CN106771264A - Kit for determining thyroid stimulating hormone and fabrication method - Google Patents

Kit for determining thyroid stimulating hormone and fabrication method Download PDF

Info

Publication number
CN106771264A
CN106771264A CN201611162058.1A CN201611162058A CN106771264A CN 106771264 A CN106771264 A CN 106771264A CN 201611162058 A CN201611162058 A CN 201611162058A CN 106771264 A CN106771264 A CN 106771264A
Authority
CN
China
Prior art keywords
rare earth
tsh
monoclonal antibody
microspheres
pad
Prior art date
Application number
CN201611162058.1A
Other languages
Chinese (zh)
Inventor
王鹏浩
宋璐琳
王文亮
刘衍亮
陈萍萍
孙宁宁
Original Assignee
威海纽普生物技术有限公司
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 威海纽普生物技术有限公司 filed Critical 威海纽普生物技术有限公司
Priority to CN201611162058.1A priority Critical patent/CN106771264A/en
Publication of CN106771264A publication Critical patent/CN106771264A/en

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72Assays involving receptors, cell surface antigens or cell surface determinants for hormones

Abstract

The invention relates to the technical field of fluorescence immunochromatography in medical immunology and particularly relates to a kit (fluorescence immunochromatography) for determining thyroid stimulating hormone (TSH) and a fabrication method. A test strip is arranged. The kit is characterized in that the test strip is sequentially provided with a PVC plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad from bottom to top, wherein a rare-earth Eu<3+> fluorescent microsphere-labelled thyroid stimulating hormone monoclonal antibody is adsorbed to the conjugate pad; the diameters of rare-earth fluorescent microspheres are 150nm; the rare-earth fluorescent microspheres contain rare-earth lanthanide Eu<3+>, are stable in a ground state, and emit fluorescence with the wavelengths of 615nm under the action of an excitation light source of 337nm; the monoclonal antibody is a purified and mixed monoclonal antibody, and is from monoclonal antibody cell lines of 2-6 different thyroid stimulating hormone epitopes. The kit has the advantages of being simple and convenient to operate, fast in reaction, high in sensitivity and high in specificity.

Description

促甲状腺激素测定试剂盒及制作方法 TSH assay kit and method for making

技术领域: FIELD:

[0001] 本发明涉及医学免疫学中荧光免疫层析技术领域,包括蛋白交联技术、膜层析技术、标记免疫分析技术等。 [0001] The present invention relates to a fluorescence immunoassay Medical Immunology BACKGROUND chromatography, including protein cross-linking, membrane chromatography, labeled immunoassay technology. 具体地说是一种能够快速准确的对血清、血浆和全血等样品中促甲状腺激素进行定量分析的促甲状腺激素测定试剂盒及制作方法。 Specifically kit and method capable of making fast and accurate sample of serum, plasma and whole blood, and the like for quantitative analysis of thyroid stimulating hormone TSH assay.

背景技术: Background technique:

[0002] 促甲状腺激素(thyrotropin,thyroid stimulating hormone,TSH)是腺垂体分泌的促进甲状腺的生长和机能的激素。 [0002] thyroid stimulating hormone (thyrotropin, thyroid stimulating hormone, TSH) is to promote the growth and function of thyroid hormone pituitary secretion. 人类的TSH为一种糖蛋白,含211个氨基酸,糖类约占整个分子的15%。 Human TSH is a glycoprotein containing 211 amino acids, sugars about 15% of the whole molecule. 整个分子由两条肽链一一a链和P链组成。 Whole molecule is composed of two peptide chains eleven chain and a P chain. 促甲状腺激素(TSH)是调控甲状腺细胞生长和甲状腺激素合成及分泌的主要因子,由垂体促甲状腺素细胞合成和分泌,并受甲状腺激素的负反馈性调节。 Thyroid stimulating hormone (TSH) is the regulation of cell growth and thyroid thyroid hormone synthesis and secretion of the main factors, thyroid stimulating hormone by the pituitary synthesis and secretion of cell, and subject to negative feedback regulation of thyroid hormones. 临床上,TSH、FT3 (Free Triiodothyronine,即游离三碘甲状腺原氨酸)和FT4(Fr ee Thyroxine,即游离甲状腺激素)联合应用,用于甲状腺功能紊乱的辅助诊断:(1) FT3、FT4升高伴TSH降低,多为甲状腺本身的疾病引起的原发性甲亢。 Clinically, TSH, FT3 (Free Triiodothyronine, i.e. three free triiodothyronine) and FT4 (Fr ee Thyroxine, i.e. free thyroid hormone) combination, for the diagnosis of thyroid dysfunction: (1) FT3, FT4 liters with lower high TSH, thyroid disease, mostly primary hyperthyroidism caused by itself. (2) FT3、 FT4升高,TSH也升高,多为下丘脑一垂体功能紊乱引起的继发性甲亢。 (2) FT3, FT4 elevated TSH also increased, mostly secondary hyperthyroidism hypothalamic-pituitary dysfunction caused. (3)FT3、FT4降低伴TSH升高,多为原发于甲状腺的机能减退。 (3) FT3, FT4 lower with increased TSH, mostly primary in the thyroid function decline. ⑷FT3、FT4降低,TSH也降低,多为下丘脑一垂体功能受损引起的继发性甲减。 ⑷FT3, FT4 decreased TSH also decreases, mostly secondary hypothyroidism hypothalamic-pituitary dysfunction caused. 甲状腺功能改变时,TSH的波动较甲状腺激素更迅速而显著, 是反映下丘脑-垂体-甲状腺轴功能的敏感指标。 Changes in thyroid function, TSH thyroid hormone fluctuations than more rapid and significant, is a reflection of the hypothalamic - pituitary - sensitive indicator of thyroid axis function. 通过检测血TSH水平可以反映甲状腺功能状态,有助于甲状腺疾病的辅助筛查、辅助诊断、治疗效果评判和预后判断。 By detecting blood TSH levels may reflect thyroid function, thyroid disease contribute to the auxiliary screening, diagnosis, prognosis and treatment evaluation.

[0003] 免疫分析技术是利用微量抗原与相应的高特异性抗体之间的免疫反应,来检测如激素、药物、蛋白质、多肽、酶、肿瘤相关抗原、微生素、病毒、细菌及金属元素等生物体内活性物质。 [0003] Immunoassay is the use of highly specific immunological reaction between the antibodies and antigens corresponding to trace, to detect such as hormones, drugs, proteins, polypeptides, enzymes, tumor associated antigen, micro-elements, viruses, bacteria and metals and other biologically active substances in vivo. 免疫分析技术包括标记免疫分析、非标记免疫分析和仪器免疫分析。 Labeled immunoassay techniques include immunoassays, immunoassays and immunoassay instruments unlabeled. 本试剂盒利用的含有稀土元素的羧基乳胶微球标记免疫分析技术是属于标记免疫分析之一种。 This kit utilizes carboxylic latex beads labeled immunoassay technique is a kind of rare earth element-containing immunoassay of the marker.

[0004] 荧光免疫分析(FIA)和放射免疫分析(RIA)自问世以来,经历了几十年的发展,但是人们越来越感觉到FIA因自然本地太高,干扰检测结果;RIA米用同位素标记,对人体有极大危害并给实验带来不便。 [0004] fluorescence immunoassay (FIA) and radioimmunoassay (RIA) since its inception, has experienced decades of development, but people are increasingly feeling the FIA ​​due to natural local too high, interference detection result; RIA meters with isotope mark, on the human body great harm and inconvenience to experiment. 酶免疫分析®IA)也因酶本身不稳定,受其他影响因素较大,推广应用受到限制。 Enzyme Immunoassay ®IA) but also because the enzyme itself is unstable, subject to greater influence other factors, the application is limited. 80年代初,人们开始研宄用稀土元素代替荧光物质和同位素标记蛋白质或抗体,将时间分辨技术引入到生物检测领域,建立了新型的超微量时间分辨荧光免疫分析技术(Time resolved Fluoroimmunoassay,简称TrFIA)。 Early 1980s, people began to study based on rare-earth element was replaced with a fluorescent substance, and isotopically labeled proteins or antibodies, a time-resolved technology into the field of biological testing, the establishment of a new ultra trace time-resolved fluorescence immunoassay technique (Time resolved Fluoroimmunoassay, referred TrFIA ). 该技术米用多学科先进技术,集结了其他免疫分析的特点,在免疫学、分子生物学、细胞学和医学等领域,取得长足的发展和广泛应用。 The multi-disciplinary technical meters with advanced technology, brings together the characteristics of other immunoassay, in the field of immunology, molecular biology, cytology and medicine, has made considerable development and wide application.

[0005] TrFIA利用了具有独特荧光特性的3价稀土离子及螯合物为示踪物代替荧光物质、 酶、同位素、化学发光物质,标记抗体、抗原、激素、多肽、蛋白质、核酸探针及生物细胞,待反应体系(如抗原抗体反应、核酸探针杂交、生物素亲和素反应以及靶细胞对效应细胞的杀伤效应等)发生后,用TrFIA检测仪测定反应产物中的荧光强度。 [0005] TrFIA use of trivalent rare earth ions and chelates with unique fluorescence characteristics instead of fluorescent tracer substance, enzyme, isotope, a chemiluminescent substance, a labeled antibody, antigen, hormone, polypeptide, protein, nucleic acid probes and biological cells, after the reaction system (e.g., antigen-antibody reactions, nucleic acid probe hybridization, biotin-avidin reaction and target cell killing effect of effector cells, and the like) and the fluorescence intensity of the reaction product was measured by TrFIA detector. 根据产物荧光强度和相对荧光强度的比值,判断反应体系中分析物的浓度,从而达到定量分析。 The ratio of the product and the relative fluorescence intensity of fluorescence intensity, determining the concentration of an analyte in the reaction system, so as to achieve quantitative analysis. 在通常的荧光测定中, 由于测试样品中含有多种荧光成分,背景荧光(来自样品中的胶体颗粒和溶剂分子引起的散射光以及血清中蛋白质和其他化合物发出的非特异性荧光)强度大、干扰强,成为荧光分析法大范围推广的瓶颈。 In a typical fluorescence measurement, since the test sample contains a plurality of fluorescent components, a fluorescent background (non-specific scattered light and fluorescence serum proteins and other compounds emitted from the sample and the colloidal particles due to solvent molecules) intensity, interference strong, become large-scale fluorescence analysis to promote the bottleneck. TrFI A之所以能够成为继EIA、RIA之后一种新的灵敏的检测方法, 主要取决于镧系元素独特的荧光特点、检测中采用的波长分辨和时间延迟技术以及解离- 增强技术。 TrFI A has been able to become after EIA, RIA A new sensitive method of detection, depending on the unique characteristics of lanthanide fluorescence wavelength detection uses the time delay and resolution techniques and dissociation - enhanced technology.

[0006] 镧系元素(lanthanide,Ln)属于稀土元素,共有17中,常用于TrFIA主要有铕(Eu)、 钐(Sm)、铽(Tb)、镝(Dy)。 [0006] lanthanides (lanthanide, Ln) belonging to the rare earth element, a total of 17, are mainly used in TrFIA europium (Eu), samarium (Sm), terbium (Tb), dysprosium (Dy). 镧系元素具有独特的荧光发光特点,与普通荧光相比,镧系离子螯合物荧光衰变时间长,为传统荧光的103-106倍。 Lanthanide fluorescence light has a unique characteristic, compared with ordinary fluorescence, fluorescence decay chelate lanthanide ions for a long time, a conventional fluorescence 103-106 times. 如镧系离子螯合物的荧光衰变时间在60-900ys,常用的Eu3+荧光衰变时间为714us,普通荧光免疫分析中荧光团的荧光衰变时间只有1-lOOus,样品中一些蛋白质的荧光衰变时间仅为1-lOus,因此利用时间分辨技术,延迟一定时间后测量,便可获得Eu3+特异性荧光信号。 The fluorescent chelate lanthanide ions decay time 60-900ys, conventional Eu3 + fluorescence decay time is 714us, ordinary fluorescence immunoassay the fluorescence decay time of a fluorophore in only 1-lOOus, sample fluorescence decay time only some protein It is 1-lOus, so the use of time-resolved techniques, after a predetermined time delay measurements can be obtained for Eu3 + specific fluorescence signal. 同时由于衰变时间长,Eu3+标记物在测量时间里可以反复被激发,每次激发后由激发态很快跃迀到基态,就有荧光发出,然后又可被重新激发,如此每秒可有1〇〇〇次激发,使得TrFIA荧光标记物的相对比活性很高。 At the same time due to the long decay time, Eu3 + marker may be repeatedly excited in the measurement period after each excitation from an excited state to a ground state quickly jump Gan, have emitted fluorescence, then in turn be re-excited, so there may be a second 〇〇〇 shot, so that a relatively high specific activity TrFIA fluorescent marker. 镧系元素荧光光谱的最大特征是激发光与发射光之间的Stokes位移较大,Eu3+激发波长为337nm,发射波长为615mn,St〇keS位移可达278nm;同时Eu3+被激发的荧光光带极窄,荧光的发射峰非常尖锐,可使仪器调整在极窄的波长范围内测定,这样就几乎完全消除了背景荧光的干扰,继而通过时间延迟和波长分辨,将强特异性荧光和背景荧光辨开(故称为时间分辨),使干扰达到几乎为零。 The maximum fluorescence spectrum characteristic of the lanthanide is between Stokes excitation and emitted light to large displacements, Eu3 + excitation wavelength 337nm, emission wavelength of 615mn, St〇keS displacement up to 278nm; Eu3 + simultaneously excited fluorescent light with pole narrow emission peak of the phosphor is sharp, adjustment and measurement instrument can in a very narrow wavelength range, thus almost completely eliminating background fluorescence interference, then by the time delay and wavelength-resolved, the strong and specific fluorescence background fluorescence Discrimination open (so called time-resolved), so that the interference reaches almost zero.

[0007] 鉴于以上标记方法及检测技术的应用,本试剂盒具有良好的检测特异性、较高的灵敏度、操作的简便性以及稳定的荧光标记物保证了检测的准确性。 [0007] In view of the application of the above labeling method and detection techniques, this kit having good detection specificity, high sensitivity, simplicity and a stable operation of the fluorescent marker to ensure the accuracy of detection.

发明内容: SUMMARY:

[0008]本发明针对现有技术中存在的缺点和不足,提出了一种利用荧光免疫层析的灵敏性,结合荧光免疫层析分析仪实现的灵敏度高、快捷简便,可以准确定量的促甲状腺激素测定试剂盒及制作方法。 [0008] The present invention addresses the prior art shortcomings and deficiencies, proposed that the sensitivity of the immunochromatography using fluorescence, the sensitivity of fluorescent binding immunochromatographic analyzer to achieve high, quick and easy, accurate quantitation can thyrotropin kit and method for manufacturing hormone determination.

[0009] 本发明可以通过以下措施达到: [0009] The present invention can be achieved by the following measures:

[0010] —种促甲状腺激素测定试剂盒,设有试纸卡,其特征在于所述试纸卡由下至上依次设有:PVC板、样品垫、结合垫、硝酸纤维素膜和吸水垫,其中结合垫上吸附有稀土Eu3+荧光微球标记的抗促甲状腺激素单克隆抗体-微球偶联复合物,所述稀土荧光微球的直径为100-250nm,稀土荧光微球含稀土镧系元素中的一种或几种,在基态下稳定,在3〇〇-4〇〇nm的激发光源作用下发射出波长范围为550-650nm的荧光;所述单克隆抗体为纯化后混合的单克隆抗体,来源于针对2-6个不同的促甲状腺激素抗原表位的单克隆抗体细胞株。 [0010] - species TSH assay kit, with the card strip, characterized in that the paper card has successively from bottom to top: PVC plate, sample pad, conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the binding adsorption pad rare earth fluorescent microspheres Eu3 + labeled anti-TSH monoclonal antibody - conjugated microsphere composite, the microspheres rare earth phosphors 100 and 250nm in diameter, a rare earth fluorescent microspheres containing a lanthanide rare earth elements one or several, in the stable ground state, emitting 550-650nm wavelength range of the fluorescence excitation light source under the action of 3〇〇-4〇〇nm; the monoclonal antibody is a monoclonal antibody purified mixed source monoclonal antibodies against cell lines 2-6 different TSH antigenic epitope.

[0011]本发明所述结合垫的稀土荧光微球的直径是l20-200nm;所述稀土荧光微球含有一种或几种稀土镧系元素;结合垫上稀土荧光微球标记的抗体优选来源于针对2个不同抗原表位的单克隆细胞细胞株。 [0011] the diameter of the bonding pads of the present invention microspheres are rare earth phosphors l20-200nm; the rare earth phosphors microspheres contain one or more rare earth lanthanides; rare earth pad fluorescent microspheres bound labeled antibody is preferably derived from monoclonal cell lines for two different epitopes.

[0012]本发明所述结合垫采用如下步骤制得:将玻璃纤维膜浸泡于i5〇mM Tris-HCL处理液中(含1.0%Triton X-100,2_5%BSA,pH7.4),4°C浸泡2小时,然后取出37。 [0012] The bonding pad of the present invention was prepared using the following procedure: a glass fiber membrane was immersed in the treatment liquid i5〇mM Tris-HCL (containing 1.0% Triton X-100,2_5% BSA, pH7.4), 4 ° C soak 2 hours, then remove 37. (:烘箱烘干4小时,备用,将玻璃纤维膜放在Bio_DotXYZ3050三维喷点平台上,用Bi〇-jet Quanti300非接触式微定量喷头将稀土荧光微球标记的抗促甲状腺激素单克隆抗体偶联复合物喷到玻璃纤维膜,37 °C烘干1小时后制得。 (: Oven dried 4 hours, standby, the glass fiber membrane Bio_DotXYZ3050 three dots on the platform, with Bi〇 non-contact micro-jet Quanti300 head quantitative rare earth fluorescent microspheres labeled anti-TSH monoclonal antibody conjugation composite sprayed onto glass fiber membrane, 37 ° C for 1 hour to give after drying.

[0013] 本发明中结合垫上的所述稀土荧光微球标记的抗促甲状腺激素单克隆抗体采用如下步骤制得: _ [0013] The present invention in conjunction with the rare earth pad fluorescent beads-labeled anti-TSH monoclonal antibody was prepared using the following procedure: _

[0014] 步骤1:单克隆抗体细胞株的获得:用促甲状腺激素纯品免疫小鼠,米用标准的单克隆抗体制备方法制备特异性高亲和力的单克隆抗体细胞株,对所获得的单抗细胞株进行配对筛选,根据配对结果和亲和力数据优选出用于试剂盒的单抗细胞株; [0014] Step 1: monoclonal antibodies obtained in cell lines: rice prepared using standard monoclonal antibody preparation of monoclonal antibodies specific for cell lines with high affinity TSH pure immunized mice, the obtained single anti pair screening cell lines, according to the result of pairing and affinity data for mAb preferably a cell line a kit;

[0015] 步骤2:单克隆抗体的制备:米用标准的腹水生广工艺制备并纯化抗促甲状腺激素单克隆抗体,分装后保存于-2〇r备用; [0015] Step 2: Preparation of monoclonal antibody: Ascites Shengguang rice prepared by standard processes and Purification of Anti-TSH monoclonal antibodies, aliquots stored at -2〇r standby;

[0016] 步骤3:稀土荧光微球的醛基化:取5mg稀土荧光微球,用20mM,pH 9.5的碳酸盐缓冲液,采用离心法洗涤3遍,离心速度为12000rpm,时间为5分钟,最后重悬于100yl的上述碳酸盐缓冲液中,加入500yl醛基化的葡聚糖,混匀,室温下暗反应4小时,采用同样的离心法洗涤和重悬到lOOyl的上述碳酸盐缓冲液中,置于4°C备用; [0016] Step 3: rare earth phosphors microspheres aldehydized: Take 5mg rare earth fluorescent microspheres with 20mM, pH 9.5 carbonate buffer, washed by centrifugation 3 times, centrifugal speed of 12000 rpm, time was 5 minutes , and finally resuspended in the above carbonate buffer 100yl was added 500yl aldehyde dextran, mixing, dark and reacted for 4 hours at room temperature, washed with the same centrifugation and resuspended in the carbonate lOOyl salt buffer and placed in 4 ° C for later use;

[0017]步骤4:稀土荧光微球标记的抗促甲状腺激素单克隆抗体的制备:选取来自2个不同抗原表位的单克隆细胞细胞株的单克隆抗体,按照质量比1:1将2mg促甲状腺激素单克隆抗体用上述碳酸盐缓冲液于4°C透析过夜,然后与上述醛基化的稀土荧光微球混合,4°C反应过夜;然后,加入硼氢化钠至终浓度5mM,4°C反应4小时;再加入等体积的封闭液(50mM Tris-HCL,pH7 • 4,含2 %BSA,5 % 蔗糖),4°C封闭过夜;然后用50mM Tris-HCL,pH7 •4的缓冲液采用离心法洗涤3遍,重悬于1 OOiU的50mM Tris-HCL缓冲液中(含1 • 2%NaCL,0 • 5 %BSA, 0.1%Tween 20),4°C避光保存备用。 [0017] Step 4: Preparation of rare earth fluorescent microspheres labeled anti-TSH monoclonal antibodies: monoclonal antibodies selected from monoclonal cell lines to two different epitopes, at a mass ratio of 1: 1 to promote 2mg thyroid hormone monoclonal antibody with the above carbonate buffer, dialyzed overnight at 4 ° C, then mixed with rare earth phosphors aldehyde of the above microspheres, 4 ° C overnight; then, sodium borohydride was added to a final concentration of 5mM, 4 ° C for 4 hours; then add an equal volume of blocking solution (50mM Tris-HCL, pH7 • 4, containing 2% BSA, 5% sucrose), 4 ° C blocked overnight; then with 50mM Tris-HCL, pH7 • 4 of buffer, washed by centrifugation 3 times, 50mM Tris-HCL buffer, resuspended in 1 OOiU (containing 1 • 2% NaCL, 0 • 5% BSA, 0.1% Tween 20), 4 ° C protected from light use.

[0018] 本发明所述包被有检测线和质控线的硝酸纤维素膜通过以下步骤制得: [0018] The present invention is coated with the test and control lines nitrocellulose membrane prepared by the following steps:

[0019] 步骤1:采用与结合垫上所用抗促甲状腺激素单克隆抗体细胞株不同的细胞株,采用标准的腹水生产工艺制备并纯化抗促甲状腺激素单克隆抗体,保存于-20°c备用; [0019] Step 1: Using the pad and binding with anti-TSH monoclonal antibody cell lines of different cell lines, prepared using standard production process ascites and purified anti-TSH monoclonal antibody stored at -20 ° c standby;

[0020] 步骤2:分别用包被稀释液将上述鼠源抗促甲状腺激素单克隆抗体和羊抗小鼠IgG 抗体调整浓度到l-3mg/ml,膜液量为0.5-3yl/Cm,将它们分别作为检测线和质控线平行喷洒于硝酸纤维素膜上进行包被,检测线和质控线间隔为3-7mm,然后置于烘箱中,37°C烘干2 小时。 [0020] Step 2: The solution is diluted with the package, respectively above the murine monoclonal anti-TSH antibody and the goat anti-mouse IgG antibody adjusted to a concentration of l-3mg / ml, an amount of a liquid film 0.5-3yl / Cm, the they are sprayed on nitrocellulose filters in parallel for a test and control line coating, test and control line spacing of 3-7mm, and then placed in an oven, 37 ° C and drying for 2 hours.

[0021] 本发明所述样品垫通过以下步骤制得:将玻璃纤维膜浸泡于含有1.0%1'1^1:〇11乂- 100,2 • 5 %BSA,0 • 15M TriS缓冲液,pH7 • 5的处理液中,于4°C浸泡4个小时,然后置于烘箱中,37 °C烘干2小时。 [0021] The sample pad of the present invention prepared by the following steps: the glass fiber membrane was immersed in 1.0% 1'1 ^ 1 comprising: 〇11 qe - 100,2 • 5% BSA, 0 • 15M TriS buffer, pH7 • 5 of the treatment liquid, soaked in 4 ° C 4 hours, and then placed in an oven, 37 ° C and drying for 2 hours.

[0022] 本发明还提供了一种如上所述试剂盒实现的促甲状腺激素制作方法,其特征在于包括以下步骤: [0022] The present invention further provides a method for manufacturing a TSH kit noted above to achieve, characterized by comprising the steps of:

[0023] 步骤i :将检测试剂及样本平衡至室温,取出试纸卡,平放; [0023] Step i: The reagents and samples equilibrated to room temperature, remove the test strip cards, flat;

[0024]步骤2:读卡:将1C卡放置在干式荧光免疫分析仪标注位置,读取相关信息,所述荧光免疫层析分析仪是一种光学检测系统,对促甲状腺激素的测定范围为0.3-120mn/mL; [0025] 步骤3:加样:血清/血浆:取1〇〇此血清/血浆样本垂直滴加至测试卡加样处;对于全血:取l5〇yL全血样本垂直滴加至测试卡加样处;取样时注意不要吸入气泡; [0024] Step 2: Reader: the card is placed in a dry 1C fluorescence immunoassay analyzer label location, read the relevant information, the fluorescent immunochromatographic analyzer is an optical detection system, the measurement range of TSH is 0.3-120mn / mL; [0025] step 3: loading: serum / plasma: take 1〇〇 this serum / plasma sample was added dropwise to a vertical test sample at Calgary; for whole blood: whole blood samples taken l5〇yL vertical added dropwise to the test sample at Calgary; also avoid inhaling air bubbles during sampling;

[0026]步骤4:检测,可采用自动测试或即时测试两种模式进行检测,自动测试:将测试卡插入干式荧光免疫分析仪的承载器上,按测试键,仪器将自动对测试卡进行扫描分析检测; 即时测试:测试卡室温放置20min后,插入干式荧光免疫分析仪的承载器上,点击即时测试。 [0026] Step 4: detecting, instant or automatic test can be used in two modes detection test, automatic test: the test card is inserted into the dry-type fluorescence immunoassay analyzer carrier, press the test button, the instrument will automatically test card scanning detection analysis; instant test: placing test card room temperature 20min, fluorescence is inserted into the dry immunoassay analyzer carrier, instant-test. [0027]本发明提供一种利用稀土羧基乳胶微球标记的荧光免疫层析技术制备的促甲状腺激素(TSH)测定试剂盒(荧光免疫层析法),同时适合血清、血浆和全血样本,并适合临床上单人份检测,相对于促甲状腺激素定性胶体金试剂,能定量检测样本中的促甲状腺激素含量,具有更明确的临床指导意义,具有操作简便、反应快速、灵敏度高、特异性强、适合^见场检测和经济实用等优点。 [0027] The present invention provides a TSH preparation fluorescence immunochromatography utilizing rare-carboxy-labeled latex beads (TSH) assay kit (fluorescence immunoassay chromatography), suitable for serum, plasma and whole blood samples, and for detection of single-clinical, with respect to the thyroid-stimulating hormone qualitative colloidal gold reagent, can quantitatively detect thyroid stimulating hormone content of the sample, have a more clear clinical significance, is simple, fast response, high sensitivity, specificity strong, ^ see for field detection and economical and practical advantages.

附图说明: BRIEF DESCRIPTION OF:

[0028]附图1是本发明中试纸卡的结构示意图。 [0028] Figure 1 is a schematic view of the test strip card according to the present invention.

[0029]附图2是本发明中实施例2的准确度分析结果示意图。 [0029] Figure 2 is the accuracy of Example 2 of the present invention in a schematic view of the analysis results.

[0030]附图3是本发明中实施例2的准确度分析结果示意图。 [0030] Figure 3 is the accuracy of Example 2 of the present invention in a schematic view of the analysis results.

[0031]附图4是本发明中实施例2的准确度分析结果示意图。 [0031] Figure 4 is the accuracy of Example 2 of the present invention in a schematic view of the analysis results.

[0032]附图标记:PVC板1、样品垫2、结合垫3、硝酸纤维素膜4、吸水垫5。 [0032] The reference numerals: PVC plates 1, 2 a sample pad, conjugate pad 3, a nitrocellulose membrane 4, an absorbent pad 5.

具体实施方式: Detailed ways:

[0033]下面结合附图和实施例对本发明作进一步的说明: Drawings and embodiments of the present invention will be further described [0033] below with:

[0034]如附图1所示,本发明首先提出了一种促甲状腺激素测定试剂盒,盒内设有试纸卡,所述试纸卡由下至上依次设有:PVC板1、样品垫2、结合垫3、硝酸纤维素膜4和吸水垫5, 其中结合垫上吸附有稀土荧光微球标记的抗促甲状腺激素单克隆抗体-微球偶联复合物, 所述稀土荧光微球的直径为150nm,稀土荧光微球含稀土镧系元素Eu3+,在基态下稳定,在337nm的激发光源作用下发射出波长615nm的荧光;所述单克隆抗体为纯化后混合的单克隆抗体,来源于针对2-6个不同的促甲状腺激素抗原表位的单克隆抗体细胞株。 [0034] As shown in Figure 1, the present invention first proposes a TSH assay kit, box with strip card, the paper card is provided by the bottom order: PVC plates 1, 2 a sample pad, 3 conjugate pad, a nitrocellulose membrane 4 and pad 5, wherein the rare earth-bound adsorption pad fluorescent microspheres labeled anti-TSH monoclonal antibody - conjugated microsphere composite, the microspheres rare earth phosphors diameter 150nm rare earth fluorescent microspheres containing rare earth lanthanide-Eu3 +, in a stable ground state, the fluorescence emission wavelength of 615nm in the excitation light source of 337nm action; the purified monoclonal antibody is a hybrid monoclonal antibodies derived against 2- six different epitopes of TSH monoclonal antibody cell line.

[0035]所述结合垫3的稀土荧光微球的直径优选是150nm;所述稀土荧光微球优选含有稀土镧系元素铕(Eu3+);结合垫上稀土荧光微球标记的抗体优选来源于针对2个不同抗原表位的单克隆细胞细胞株。 [0035] The diameter of the bonding pads 3 of a rare earth fluorescent microspheres is preferably 150 nm; preferably a rare earth phosphor containing a rare microspheres lanthanide europium (Eu3 +); rare earth pad fluorescent microspheres bound labeled antibody is preferably derived from 2 against different epitopes of monoclonal cell lines.

[0036] 实施例1: [0036] Example 1:

[0037] 促甲状腺激素测定试剂盒中试纸卡的各组成部分可以通过以下措施制得: [0037] TSH measured in each of the components of the kit may be obtained paper card manufactured by the following measures:

[0038] 1、样品垫2的制备: [0038] 1, 2 sample pad prepared:

[0039] 将玻璃纤维膜浸泡于含有l_0%Triton X-100,2.5%BSA,0.15M Tris缓冲液, PH7.5的处理液中,于4°C浸泡4个小时,然后置于烘箱中,37°C烘干2小时。 [0039] The film was immersed in a glass fiber containing l_0% Triton X-100,2.5% BSA, 0.15M Tris buffer solution treatment, the pH 7.5, soaked 4 hours at 4 ° C, and then placed in an oven, drying 37 ° C for 2 hours.

[0040] 2、吸附荧光微球标记抗体的结合垫3的制备: [0040] 2, adsorption fluorescent microspheres bound labeled antibody pad of Preparation 3:

[0041] 将玻璃纤维膜浸泡于150mM Tris-HCL处理液中(含1.0%Triton X-100,2.5% BSA,pH7.4),4°C浸泡2小时,然后取出37°C烘箱烘干4小时,备用,将玻璃纤维膜放在Bio-DotXYZ3050三维喷点平台上,用Bio-Jet Quanti300非接触式微定量喷头将稀土Eu3+荧光微球标记的抗促甲状腺激素单克隆抗体偶联复合物喷到玻璃纤维膜,37°C烘千1小时后制得。 [0041] The glass fiber membrane was immersed in 150mM Tris-HCL treatment solution (containing 1.0% Triton X-100,2.5% BSA, pH7.4), 4 ° C soak 2 hours, then remove 37 ° C oven dried 4 hours, standby, the glass fiber membrane on Bio-DotXYZ3050 three dots platform, with Bio-jet Quanti300 non-contact micro nozzle quantitative rare earth fluorescent microspheres Eu3 + labeled anti-TSH monoclonal antibody conjugate complex sprayed glass fiber membrane, 37 ° C 1 hour after drying to prepare one thousand. [0042] 稀土荧光纳米微球的醛基化:取5mg稀土荧光纳米微球,用20mM,pPi9.5的碳酸盐缓冲液,米用尚心法洗漆3遍,尚心速度为l2〇〇Orpm,时间为5分钟,最后重悬于lOOwl的上述碳酸盐缓冲液中,加入500U1醛基化的葡聚糖,混匀,室温下暗反应4小时,采用同样的离心法洗涤和重悬到100U1的上述碳酸盐缓冲液中,置于4°C备用; [0042] The aldehyde group of rare earth phosphors Nanospheres: Take 5mg nanosphere rare earth phosphors with 20mM, pPi9.5 carbonate buffer, washed rice with Heart paint still 3 times, the heart rate is still l2〇 〇Orpm, time was 5 minutes, and finally resuspended in the above carbonate buffer lOOwl was added 500U1 aldehyde dextran, mixing, dark and reacted for 4 hours at room temperature, washed with the same weight and centrifugation 100U1 suspended to the carbonate buffer and placed in 4 ° C for later use;

[0043] 稀土Eu3+荧光微球标记的抗促甲状腺激素单克隆抗体的制备:选取来自2个不同抗原表位的单克隆细胞细胞株的单克隆抗体,按照质量比1:1将2mg抗促甲状腺激素单克隆抗体用上述碳酸盐缓冲液于4°C透析过夜,然后与上述醛基化的稀土荧光微球混合,4-C反应过夜;然后,加入硼氢化钠至终浓度5mM,4°C反应4小时;再加入等体积的封闭液(50mM 1'1^-11〇^耵.4,含2%834,5%蔗撤,4°(:封闭过夜;然后用5011^1'1^-呢1^耵.4的缓冲液采用离心法洗涤3遍,重悬于1 OOul的50mM Tri s-HCL缓冲液中(含1 • 2 % NaCL,0.5 % BSA, 0.1%Tween 20),4°C避光保存备用。 [0043] The rare earth fluorescent microspheres Eu3 + labeled anti-thyroid stimulating hormone preparation of monoclonal antibodies: a monoclonal antibody selected from the monoclonal cell lines to two different epitopes, at a mass ratio of 1: 1 to 2mg of anti-thyroid stimulating hormones monoclonal antibodies with the above carbonate buffer, dialyzed overnight at 4 ° C, then mixed with rare earth phosphors aldehyde of the above microspheres, 4-C overnight; then, sodium borohydride was added to a final concentration of 5mM, 4 ° C for 4 hours; then a blocking solution was added an equal volume (5OmM -11〇 1'1 ^ .4 ^ Ding, containing 2% 834,5% cane withdrawal, 4 ° (: blocked overnight; followed by 5011 ^ 1'1 ^ - ^ 1 it Ding buffer .4 washed 3 times by centrifugation, resuspended in 50mM Tri s-HCL buffer, 1 OOul (including 1 • 2% NaCL, 0.5% BSA, 0.1% Tween 20), 4 ° C protected from light use.

[0044] 3、包被有检测线和质控线的硝酸纤维素膜4的制备: [0044] 3, was prepared with a package test and control lines nitrocellulose membrane 4:

[0045]采用与结合垫上所用抗促甲状腺激素单克隆抗体细胞株不同的细胞株,采用标准的腹水生产工艺制备并纯化抗促甲状腺激素单克隆抗体,保存于-20°C备用; [0045] The pad in conjunction with the anti-TSH monoclonal antibodies with different cell lines cell lines, prepared using standard production process ascites and purified monoclonal anti-TSH antibody and stored at -20 ° C for later use;

[0046] 分别用包被稀释液将上述鼠源抗促甲状腺激素单克隆抗体和羊抗小鼠IgG抗体调整浓度到2mg/ml,膜液量为2yl/cm,将它们分别作为检测线和质控线平行喷洒于硝酸纤维素膜上进行包被,检测线和质控线间隔为4mm,然后置于烘箱中,37°C烘干2小时。 [0046] The solution is diluted with the package, respectively above the murine anti-TSH monoclonal antibodies and goat anti-mouse IgG antibody adjusted to a concentration of 2mg / ml, an amount of a liquid film 2yl / cm, respectively, as the quality test line and spray control line parallel to the nitrocellulose membrane were coated, test and control line spacing of 4mm, and then placed in an oven, 37 ° C and drying for 2 hours.

[0047] 试纸卡的组装:在PVC板1上依次粘贴经过处理的样品垫2、吸附有稀土荧光标记的抗体的结合垫3、包被有检测线和质控线的硝酸纤维素膜4和吸水垫5,组装后得到试纸大板,按照要求切割成4mm宽,将试纸装入塑料卡内形成试纸卡。 Assembly [0047] The test strip card: 1 on the PVC sheet successively treated paste sample pad 2, is adsorbed fluorescent labeled antibody bound rare earth pad 3 coated with the test and control lines 4 and nitrocellulose membrane absorbent pad 5, after assembly to give a large paper sheet, cut into 4mm wide as required, the paper strip is formed is loaded within the card plastic card.

[0048]上述各步骤中选用的设备及原料优选以下原料: [0048] The equipment and raw materials in each of the above steps is preferably chosen the following ingredients:

[0049] 促甲状腺激素特异性配对抗体;促甲状腺激素质控品:英国朗道实验诊断有限公司;稀土荧光微球:上海甄准生物科技有限公司;硝酸纤维素(NC)膜:Millipore公司产品; 牛血清白蛋白(BSA),聚乙二醇roG20000,水解酪蛋白:Sigma产品,其它常用试剂均为分析纯试剂。 [0049] TSH antibody specific pairing; thyroid stimulating hormone control materials: UK Long Road laboratory diagnosis Ltd; rare earth fluorescent microspheres: Shanghai Zhen quasi Biotechnology Co., Ltd.; cellulose (NC) nitrate film: Millipore Company Products ; bovine serum albumin (BSA), polyethylene glycol roG20000, casein hydrolyzate: Sigma product, other conventional reagents were of analytical grade.

[0050] 实施例2:准确度试验 Accuracy test: [0050] Example 2

[0051] 选用上述试纸卡以及荧光免疫层析分析仪(型号:NE0_007),荧光免疫分析仪参数的设定:在荧光免疫分析仪上设定好试纸卡工艺参数后,取上述组装好的试纸卡,分别用0 • 4、4、10、20、40、80、100mIU/L的促甲状腺激素校准品,用试纸卡进行测定,得到各校准品的荧光强度值,将结果输入到分析仪的参数中,完成分析仪的参数的设定。 [0051] mentioned above are selected and a fluorescent immunochromatographic test strip card analyzer (Model: NE0_007), fluorescence immunoassay analyzer parameter configuration: paper card process parameters are set on a fluorescent immunoassay analyzer after taking the above-described assembled strip cards, respectively 0 • 4,4,10,20,40,80,100mIU / L TSH calibrators, measured by paper card, to obtain a fluorescence intensity value of each calibrator, the result is input to the analyzer parameters, the parameters are set to complete the analyzer.

[0052] 主要检测材料:临床样本由相关医院获得,共300份胶乳增强免疫比浊法定值样本,其中血清样本100份,血浆样本100份,全血样本100份,促甲状腺激素含量分布区间为0.3-120mIU/L 之间。 [0052] The main detection material: clinical samples obtained from a hospital or clinic, a total of 300 parts of a latex enhanced turbidimetric values ​​of the samples immune ratio, wherein 100 parts of serum samples, 100 parts of a plasma sample, 100 parts of a whole blood sample, thyroid stimulating hormone content distribution interval 0.3-120mIU / between L.

[0053] 检测方法: [0053] Test Method:

[0054] 步骤1:将检测试剂及样本平衡至室温,取出试纸卡,平放; [0054] Step 1: The reagents and samples equilibrated to room temperature, remove the test strip cards, flat;

[0055] 步骤2:读卡:将1C卡放置在干式荧光免疫分析仪标注位置,读取相关信息; [0055] Step 2: Reader: the card is placed in a dry 1C fluorescence immunoassay analyzer label position, reads the relevant information;

[0056] 步骤3:加样:血清/血浆:取100UL血清/血浆样本垂直滴加至测试卡加样处,对于全血样本,取150UL全血样本垂直滴加至测试卡加样处,取样时注意不要吸入气泡; [0056] Step 3: loading: Serum / Plasma: Take 100UL serum / plasma sample was added dropwise to a vertical test card with the sample, for a whole blood sample, whole blood samples taken perpendicular 150UL added dropwise to the test card with the sample, the sample Be careful not to inhale when the bubble;

[0057] 步骤4:检测:可采用自动测试或即时测试两种模式进行检测,其中自动测试:将测试卡插入干式荧光免疫分析仪的承载器上,按测试键,仪器将自动对测试卡进行扫描分析检测;即时测试:测试卡室温放置20min后,插入干式荧光免疫分析仪的承载器上,点击即时测试。 [0057] Step 4: Detection: immediate test or automatic test can be used in two modes is detected, wherein the automatic test: the test card is inserted into the dry fluorescence immunoassay analyzer carrier, press the test button, the instrument will automatically test card scanning analysis and detection; instant test: after the test card room temperature for 20min, fluorescence is inserted into the dry immunoassay analyzer carrier, instant-test.

[0058] 试验结果分析: [0058] Analysis of test results:

[0059] 临床样本检测试剂制备完成后,按检测方法对所有临床样本进行检测,并分析检测结果。 [0059] After the completion of the preparation of clinical samples detection reagent, the detection method by the detection of all clinical samples, and to analyze test results.

[0060]试验结果: [0060] Test Results:

[0061]如附图2所示,以实验系统的检测值为Y轴,以对照系统的测验值为X轴,绘制散点图,并进行相关性分析。 [0061] As shown in the drawings, for detecting the value of the Y-axis experimental system to test the value of the X-axis control system, plotted scattergram 2, and correlation analysis. 临床样本检测对300份临床定值样本检测,样本平均偏差值小于1〇%,最大偏差小于20%,R2>0.98,一致性系数>0.90。 Clinical samples of 300 parts of test sample in clinical detection value, the sample value is less than the mean deviation 1〇%, the maximum deviation is less than 20%, R2> 0.98, consistency factor> 0.90. 检测结果表明制备的检测试剂盒性能良好,适合用于临床检测,满足不同客户不同检测场合的差异化需要。 Test results show that good performance preparing test kit suitable for use in clinical testing, to meet the different needs of different customers different detection applications.

[0062] 本发明提供一种利用稀土荧光免疫层析技术制备的促甲状腺激素快速定量免疫层析检测试剂盒,同时适合血清、血浆和全血样本,并适合临床上单人份检测,相对于促甲状腺激素定性胶体金试剂,能定量检测样本中的促甲状腺激素含量,具有更明确的临床指导意义,具有操作简便、反应快速、灵敏度高、特异性强、适合现场检测和经济实用等优点。 [0062] The present invention provides a process for preparing rare earth fluorescence immunochromatography using thyrotropin rapid quantitative immunochromatographic assay kit, suitable for serum, plasma and whole blood samples and for detection of single-clinical, with respect to thyroid stimulating hormone qualitative colloidal gold reagent, can quantitatively detect thyroid stimulating hormone content of the sample, have a more clear clinical significance, is simple, fast response, high sensitivity and specificity, suitable for field testing and economical and practical advantages.

Claims (8)

1.一种促甲状腺激素测定试剂盒,设有试纸卡,其特征在于所述试纸卡由下至上依次设有:PVC板、样品垫、结合垫、硝酸纤维素膜和吸水垫,其中结合垫上吸附有稀土Eu3+荧光微球标记的抗促甲状腺激素单克隆抗体-微球偶联复合物,所述稀土荧光微球的直径为100 一250nm,稀土荧光微球含稀土镧系元素中的一种或几种,在基态下稳定,在300_400nm的激发光源作用下发射出波长范围为55〇_650nm的焚光;所述单克隆抗体为纯化后混合的单克隆抗体,来源于针对2_6个不同的促甲状腺激素抗原表位的单克隆抗体细胞株。 A TSH assay kit, with the card strip, characterized in that the strip is provided with cards successively from bottom to top: PVC plate, sample pad, conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the pad binding adsorption rare earth fluorescent microspheres Eu3 + labeled anti-TSH monoclonal antibody - conjugated microsphere composite, the microspheres diameter rare earth phosphors 100 a 250 nm, fluorescent microspheres containing rare earth lanthanide rare earth elements or more, in a stable ground state, emit light at an excitation wavelength range for the effect of burning 300_400nm 55〇_650nm of light; the monoclonal antibody is a monoclonal antibody mix purified, derived for different 2_6 thyroid stimulating hormone epitope monoclonal antibody cell lines.
2. 根据权利要求1所述的一种促甲状腺激素测定试剂盒,其特征在于所述结合垫的稀土焚光微球的直径是l2〇-200nm;结合塾上稀土荧光微球标记的抗体来源于针对2个不同抗原表位的单克隆细胞细胞株;结合垫上吸附有稀土Eu3+荧光微球标记的抗促甲状腺激素单克隆抗体-微球偶联复合物。 The claim requires a TSH assay kit of claim 1, wherein the diameter of said bonding pads of the light rare earth burning microspheres is l2〇-200nm; Sook antibody binding source of rare earth fluorescent microspheres labeled to for two different epitopes of the monoclonal cell lines; conjugate pad adsorbed with a rare earth fluorescent microspheres Eu3 + labeled anti-TSH monoclonal antibody - conjugated microsphere composite.
3. 根据权利要求1所述的一种促甲状腺激素测定试剂盒,其特征在于稀土荧光微球的直径为150nm,稀土荧光微球含稀土镧系元素Eu3+,在基态下稳定,在337nm的激发光源作用下发射出波长615nm的荧光。 3. The TSH assay kit according to claim 1, characterized in that the diameter of the rare earth phosphors microspheres 150nm, fluorescent microspheres containing a rare earth lanthanide rare earth Eu3 +, stable ground state, the excited at 337nm under the action of light emitted fluorescence wavelength of 615nm.
4. 根据权利要求1所述的一种促甲状腺激素测定试剂盒,其特征在于所述结合垫的稀土荧光微球的直径是200nm;所述稀土荧光微球掺杂有稀土镧系元素Eu3+。 4. According to a TSH assay kit according to claim 1, wherein said bonding pad has a diameter microspheres rare earth phosphors is 200 nm; fluorescent beads of the rare earth lanthanide rare earth-doped Eu3 +.
5.根据权利要求1所述的一种促甲状腺激素测定试剂盒,其特征在于所述结合垫采用如下步骤制得:将玻璃纤维膜浸泡于150mM Tris-HCL处理液中,Tris-HCL处理液含1.0% Tri ton X-100,2.5 % BSA,pH7.4,4°C浸泡2小时,然后取出37°C烘箱烘干4小时,备用,将玻璃纤维膜放在Bio_DotXYZ3050三维喷点平台上,用Bio-Jet Quanti300非接触式微定量喷头将稀土Eu3+荧光微球标记的抗促甲状腺激素单克隆抗体偶联复合物喷到玻璃纤维膜,37 °C烘千1小时后制得。 According to claim one kind TSH assay kit of claim 1, wherein said bonding pads prepared using the following procedure: a glass fiber membrane was immersed in 150mM Tris-HCL treatment solution, Tris-HCL treatment liquid containing 1.0% Tri ton X-100,2.5% BSA, pH7.4,4 ° C soak 2 hours, then remove 37 ° C drying oven for four hours, the standby, the glass fiber membrane on the three-dimensional dots Bio_DotXYZ3050 platform, Bio-jet Quanti300 with a non-contact micro nozzle quantitative rare earth fluorescent microspheres Eu3 + labeled anti-TSH monoclonal antibody conjugate complexes sprayed onto glass fiber membrane, 37 ° C 1 hour after drying to prepare one thousand.
6.根据权利要求1所述的一种促甲状腺激素测定试剂盒,其特征在于结合垫上的所述稀土荧光微球标记的促甲状腺激素单克隆抗体采用如下步骤制得: 步骤1:单克隆抗体细胞株的获得:用促甲状腺激素纯品免疫小鼠,采用标准的单克隆抗体制备方法制备特异性高亲和力的单克隆抗体细胞株,对所获得的单抗细胞株进行配对筛选,根据配对结果和亲和力数据优选出用于试剂盒的单抗细胞株; 步骤2:单克隆抗体的制备:采用标准的腹水生产工艺制备并纯化促甲状腺激素单克隆抗体,分装后保存于-20°C备用; 步骤3:稀土荧光微球的醛基化:取5mg稀土荧光微球,用20mM,pH9.5的碳酸盐缓冲液, 采用离心法洗涤3遍,离心速度为12000rpm,时间为5分钟,最后重悬于100ul的上述碳酸盐缓冲液中,加入500yl醛基化的葡聚糖,混匀,室温下暗反应4小时,采用同样的离心法洗涤 6. According to a TSH assay kit according to claim 1, wherein the rare earth pad binding fluorescent beads labeled TSH monoclonal antibody was prepared using the following procedure: Step 1: monoclonal antibody cell lines obtained: the preparation of specifically with high affinity TSH pure immunized mice using standard monoclonal antibody preparation method a monoclonal antibody cell lines, of the obtained monoclonal cell lines screened pair, according to the result of pairing and affinity data for the kit is preferably a monoclonal cell line; step 2: preparation of monoclonal antibodies: production process of preparing ascites and purified using standard TSH monoclonal antibodies, aliquots stored at -20 ° C standby ; step 3: rare earth phosphors microspheres aldehydized: take 5mg rare earth fluorescent microspheres, carbonate buffer 20mM, pH9.5, and washed by centrifugation 3 times, centrifugal speed of 12000 rpm, for 5 minutes. finally resuspended in 100ul of the aforementioned carbonate buffer were added 500yl aldehyde dextran, mixing, dark and reacted for 4 hours at room temperature, washed with the same centrifugation 重悬到lOOyl的上述碳酸盐缓冲液中,置于4°C备用; 步骤4:稀土荧光微球标记的促甲状腺激素单克隆抗体的制备:选取来自2个不同抗原表位的单克隆细胞细胞株的单克隆抗体,按照质量比1:1将2mg促甲状腺激素单克隆抗体用上述碳酸盐缓冲液于4°C透析过夜,然后与上述醛基化的稀土荧光微球混合,4°C反应过夜; 然后,加入硼氢化钠至终浓度5mM,4°C反应4小时;再加入等体积的封闭液(50mM Tris-HCL, pH7.4,含2%BSA,5%蔗撤,4°C封闭过夜;然后用50mM Tris-HCL,pH7.4的缓冲液采用离心法洗涤3遍,重悬于1 OOul 的50mM Tr i s-HCL缓冲液中(含1 • 2 °乂NaCL,0 • 5 % BSA,0 • 1 % Tween 20),4 °C避光保存备用。 Above and resuspended in carbonate buffer lOOyl, placed in 4 ° C standby; Step 4: Preparation of rare earth fluorescent microspheres labeled TSH monoclonal antibodies: selecting from two different monoclonal cell epitopes monoclonal antibody cell lines, a mass ratio of 1: 1 to 2mg TSH monoclonal antibodies with the above carbonate buffer, dialyzed overnight at 4 ° C, then mixed with rare earth phosphors aldehyde of the above microspheres, 4 ° C overnight; then, sodium borohydride was added to a final concentration of 5mM, 4 ° C for 4 hours; then the addition of an equal volume of blocking solution (50mM Tris-HCL, pH7.4, containing 2% BSA, 5% sucrose withdrawal, 4 blocked overnight ° C; then with 50mM Tris-HCL, pH7.4 buffer using centrifugation and washed 3 times, resuspended in 50mM Tr i s-HCL buffer, 1 OOul of (1 • 2 ° qe containing NaCL, 0 • 5% BSA, 0 • 1% Tween 20), 4 ° C protected from light use.
7. 根据权利要求1所述的一种促甲状腺激素测定试剂盒,其特征在于所述包被有检测线和质控线的硝酸纤维素膜通过以下步骤制得: 步骤1:采用与结合垫上所用促甲状腺激素单克隆抗体细胞株不同的细胞株,采用标准的腹水生产工艺制备并纯化促甲状腺激素单克隆抗体,保存于-2(TC备用; 步骤2:分别用包被稀释液将上述鼠源抗促甲状腺激素单克隆抗体和羊抗小鼠IgG抗体调整浓度到2mg/tnl,膜液量为2ul/cm,将它们分别作为检测线和质控线平行喷洒于硝酸纤维素膜上进行包被,检测线和质控线间隔为4mm,然后置于烘箱中,37°C烘干2小时。 7. According to a TSH assay kit according to claim 1, wherein said coated with test and control lines nitrocellulose membrane prepared by the following steps: Step 1: using the pad binding as used TSH monoclonal antibody cell lines of different cell lines, prepared using standard production process ascites and purified TSH monoclonal antibody stored at -2 (TC standby; step 2: the solution is diluted with the package, respectively above the murine anti TSH monoclonal antibodies and goat anti-mouse IgG antibody adjusted to a concentration of 2mg / tnl, the liquid film amount 2ul / cm, they were sprayed on nitrocellulose filters as parallel test and control lines for packet is, test and control line spacing of 4mm, and then placed in an oven, 37 ° C and drying for 2 hours.
8. 根据权利要求1所述的一种促甲状腺激素测定试剂盒,其特征在于所述样品垫通过以下步骤制得:将玻璃纤维膜浸泡于含有1 • 〇%Triton X-100,2• 5%BSA,0• 15M Tris缓冲液,PH7.5的处理液中,于4。 8. According to a TSH assay kit according to claim 1, wherein said sample pad prepared by the following steps: the glass fiber membrane was immersed in 1 • square containing% Triton X-100,2 • 5 % BSA, 0 • 15M Tris buffer, PH7.5 in the treatment liquid, at 4. (:浸泡4个小时,然后置于烘箱中,37。(:烘干2小时。 (: Soak 4 hours, then placed in an oven at 37 (: drying for 2 hours.
CN201611162058.1A 2016-12-15 2016-12-15 Kit for determining thyroid stimulating hormone and fabrication method CN106771264A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611162058.1A CN106771264A (en) 2016-12-15 2016-12-15 Kit for determining thyroid stimulating hormone and fabrication method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611162058.1A CN106771264A (en) 2016-12-15 2016-12-15 Kit for determining thyroid stimulating hormone and fabrication method

Publications (1)

Publication Number Publication Date
CN106771264A true CN106771264A (en) 2017-05-31

Family

ID=58892634

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611162058.1A CN106771264A (en) 2016-12-15 2016-12-15 Kit for determining thyroid stimulating hormone and fabrication method

Country Status (1)

Country Link
CN (1) CN106771264A (en)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136549A (en) * 1999-10-15 2000-10-24 Feistel; Christopher C. systems and methods for performing magnetic chromatography assays
JP2009115822A (en) * 2009-02-23 2009-05-28 Furukawa Electric Co Ltd:The Label silica nano-particle for immuno-chromatography reagent, immuno-chromatography reagent, test strip for immuno-chromatography using it, and fluorescence detection system for immuno-chromatography
CN102890155A (en) * 2012-09-12 2013-01-23 暨南大学 Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip
CN202794191U (en) * 2012-08-07 2013-03-13 天津中新科炬生物制药有限公司 Thyroid stimulating hormone (TSH) quick quantitative immunochromatographic detection kit
CN103172941A (en) * 2011-12-26 2013-06-26 苏州新波生物技术有限公司 Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof
CN104714033A (en) * 2014-11-28 2015-06-17 威海纽普生物技术有限公司 Procalcitonin detection kit and detection method
CN104730245A (en) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 D-dimer detection kit and D-dimer detection method
CN104880414A (en) * 2015-05-27 2015-09-02 广州华弘生物科技有限公司 TSH immunochromatography kit and manufacturing method thereof
CN105717303A (en) * 2016-01-29 2016-06-29 山东康力医疗器械科技有限公司 Method and reagent kit for detecting phosphatidylinositol proteoglycan 3 with fluorescence immunochromatographic method

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136549A (en) * 1999-10-15 2000-10-24 Feistel; Christopher C. systems and methods for performing magnetic chromatography assays
JP2009115822A (en) * 2009-02-23 2009-05-28 Furukawa Electric Co Ltd:The Label silica nano-particle for immuno-chromatography reagent, immuno-chromatography reagent, test strip for immuno-chromatography using it, and fluorescence detection system for immuno-chromatography
CN103172941A (en) * 2011-12-26 2013-06-26 苏州新波生物技术有限公司 Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof
CN202794191U (en) * 2012-08-07 2013-03-13 天津中新科炬生物制药有限公司 Thyroid stimulating hormone (TSH) quick quantitative immunochromatographic detection kit
CN102890155A (en) * 2012-09-12 2013-01-23 暨南大学 Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip
CN104714033A (en) * 2014-11-28 2015-06-17 威海纽普生物技术有限公司 Procalcitonin detection kit and detection method
CN104730245A (en) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 D-dimer detection kit and D-dimer detection method
CN104880414A (en) * 2015-05-27 2015-09-02 广州华弘生物科技有限公司 TSH immunochromatography kit and manufacturing method thereof
CN105717303A (en) * 2016-01-29 2016-06-29 山东康力医疗器械科技有限公司 Method and reagent kit for detecting phosphatidylinositol proteoglycan 3 with fluorescence immunochromatographic method

Similar Documents

Publication Publication Date Title
Soukka et al. Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology
AU614109B2 (en) Test method and reagent kit therefor
US4659678A (en) Immunoassay of antigens
CN100464189C (en) Magnetic separating direct chemical illuminating reagent and testing method using the same reagent
EP0427984B2 (en) Articles for use in enzyme immuno-assay techniques, and methods of making and using such articles
JP3535158B2 (en) The method for improving measurement accuracy in depleting wave optical biosensor analysis
CN1945331B (en) Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds
US9594078B2 (en) Chromatographic assay system
CN101377490B (en) Magnetic microparticle separating chemiluminescence immune analysis determination reagent kit for detecting related sign object and preparing method thereof
NL8702776A (en) Method for determining antigens by means of two monoclonal antibodies.
EP0399184A3 (en) Reagents, methods and kits for an amphetamine-class fluorescence polarization immunoassay
CN102192983B (en) Time-resolved immunoflourometric chromatographic test strip for quantitative detection as well as preparation method and application thereof
CN101377491A (en) Magnetic granule competing method chemiluminescence immune analysis determination reagent kit for detecting hormone and preparing method thereof
WO1985000663A1 (en) Immunometric assay using polyclonal and monoclonal antibodies and a kit for use therein
CN103901203B (en) A primal quantitative chemiluminescence detection kit and a detection method and a preparation method calcitonin
CN102879559B (en) Now a time-resolved fluorescence quantitative detection reagents and methods immunochromatographic
CN103048460B (en) Method for detecting by using quantum dot fluorescence immunochromatographic test strips
JP4236629B2 (en) Sandwich assay test to determine the NT-proBNP
Gribnau et al. Particle-labelled immunoassays: a review
EP1448990B1 (en) Particle-based ligand assay with extended dynamic range
CN102323422A (en) Immunochromatographic test strip for semi-quantitatively and simultaneously detecting cTnI and Myo and preparation method thereof
KR920000057B1 (en) Process and reagent for the determination of a specifically bindable substance
CN101493460B (en) Method for producing fluorescent microballoons immune chromatography test paper stripe and quantitative determination method
CN104897901A (en) Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4
CN102520192A (en) Fluorescence immunochromatographic assay and kit for quantitative detection of troponin I/creatine kinase isoenzyme/myohemoglobin

Legal Events

Date Code Title Description
PB01
SE01