰程度依然在5.5%以内,说明本申请所制真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒在检测临床样本的过程中抗干扰能力很强,不会发生因干扰物引起的假阳性结果,能为临床提供更精准的检测数据。
显然,所描述的实施例仅仅是本申请的一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
Claims (10)
- 一种抗真菌(1,3)-β-D葡聚糖单克隆抗体,其特征在于,所述抗真菌(1,3)-β-D葡聚糖单克隆抗体包括轻链可变区决定簇互补区和重链可变区决定簇互补区;所述轻链可变区决定簇互补区的氨基酸序列包括:SEQ ID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2及SEQ ID NO:3所示的VL-CDR3;所述重链可变区决定簇互补区的氨基酸序列包括:SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2及SEQ ID NO:6所示的VH-CDR3。
- 权利要求1所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体,其特征在于,所述抗真菌(1,3)-β-D葡聚糖单克隆抗体的轻链可变区决定簇互补区的氨基酸序列为SEQ ID NO:7;重链可变区决定簇互补区的氨基酸序列为SEQ ID NO:8。
- 一种编码权利要求1或2所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因,其特征在于,所述轻链可变区决定簇互补区的核苷酸序列为SEQ ID NO:9,所述重链可变区决定簇互补区的核苷酸序列为SEQ ID NO:10。
- 一种编码权利要求1所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因,其特征在于,所述轻链可变区决定簇互补区的核苷酸序列如SEQ ID NO:11-13所示,所述重链可变区决定簇互补区的核苷酸序列如SEQ ID NO:14-16所示。
- 包含权利要求3或4所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组载体。
- 包含权利要求3或4所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组表达细胞。
- 一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,其特征在于,含有权利要求1或2所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体。
- 如权利要求7所述的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,其特征在于,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中含有采用生物素标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
- 如权利要求7或8所述的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,其特征在于,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中还含有采用化学发光基团标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
- 一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测方法,其特征在于,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测方法是利用权利要求7-9中任一项所述的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,采用磁微粒化学发光法进行检测。
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