New! View global litigation for patent families

CN1271409C - Raid quantitative determination of cardiac muscle troponin I by three-anti method - Google Patents

Raid quantitative determination of cardiac muscle troponin I by three-anti method Download PDF

Info

Publication number
CN1271409C
CN1271409C CN 03131931 CN03131931A CN1271409C CN 1271409 C CN1271409 C CN 1271409C CN 03131931 CN03131931 CN 03131931 CN 03131931 A CN03131931 A CN 03131931A CN 1271409 C CN1271409 C CN 1271409C
Authority
CN
Grant status
Grant
Patent type
Prior art keywords
troponin
quantitative
method
determination
anti
Prior art date
Application number
CN 03131931
Other languages
Chinese (zh)
Other versions
CN1470875A (en )
Inventor
苏恩本
Original Assignee
南京医科大学第一附属医院
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
Grant date

Links

Abstract

本发明专利涉及一种新的根据酶联免疫原理建立的快速定量测定人体外周血或组织心肌肌钙蛋白I含量的方法。 The present invention patent relates to establishing a new rapid quantitative enzyme immunoassay according to the principle of human peripheral blood or a tissue levels of cardiac troponin I assay. 因心肌肌钙蛋白I在血中含量极微,目前普通酶联法测定的灵敏度较低,最低检测灵敏度约为0.2ng/ml,很难达到0.1ng/ml以下,测定速度也较慢,需1小时以上。 Due to cardiac troponin I content in blood is minimal, current low sensitivity of the assay ELISA kits general, the minimum detection sensitivity is about 0.2ng / ml, difficult to achieve 0.1ng / ml or less, the measurement speed is slower, need 1 hour or more. 为提高测定速度及测定灵敏度,本发明成功研制出三抗法心肌肌钙蛋白I快速定量测定试剂盒,用二株单克隆抗体替代传统方法中的一株单克隆抗体为捕获抗体,提高了抗体捕获能力,同时结合链亲和素-生物素放大系统,有效地缩短了检测时间,提高了测定灵敏度,使在普通酶联仪等仪器上快速定量检测心肌肌钙蛋白I能达到检验与临床要求。 To improve the measurement speed and measurement sensitivity, the present invention successfully developed a method of three anti-cardiac troponin I rapid quantitative assay kit, an alternative to the monoclonal antibody is a monoclonal antibody using conventional methods for the capture antibody two, antibody increased capturing ability, while binding streptavidin - biotin amplification system, effectively shortening the detection time, improved measurement sensitivity, so that the quantitative analysis of cardiac troponin in normal enzyme-linked and other equipment inspection and I can reach the clinical requirements .

Description

三抗法快速定量测定心肌肌钙蛋白I Rapid quantitative determination of three anti-cardiac troponin I

技术领域 FIELD

本发明专利涉及一种新的根据酶联免疫原理建立的快速定量测定人体外周血或组织心肌肌钙蛋白I含量的方法。 The present invention patent relates to establishing a new rapid quantitative enzyme immunoassay according to the principle of human peripheral blood or a tissue levels of cardiac troponin I assay.

背景技术 Background technique

心肌缺血性损伤,特别是急性心肌梗塞(AMI)是威协人类生命的主要疾病之一,临床化学家和临床科学家一直努力寻找和探索特异性好、灵敏度高的血清指标。 Myocardial ischemic injury, particularly acute myocardial infarction (AMI) is one of the major diseases that threaten human life, clinical chemists and clinical scientists have been trying to find and explore good specificity, high sensitivity of serum markers. 心肌肌钙蛋白I(cardiac troponin I,cTnI)具有高度的心肌特异性,当心肌细胞受损时,血液中cTnI出现时间早,持续时间长,与心肌损伤程度及预后密切相关。 Cardiac troponin I (cardiac troponin I, cTnI) is highly cardiac specific, when the damaged myocardial cells, blood cTnI appear earlier, lasted for a long time, closely related to the extent of myocardial injury and prognosis. 因此,它可作为心肌损伤的一种特异性指标,在心脏病的诊断及预后判断中具有十分重要的意义。 Therefore, it can be used as a specific marker of myocardial injury, it is of great importance in the diagnosis and prognosis of heart disease.

心肌肌钙蛋白I测定可分为定性与定量测定两种方法,定性测定主要运用层析原理,制作试纸片完成,目前已开展临床应用,其优点是速度快、操作方便,但其缺点是仅能显示阳性、阴性结果,无具体数值,不能观察其动态变化和根据数值判断梗死时间、面积及预后,且敏感性低,易受多种因素干扰,即使改进的半定量小型仪器测定方法同样也有上述缺点。 Determination of cardiac troponin I can be divided into two qualitative and quantitative determination, the main use of chromatographic qualitative measurement principle, the production is completed dipstick, clinical application has been carried out, the advantage of fast, easy to operate, but its drawback is only to show a positive, negative results, no specific values ​​can not be observed and its dynamic changes according to the value of infarct time, area and prognosis, and low sensitivity to a variety of factors susceptible to interference, even if an improved method of semi-quantitative determination of small instruments also have the above-mentioned drawbacks. 定量测定主要依据酶联免疫与化学发光原理设计,绝大部分运用了二株单克隆抗体(少部分仅用一株单克隆抗体或其中一株用多克隆抗体),一株单克隆抗体为捕获抗体,另一株单克隆抗体为标记抗体,形成酶联夹心法测定。 Quantified mainly based chemiluminescent enzyme immunoassay principles of design, most of the use of two monoclonal antibodies (only a small portion of which a monoclonal antibody or polyclonal antibody), a monoclonal antibody to capture antibody, the other was labeled antibody is a monoclonal antibody, forming a sandwich enzyme-linked assay. 测定中依靠与辅助的仪器有自动化与非自动化的化学发光仪、荧光测定仪及酶联仪等,其中酶联仪价格低,在数千元至数万元之间,国内大中小型医院较为普及,而化学发光仪与荧光测定仪等价格昂贵,数十万元至数佰万元,国外已有数家大公司生产大型自动化化学发光或荧光仪完成定量测定,但其价格昂贵,且需配套试剂,国内大部分医院尚难以推广应用。 Assays rely on auxiliary instruments have automated and non-automated chemiluminescence, fluorescence analyzer and enzyme-linked instrument, in which low enzyme-linked instrument price, between a few thousand dollars to tens of thousands of domestic large and small hospitals more popular, and chemiluminescent analyzer with fluorescence detector, and so expensive, several hundred thousand dollars to several million Bai, abroad several large companies producing large-scale automated chemiluminescence or fluorescence analyzer to complete the quantitative determination, but its price is expensive, and need support reagents, most domestic hospitals still difficult application. 故依靠酶联仪建立心肌肌钙蛋白I定量酶联法测定在国内非常必要,但普通酶联法建立的心肌肌钙蛋白I测定的灵敏度较低,最低检测灵敏度约为0.2ng/ml,很难达到0.1ng/ml以下,测定速度也较慢,需1小时以上。 So the determination of the domestic device is necessary to rely on enzyme-linked establish quantitative ELISA kits cardiac troponin I, but lower sensitivity to establish normal ELISA kits cardiac troponin I, the minimum detection sensitivity was about 0.2ng / ml, was inaccessibility 0.1ng / ml or less, measured speed is slow, takes more than one hour.

发明内容 SUMMARY

因心肌肌钙蛋白I在血中含量极微,为提高测定速度及测定灵敏度,本发明成功研制出三抗法心肌肌钙蛋白I快速定量测定试剂盒,用二株单克隆抗体替代传统方法中的一株单克隆抗体为捕获抗体,提高了抗体捕获能力,同时结合链亲和素-生物素放大系统,有效地缩短了检测时间,提高了测定灵敏度,使在普通酶联仪等仪器上快速定量检测心肌肌钙蛋白I达到检验与临床要求。 Minimal due to cardiac troponin I content in blood, in order to improve measurement sensitivity and the measurement speed, the present invention successfully developed a method of three anti-cardiac troponin I rapid quantitative assay kit, with two alternative to traditional methods of monoclonal antibodies the capture antibody is a monoclonal antibody, to improve the ability to capture antibody, while binding streptavidin - biotin amplification system, effectively shortening the detection time, improved measurement sensitivity, so that in the normal rapid enzyme-linked and other equipment quantitative detection of cardiac troponin I reaches inspection and clinical requirements.

本发明技术方案:二株作用于心肌肌钙蛋白I的单克隆抗体为捕获抗体,简称为Ab1+Ab2,联结生物素或长链生物素后,简称为Ab1-Bio+Ab2-Bio。 Aspect of the present invention: the role of two monoclonal antibodies to cardiac troponin I capture antibody, referred to as Ab1 + Ab2, coupled to biotin or biotin long chain, referred to as Ab1-Bio + Ab2-Bio. 另一株作用于心肌肌钙蛋白I的单克隆抗体为标记抗体,简称为Ab3,联结辣根过氧化物酶后,简称为Ab3-POD,联结生物素或长链生物素后,简称为Ab3-Bio。 The other was applied to monoclonal antibodies to cardiac troponin I labeled antibody, referred to as Ab3, the coupling horseradish peroxidase, referred to as Ab3-POD, coupled biotin or biotin long chain, referred to as Ab3 -Bio. Ab1、Ab2、Ab3要求作用于cTnI抗原的不同位点,Ab1+Ab2与Ab3配对后对cTnI抗原有较好的结合力及较高的特异性,单克隆抗体及其它原料可来源于合格商用产品(如Sigma,Roch公司等)。 AbI, Ab2, Ab3 claim cTnI acting on different sites of the antigen, Ab1 + Ab2 and Ab3 after pairing has good adhesion and high antigen specificity for cTnI, monoclonal antibodies, and other materials may be derived from commercial products qualifying (e.g., Sigma, Roch companies, etc.). 运用酶联免疫原理其反应途径具体包括如下几种方案:方案一:(1).96孔酶联板、酶联条或其它固项支持物联结好链亲和素或亲和素(简称为S),白蛋白或其它封闭液封闭。 Application of the principle of enzyme immunoassay which comprises the following reaction pathway several options: Option one: (1) .96 holes ELISA plate, bar or other solid items enzyme-linked support good coupling streptavidin or avidin (hereinafter referred to as S), albumin or other blocking solution.

(2).Ab1-Bio+Ab2-Bio与S结合,心肌肌钙蛋白I抗原(包括已知的心肌肌钙蛋白I标准品、质控品与待测样品中的心肌肌钙蛋白I,简称为cTnI)与Ab1-Bio+Ab2-Bio结合,cTnI与Ab3-POD结合。 (2) .Ab1-Bio + Ab2-Bio combined with S, cardiac troponin I antigen (including known cardiac troponin I standards, quality control materials with a test sample of cardiac troponin I, Acronym of cTnI) Ab1-Bio + Ab2-Bio binding, cTnI and Ab3-POD binding. 先后形成S~Ab1-Bio+Ab2-Bio~cTnI~Ab3-POD反应链。 Successively forming S ~ Ab1-Bio + Ab2-Bio ~ cTnI ~ Ab3-POD reaction chain.

(3).POD使显色底物(如四甲基联苯胺TMB等)显色或化学发光底物发光,再通过相应仪器测定,根据标准曲线计算待测样品心肌肌钙蛋白I含量。 (3) .POD that the chromogenic substrate (such as tetramethyl benzidine TMB etc.) chromogenic or chemiluminescent substrate to emit light, and then through respective measuring instruments, calculating cardiac troponin I content of the sample to be tested according to the standard curve.

方案二:(1).96孔酶联板、酶联条或其它固项支持物联结好Ab1+Ab2,白蛋白或其它封闭液封闭。 Option II: (1) .96 holes ELISA plate, bar or other solid items enzyme-linked support links good Ab1 + Ab2, albumin or other blocking solution.

(2).Ab1+Ab2与cTnI结合,cTnI与Ab3-POD结合,先后形成Ab1+Ab2~cTnI~Ab3-POD反应链。 (2) .Ab1 + Ab2 binding and cTnI, cTnI and Ab3-POD binding, it has formed Ab1 + Ab2 ~ cTnI ~ Ab3-POD reaction chain.

(3).同方案一中(3)。 (3). In a different embodiment (3).

方案三:(1).同方案二中1。 Program III: (1) 1 with Scheme II.

(2).Ab1+Ab2与cTnI结合,cTnI与Ab3-Bio结合,Ab3-Bio与链亲和素辣根过氧化物酶(简称S-POD)结合,先后形成Ab1+Ab2~cTnI~Ab3-Bio~S-POD反应链。 (2) .Ab1 + Ab2 binding and cTnI, cTnI and binding Ab3-Bio, Ab3-Bio with streptavidin horseradish peroxidase (abbreviated S-POD) binding, has formed Ab1 + Ab2 ~ cTnI ~ Ab3- Bio ~ S-POD reaction chain.

(3).同方案一中(3)。 (3). In a different embodiment (3).

经上述创新后,本法测定时间可缩短至35分钟左右,其最低检测灵敏度为0.05ng/ml,等同或超过国外同类试剂盒水平,且仅需普通酶联仪即能测定,快速出具检测结果,能灵活分次用于急诊与常规不同病例数的检测,更加方便了临床应用,适合于大、中、小型医院广泛推广应用,这对进一步提高国内心血管病诊断水平有不可估量的推动作用。 After the above innovation, applied to the determination time can be shortened to about 35 minutes, the minimum detection sensitivity was 0.05ng / ml, equal or exceed the level of similar foreign kit, and that can only ordinary enzyme-linked assay analyzer, rapid detection result issued , can be flexibly divided for the detection of different emergency and regular number of cases, more convenient clinical application, suitable for large, widely promoted in small hospitals application, which is immeasurable boost to further improve the domestic level of cardiovascular disease diagnosis .

具体实施方式 detailed description

按上述三种方案途径具体实施方式为:方案一:(1).96孔酶联板或酶联条联结好链亲和素,0.5~1%白蛋白或其它封闭液封闭好备用。 DETAILED above three schemes way as Embodiment: a program: (1) .96 holes or enzyme-linked ELISA plate strip a good coupling streptavidin, 0.5 to 1% albumin or other well blocking solution for use.

(2).加入一定量的Ab1+Ab2-Bio,37℃孵育一定时间(优选60分钟)或4℃过夜,密封后包装在试剂盒内,样本测定时打开应用。 (2) adding an amount of Ab1 + Ab2-Bio, 37 ℃ incubated for a predetermined time (preferably 60 minutes) or overnight at 4 ℃, sealed packaged within the kit, when applied to open the sample measurement.

(3).测定时孔内已知的心肌肌钙蛋白I标准品或待测样品(已知的心肌肌钙蛋白I标准品也可预先冷冻在设置的标准孔内,检测时加标准稀释液溶解即可),再加入一定量的Ab3-POD反应液,轻振后37℃孵育一定时间(优选20分钟)。 (3) When the hole was measured cardiac troponin I known standard or sample to be tested (of known cardiac troponin I standards may also be pre-frozen in a standard bore set, the standard dilution plus detection be dissolved), and then adding an amount of Ab3-POD reaction liquid was incubated at 37 ℃ light transducer a predetermined time (preferably 20 minutes).

(4).倾去孔内液体,洗液(PBS,含0.05%Tween-20)洗数次,每次每孔加洗液量最适为250~300μl。 (4) Pour hole liquid, wash (PBS, containing 0.05% Tween-20) and washed several times, each wash added to each well optimal amount of 250 ~ 300μl.

(5).加入显色液四甲基联苯胺(TMB),37℃孵育一定时间(优选15分钟)。 (5) color reagent was added tetramethylbenzidine (TMB), 37 [deg.] C incubation predetermined time (preferably 15 minutes).

(6).每孔加终止液(0.5~2M H2SO4)50μl或滴注一滴,轻轻振匀10分钟内酶联仪450nm阅读OD值。 (6) Each well of stop solution (0.5 ~ 2M H2SO4) 50μl drop or infusion, gently homogenized in 10 minutes enzyme-linked vibration meter reading OD 450nm values.

(7).根据标准曲线计算待测样品心肌肌钙蛋白I含量。 (7) cardiac troponin I content of the sample to be tested is calculated from the standard curve.

方案二:(1).96孔酶联板或酶联条内加入一定量的Ab1与Ab2预包被,37℃孵育一定时间(优选60分钟)或4℃过夜,0.5~1%白蛋白或其它封闭液封闭好备用。 Option II: (1) .96 bore or enzyme-linked ELISA plate section adding an amount of Ab1 and Ab2 is precoated, certain incubation time 37 [deg.] C (preferably 60 minutes) or overnight at 4 ℃, or 0.5 to 1% albumin other alternate blocking solution well.

(2).以下步骤同方案一中(3)至(7)。 (2) a step with the following scheme (3) to (7).

方案三:(1).同方案二中(1)。 Scheme III: (1) the same scheme II (1).

(2).测定时孔内已知的心肌肌钙蛋白I标准品或待测样品(已知的心肌肌钙蛋白I标准品也可预先冷冻在设置的标准孔内,检测时加标准稀释液溶解即可),再加入一定量的Ab3与S-POD的混合反应液,轻振后37℃孵育一定时间(优选20分钟)。 (2) measurement of cardiac troponin I known hole standard or sample to be tested (of known cardiac troponin I standards may also be pre-frozen in a standard bore set, the standard dilution plus detection be dissolved), then added to the reaction mixture an amount of Ab3 and S-POD solution and incubated at 37 ℃ light transducer a predetermined time (preferably 20 minutes).

(3).以下步骤同方案一中(3)至(7)。 (3). In a different embodiment the step (3) to (7).

上述所述方法中反应液PH在6.5~7.5之间,优选于6.8~7.2。 The above-described method, the reaction solution between 6.5 PH 7.5, preferably at 6.8 to 7.2. 所用洗液PH6.5~7.5,优选6.8~7.2;温度在2~37℃,优选低于15℃;洗液洗3~6次,优选洗5次。 As used wash PH6.5 ~ 7.5, preferably 6.8 to 7.2; temperature 2 ~ 37 ℃, preferably less than 15 deg.] C; ~ 6 washings were washed 3 times, preferably 5 times washed.

按方案一加工的三抗法心肌肌钙蛋白I快速定量测定试剂盒组成及操作说明书:试剂盒组成1、酶联架1块。 According to the program processing method of the three anti-cardiac troponin I assay kit rapid quantitative composition and operating instructions: kit 1, an enzyme-linked carrier.

2、酶联条12条,其中标准条6条,样品条6条(试用品标准条与样品条各为2条)。 2, ELISA strip 12 wherein a standard strip 6, 6 sample strip (standard strip and the free sample strips of each sample is 2). 标准条第一孔为空白对照,2~6孔为标准点(心肌肌钙蛋白I浓度分别为3.2、1.6、0.8、0.4、0.2ng/ml,已冷冻干燥在孔内),第7孔为样品孔,可用作急诊或常规病例检测。 Standard blank of first holes, the hole 2 to 6 as a standard point (cardiac troponin I concentration were 3.2,1.6,0.8,0.4,0.2ng / ml, was lyophilized in the hole), hole 7 sample well, or can be used as a conventional emergency case detection. 样品条第2~7孔为检测样品孔。 Article Sample 2-7 to detect a sample well aperture. 标准条与样品条数量比例可根据用户需要特别订购,仍以测定孔数目为据,不另收费用。 Article number of standard sample strip ratio can special order according to user needs, the number of holes is still measured data, no additional cost.

3、标准稀释液10ml(白瓶蓝盖)。 3, 10ml standard dilutions (blue cap bottles of white).

4、4.洗液200ml(大瓶)。 4,4. Lotion 200ml (large bottle).

5、反应液10ml(白瓶红盖)。 5, the reaction solution 10ml (bottles of white red top).

6、6.显色液A(白瓶黑盖)。 6,6. Coloring solution A (black and white bottle cap).

7、显色液B 20ml(白瓶紫盖)。 7, color liquid B 20ml (bottles of white purple cap).

8、终止液10ml(滴瓶)。 8, stop solution 10ml (dropper).

9、说明书1份。 9, 1 part of the specification.

操作说明书1.原理以三株高特异性与高敏感性抗人心肌肌钙蛋白I单克隆抗体,其中两株为捕获抗体,一株为标记抗体,结合链亲和素—生物素放大系统,应用酶联免疫原理检测人血或其它样品中心肌肌钙蛋白I(cTnI)的含量,用于多种疾病引起心肌损伤的诊断。 1. Principle operating instructions with three height with high sensitivity and specificity of the anti-human cardiac troponin I monoclonal antibody, wherein the two capture antibodies, one antibody is labeled, bound streptavidin - biotin amplification system, detecting the content of human blood samples or other cardiac troponin I (cTnI) is enzyme-linked immunosorbent principle, for the diagnosis of various diseases induced myocardial injury.

2.样品采集及处理干燥管或肝素抗凝管采血2ml。 2. Sample collection and handling drying tube or heparin blood collection tubes 2ml. 干燥管需室温放置30分钟后离心(2000rpm,5分钟),取血清用于检测。 Drying tube needs to be placed at room temperature for 30 minutes centrifugation (2000rpm, 5 minutes), the serum used for detection. 肝素抗凝管可即刻离心(同上)后,取血浆用于检测,样品如需长期保存,需分离血浆或血清置-20℃以下保存。 After centrifugation tube may heparin (supra) immediately taken for detecting plasma samples For long-term storage, plasma or serum to be isolated in the following set at -20 ℃.

3.仪器酶联仪 微量加样器 37℃恒温水浴箱4.操作步骤(1)近期所用试剂盒可提前一天置2~8℃解冻或室温快速解冻。 Enzyme-linked instrument 3. Instrument micro pipette water bath at 37 ℃ 4. Procedure (1) Recent kit may be used one day in advance set 2 ~ 8 ℃ thawed at room temperature or rapidly thawed. 检测前取出所用酶联条,打开并去除标准条第7孔封口膜后压到酶联架上,以备检测。 After detection by enzyme-linked before removing the article, and removal of the standard article open hole sealing film 7 pressed to the enzyme-linked frame, to prepare test. 取洗液6ml加入反应液瓶(红盖)中并充分混匀(切勿再冻融,用后置2~8℃保存)。 Take the reaction solution was added 6ml wash bottle (red top) and mixed well (not to freezing and thawing and then, after a set 2 ~ 8 ℃ stored).

注意:①试剂盒解冻后放置2~8℃保存,2~3周内用完,避免反复冻融;②不用作本次检测的酶联条不要拆封;③试剂应用后仍置2~8℃保存。 Note: Place the thawed kit ① 2 ~ 8 ℃ stored, run 2 to 3 weeks, to avoid repeated freezing and thawing; ② not detected this time for the enzyme-linked article not opened; 2 to 8 still set after reagent application ③ ℃.

(2)标准条1~6孔加入标准稀释液100μl。 (2) Standard Article 1 ~ 6 dilutions standard well was added 100μl.

标准条第7孔与样品条第2~7检测孔加入待测样品血浆或血清100μl。 Article 7 standard sample aperture 2-7 article detecting holes plasma or serum test sample was added 100μl.

(3)每孔内加入反应液50μl,振荡10~30秒,置37℃水浴20分钟(酶联条底部接触水面或部分浸入水中)。 (3) 50 l reaction solution was added to each well, shaken for 10 to 30 seconds (contact surface immersed in the water or enzyme-linked bottom bar) at 37 ℃ water bath for 20 minutes. 注意:第(2)(3)步加样时间间隔<3分钟。 Note: (2) (3) step of sample time interval of <3 minutes.

(4)倾去孔内液体,洗液洗5次,每次每孔加洗液量最适为250~300μl,如洗液用量较多,可稀释1倍后使用(不推荐使用,因各地水质不能保证)。 (4) hole liquid decanted, washed five times wash, each wash added per well, the optimum was 250 ~ 300μl, such as lotions large amount, may be used after diluted 1-fold (not recommended, because around water quality is not guaranteed).

(5)以1∶20体积比取所需显色液A与显色液B并充分混匀,每孔加150μl,37℃水浴15分钟。 (5) at a volume ratio of 1:20 to take the desired color reagent A and color reagent B and mix well, each well was added 150μl, 37 ℃ water bath for 15 minutes.

(6)每孔加终止液50μl或滴注一滴,以标准条第一孔为空白对照,轻轻振匀10分钟内酶联仪450nm阅读OD值。 (6) 50μl stop solution per well drip or drop to the first standard is the control bar, vibration gently homogenized in 10 minutes enzyme-linked meter reading OD 450nm values.

5.计算结果标准计算:根据标准孔2~6孔OD值与对应的cTnI浓度(3.2、1.6、0.8、0.4、0.2ng/ml)分别为X轴与Y轴,计算出直线回归方程Y=aX±b,Y为cTnI浓度,X为OD值),由此公式代入样品OD值,算出样品的cTnI浓度(ng/ml),有条件者可用计算机或酶联仪自动化处理。 The standard calculation results: The cTnI concentration (3.2,1.6,0.8,0.4,0.2ng / ml) standard hole 2-6 hole OD value corresponding to X and Y axes, respectively, to calculate the linear regression equation Y = aX ± b, Y a concentration of cTnI, X is OD value), substituting this equation sample OD, the sample was calculated cTnI concentration (ng / ml), or enzyme-linked conditions are available computer automated processing instrument.

简捷计算:选待测样品与标准点OD相近者直接比较计算样品cTnI浓度(ng/ml)=(标准点浓度×待测样品OD值)/标准点OD值。 Simplified Calculation: test sample is selected from a standard point close to directly compare the calculated OD sample cTnI concentration (ng / ml) = (standard concentration × point test sample OD value) / OD of the standard point.

6.结果判断200例健康人测定统计分析示,正常参考值<0.15ng/ml,≥0.15ng/ml提示cTnI升高,0.15~0.5ng/ml提示轻度心肌损伤,≥0.5ng/ml提示急性心肌梗死或严重的心肌损伤。 6. Analyzing the results of 200 healthy people measured statistical analysis shows normal reference value <0.15ng / ml, ≥0.15ng / ml prompt elevated cTnI, 0.15 ~ 0.5ng / ml prompted mild myocardial injury, ≥0.5ng / ml prompted acute myocardial infarction or severe myocardial damage. <0.15ng/ml为正常。 <0.15ng / ml is normal. 检测范围0.05~4.0ng/ml,最低检测值为0.05ng/ml,如结果>4.0ng/ml,需用阴性血清稀释适当倍数后检测。 The detection range of 0.05 ~ 4.0ng / ml, the minimum detection value of 0.05ng / ml, as a result> 4.0ng / ml, the detection of negative serum dilution required appropriate factor.

Claims (3)

1.一种以三株抗人体心肌肌钙蛋白I单克隆抗体为基础建立快速定量测定心肌肌钙蛋白I含量的方法,其特征是以二株作用于心肌肌钙蛋白I的单克隆抗体为捕获抗体,以另一株作用于心肌肌钙蛋白I的单克隆抗体为标记抗体,结合链亲和素-生物素放大系统,运用酶联免疫原理,建立心肌肌钙蛋白I快速定量检测的方法。 An anti-human to three cardiac troponin I monoclonal antibody rapid quantitative method to establish cardiac troponin I content is determined to be based, is characterized by two applied to a monoclonal antibody for cardiac troponin I capture antibody, in another strain acting on cardiac troponin I monoclonal antibody as the labeled antibody, binding streptavidin - biotin amplification system, using enzyme immunoassay principle, cardiac troponin I establish a method of rapid quantitative detection of .
2.根据权利要求1所述方法,二株捕获抗体,分别简称为Ab1和Ab2,联结生物素或长链生物素后,分别简称为Ab1-Bio和Ab2-Bio;另一株标记抗体简称为Ab3,联结辣根过氧化物酶后,简称为Ab3-POD,联结生物素或长链生物素后,简称为Ab3-Bio;链亲和素或亲和素简称为S;链亲和素辣根过氧化物酶简称S-POD;心肌肌钙蛋白I抗原包括已知的心肌肌钙蛋白I标准品、质控品与待测样品中的心肌肌钙蛋白I,简称为cTnI;三抗法快速定量测定心肌肌钙蛋白I方法的反应途径具体包括如下几种方案:方案一:(1)96孔酶联板、酶联条或其它固项支持物联结好S,白蛋白或其它封闭液封闭;(2)Ab1-Bio及Ab2-Bio与S结合,cTnI与Ab1-Bio及Ab2-Bio结合,cTnI与Ab3-POD结合,形成 2. The method according to claim 1, two capture antibody, simply referred to as Ab1 and Ab2, or coupling a long-chain biotin biotin, simply referred to as Ab1-Bio and Ab2-Bio; the other was referred to as the labeled antibody AB3, after coupling horseradish peroxidase, hereinafter simply referred to as Ab3-POD, coupled biotin or biotin long chain, referred to as Ab3-bio; streptavidin or avidin abbreviation is S; streptavidin spicy peroxidase abbreviated S-POD; cardiac troponin I known antigen comprising a cardiac troponin I standards, quality control materials with a test sample of cardiac troponin I, referred to as of cTnI; third antibody method rapid reaction pathway method of quantitative determination of cardiac troponin I specifically include the following schemes: scheme I: (1) 96-well ELISA plate, enzyme-linked strip or other solid support coupled good items S, albumin or other blocking solution closed; (2) Ab1-Bio and Ab2-Bio and S binding, binding to Ab1-Bio of cTnI and Ab2-Bio, cTnI and Ab3-POD, forming 反应链;(3)POD使显色底物显色或使化学发光底物发光,再通过相应仪器测定,根据标准曲线计算待测样品心肌肌钙蛋白I含量;方案二:(1)96孔酶联板、酶联条或其它固项支持物联结好Ab1及Ab2,白蛋白或其它封闭液封闭;(2)Ab1及Ab2与cTnI结合,cTnI与Ab3-POD结合,形成 The reaction chain; (3) POD chromogenic substrate that the chromogenic or chemiluminescent substrate to emit light, and then measured by the corresponding instrument, the test sample is calculated cardiac troponin I content of the standard curve; Option II: (1) 96 ELISA plate, enzyme-linked strip or other solid support coupled good items Ab1 and Ab2, albumin or other blocking solution; (2) binding to Ab1 and Ab2 cTnI, cTnI and Ab3-POD, forming 反应链;(3)POD使显色底物显色或使化学发光底物发光,再通过相应仪器测定,根据标准曲线计算待测样品心肌肌钙蛋白I含量;方案三:(1)96孔酶联板、酶联条或其它固项支持物联结好Ab1及Ab2,白蛋白或其它封闭液封闭;(2)Ab1+Ab2与cTnI结合,cTnI与Ab3-Bio结合,Ab3-Bio与S-POD结合,形成 The reaction chain; (3) POD chromogenic substrate that the chromogenic or chemiluminescent substrate to emit light, and then measured by the corresponding instrument, the test sample is calculated cardiac troponin I content of the standard curve; three programs: (1) 96 ELISA plate, enzyme-linked strip or other solid support coupled good items Ab1 and Ab2, albumin or other blocking solution; (2) Ab1 + Ab2 binding and cTnI, cTnI and binding Ab3-Bio, and S- Ab3-Bio POD, forming 反应链;(3)POD使显色底物显色或使化学发光底物发光,再通过相应仪器测定,根据标准曲线计算待测样品心肌肌钙蛋白I含量。 The reaction chain; (3) POD chromogenic substrate that the chromogenic or chemiluminescent substrate to emit light, and then measured by the respective instrument, calculating cardiac troponin I content of the sample to be tested according to the standard curve.
3.根据权利要求2所述的方法,其特征在于显色底物为四甲基联胺TMB。 3. The method according to claim 2, characterized in that the chromogenic substrate tetramethylbenzidine amine is TMB.
CN 03131931 2003-06-19 2003-06-19 Raid quantitative determination of cardiac muscle troponin I by three-anti method CN1271409C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 03131931 CN1271409C (en) 2003-06-19 2003-06-19 Raid quantitative determination of cardiac muscle troponin I by three-anti method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 03131931 CN1271409C (en) 2003-06-19 2003-06-19 Raid quantitative determination of cardiac muscle troponin I by three-anti method

Publications (2)

Publication Number Publication Date
CN1470875A true CN1470875A (en) 2004-01-28
CN1271409C true CN1271409C (en) 2006-08-23

Family

ID=34153918

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 03131931 CN1271409C (en) 2003-06-19 2003-06-19 Raid quantitative determination of cardiac muscle troponin I by three-anti method

Country Status (1)

Country Link
CN (1) CN1271409C (en)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1332202C (en) * 2005-11-30 2007-08-15 首都医科大学 Protein interaction research method utilizing protein chip
CN101432626B (en) 2006-04-04 2013-06-19 神谷来克斯公司 Highly sensitive system and methods for analysis of troponin
US7838250B1 (en) 2006-04-04 2010-11-23 Singulex, Inc. Highly sensitive system and methods for analysis of troponin
CN101105497B (en) 2006-07-13 2011-10-19 上海凯创生物技术有限公司 Cardiac muscle troponin I detection reagent kit and its preparation method
CN103940986B (en) * 2014-03-24 2015-10-21 安徽省煦棠医疗科技有限公司 I troponin specific site of an antibody preparation and detection kit
CN104165992A (en) * 2014-07-28 2014-11-26 武汉璟泓万方堂医药科技股份有限公司 Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia
CN105572358A (en) * 2014-10-09 2016-05-11 苏州新波生物技术有限公司 Hepatitis B virus e antigen quantitative detection kit, preparation method and detection method thereof
CN105628938A (en) * 2016-04-07 2016-06-01 天津金虹生物科技开发有限公司 Detection kit for NS3 antigen of hepatitis C virus (HVC)
CN105785044B (en) * 2016-04-12 2018-01-19 河北特温特生物科技发展有限公司 Application of bladder cancer detection kit, and a method for detecting human complement factor related protein in which h

Also Published As

Publication number Publication date Type
CN1470875A (en) 2004-01-28 application

Similar Documents

Publication Publication Date Title
de Jager et al. Solid-phase and bead-based cytokine immunoassay: a comparison
US5770457A (en) Rapid oneside single targeting (ROST) immunoassay method
US6162645A (en) Determination of % glycated hemoglobin
Kuhn et al. Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma
Kroot et al. Immunochemical and mass-spectrometry–based serum hepcidin assays for iron metabolism disorders
US4016043A (en) Enzymatic immunological method for the determination of antigens and antibodies
Nemzek et al. Development and optimization of cytokine ELISAs using commercial antibody pairs
US20050244904A1 (en) Diagnostics based on signal peptide detection
US4504585A (en) Affinity immunoassay system
US20020106708A1 (en) Assays reagents and kits for detecting or determining the concentration of analytes
Hudson et al. Cardiac markers: point of care testing
Choi et al. A rapid, simple measurement of human albumin in whole blood using a fluorescence immunoassay (I)
WO2004090546A1 (en) Identifying a midregional proadrenomedullin partial peptide in biological liquids for diagnostic purposes, and immunoassays for conducting an identification of this type
Eriksson et al. Comparison of cardiac troponin I immunoassays variably affected by circulating autoantibodies
Lin et al. Development of a sensitive, rapid, biotin–streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone
Käkönen et al. Development and evaluation of three immunofluorometric assays that measure different forms of osteocalcin in serum
CN1844926A (en) ELISA kit for detecting Sudan red medicines and detection method thereof
Kemeny Titration of antibodies
US5639627A (en) Method for assaying specific antibody
CN101750492A (en) Self-immunity hepatitis detection protein chip and kit thereof
Ellerbe et al. A comparison of results for cholesterol in human serum obtained by the Reference Method and by the Definitive Method of the National Reference System for cholesterol.
Oda et al. Development and evaluation of a practical ELISA for human urinary lipocalin-type prostaglandin D synthase
CN103901203A (en) Chemiluminescence quantitative detection kit for procalcitonin, and preparation method and detection method thereof
Tanaka et al. A sol particle homogeneous immunoassay for measuring serum cystatin C
CN101639481A (en) Magnetic particle chemiluminescence immunoassay kit of free thyroxine

Legal Events

Date Code Title Description
C06 Publication
C10 Request of examination as to substance
ASS Succession or assignment of patent right

Owner name: NO.1 ATTACHED HOSPITAL, NANJING MEDICAL UNIV.

Effective date: 20041224

Free format text: FORMER OWNER: SU ENBEN

C41 Transfer of the right of patent application or the patent right
C14 Granted
C19 Lapse of patent right due to non-payment of the annual fee