CN103018441A - Multicomponent immunoassay method based on time-resolved chemiluminescence - Google Patents

Multicomponent immunoassay method based on time-resolved chemiluminescence Download PDF

Info

Publication number
CN103018441A
CN103018441A CN2012105891569A CN201210589156A CN103018441A CN 103018441 A CN103018441 A CN 103018441A CN 2012105891569 A CN2012105891569 A CN 2012105891569A CN 201210589156 A CN201210589156 A CN 201210589156A CN 103018441 A CN103018441 A CN 103018441A
Authority
CN
China
Prior art keywords
chemiluminescence
time
different
antibody
resolved
Prior art date
Application number
CN2012105891569A
Other languages
Chinese (zh)
Inventor
付志锋
韩静
高鸿飞
Original Assignee
西南大学
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 西南大学 filed Critical 西南大学
Priority to CN2012105891569A priority Critical patent/CN103018441A/en
Publication of CN103018441A publication Critical patent/CN103018441A/en

Links

Abstract

The invention discloses a multicomponent immunoassay method based on time-resolved chemiluminescence. The method comprises the following steps of: respectively labeling different antigens or antibodies by using chemiluminescent labels with different chemiluminescence reaction kinetic natures, and then simultaneously adding the labeled different antigens or antibodies and substances to be tested to ELISA (Enzyme-Linked Immunosorbent Assay) plate reaction holes coated with corresponding antigens or antibodies, thereby carrying out immunoreactions; and adding a co-reagent, which is used for initiating chemiluminescence, into the reaction holes, and finally, respectively detecting different chemiluminescence signals at different time windows. According to the multicomponent immunoassay method based on the time-resolved chemiluminescence, the immunoassay on different components is realized in a manner that chemiluminescence signals of the different components are respectively detected at the different time windows through time resolution, a precise fluorescence lifetime measurement system is not required for being used, and the detection on multiple components can be completed by only adopting a conventional chemiluminescence analyzer and an ordinary timing tool.

Description

基于时间分辨化学发光的多元免疫分析方法 Multivariate analysis of time-resolved immunoassay based on chemiluminescence

技术领域 FIELD

[0001] 本发明涉及化学发光免疫分析领域,特别涉及基于时间分辨化学发光的多元免疫分析方法。 [0001] The present invention relates to chemiluminescent immunoassays, and more particularly relates to a multivariate analysis method based on time-resolved immunoassay chemiluminescence.

背景技术 Background technique

[0002] 化学发光免疫分析法(CLIA)是将化学发光分析与免疫反应结合起来建立的方法,它既具有化学发光分析的高灵敏度,又具有免疫分析的高特异性。 [0002] chemiluminescence immunoassay (CLIA) is the combination of chemiluminescent reactions and immunological methods established together, both with high sensitivity chemiluminescent analysis, but also has high specific immunoassay. 早在1977年,Halman等就创建了CLIA,并用于检测抗原或抗体。 As early as 1977, Halman and so we created the CLIA, and for the detection of antigen or antibody. 由于CLIA具有灵敏度高、检测范围宽、操作简便、检测迅速,并且容易实现自动化检测,替代了放射性同位素,避免了放射性污染等优势,在临床诊断、环境检测、食品安全等领域得到了日益广泛的应用。 Since CLIA high sensitivity, wide detection range, easy operation, fast detection, and easy to implement automated testing, substitution of radioisotopes, radioactive contamination is avoided and other advantages, has been in the field of clinical diagnostics, environmental testing, food safety an increasingly wide range of application.

[0003] 随着免疫分析技术的高速发展,对单一组分的检测往往不能满足对复杂生物样品检测的实际需要,多组分免疫分析的出现引起了人们的广泛关注。 [0003] With the rapid development of immunoassay techniques, the detection of a single component often can not meet the actual needs of complex biological sample to be tested, multi-component appears immunoassay has aroused extensive attention. 目前,已有的多组分免疫分析方法主要可归为两大类:阵列模式和多标记物模式。 At present, immunoassay methods have been multicomponent mainly grouped into two categories: an array mode and multi-label mode. 阵列模式通常是在免疫反应器的不同阵列区域来实现多个组分的同时检测,如采用阵列芯片或传感器阵列。 Pattern detecting array is typically simultaneously in different array regions to achieve an immune response in a plurality of components, such as array chips or using a sensor array. 该方法需要复杂的仪器做支撑,并且通常需采用昂贵的阵列型检测器,如C⑶和多通道电化学工作站。 This method requires complex instrumentation do support, and typically requires the use of expensive array detector, such as a multi-channel electrochemical workstation and C⑶. 多标记物模式是另一种常见的多组分免疫分析模式,其基本原理是以不同的信号探针标记不同组分,通过识别不同探针的信号来检测不同组分:如在光谱法中以波长识别,电化学法中以电位识别,质谱法中以荷质比识别,放射法中以射线能量识别。 Multi-mode tag is another common multicomponent immunoassay mode, the basic principle is different signal components of the different labeled probes, a signal identifying the different probes to detect different components: As spectrometry to identify the wavelength, the electrochemical method to identify potential, mass spectrometry to identify the charge to mass ratio, the radiation of energy rays to the identification process.

[0004] 时间分辨突光多组分免疫分析是一种典型的基于多标记物模式的技术,该技术米用具有不同荧光寿命的稀土金属配合物作为探针,分别标记不同组分,然后通过荧光寿命的差异在不同时间窗口实现不同组分的检测。 Multi-component light projecting immunoassay [0004] Time-resolved technique is based on a typical multi-mode tag, the technique rice as a probe labeled with different components having different rare earth metal complex fluorescent lifetime, then enable detection of differences in fluorescence lifetime of the different components at different time windows. 由于荧光寿命短,利用时间分辨荧光需要精密的荧光寿命测量系统,价格昂贵。 Due to the short lifetime of the fluorescence, time-resolved fluorescence using a fluorescence lifetime require sophisticated measuring system expensive. 化学发光反应中存在着类似现象,很多化学发光反应有着截然不同的动力学特征:有的是闪光型化学发光反应,即信号迅速达到峰值,之后迅速衰减,发光时间很短,只有零点几秒到几秒;有的是辉光型化学发光反应,即发光试剂混合时初始发光信号极低,但随着时间延长发光信号逐渐增强,发光时间从几分钟到几十分钟,或几小时至更久;有的还是震荡反应,即随时间变化,化学发光信号发生周期性增强和衰减。 Chemiluminescent reaction, there is a similar phenomenon, has many distinct chemiluminescent reaction kinetics: some flash chemiluminescence reaction, i.e., the signal rapidly reaches a peak, followed by rapid decay, the light emission time is very short, only a few tenths to a few seconds ; some glow type chemiluminescence reaction, i.e., an initial low light emission signal when the luminescence reagent mixture, but with time gradually increased emission signal, the light emission time from several minutes to several tens of minutes to several hours or longer; or some shock reaction, i.e. changes with time, a chemiluminescent signal generating periodicity enhancement and attenuation. 但是,目前尚未见到有报道将这种现象用于化学发光免疫分析,以实现多种组分的免疫测定。 However, it has yet to see this phenomenon has been reported for CLIA, in order to achieve a variety of immunoassay components.

发明内容 SUMMARY

[0005] 本发明的目的之一在于提供基于时间分辨化学发光的多元免疫分析方法,以实现通过时间分辨化学发光的多组分。 [0005] One object of the present invention to provide a method of multivariate analysis based on time-resolved immunoassay chemiluminescence to be achieved by time-resolved multicomponent chemiluminescent.

[0006] 为实现上述发明目的,技术方案为: [0006] In order to achieve the above object, technical scheme:

基于时间分辨化学发光的多元免疫分析方法,包括如下步骤: Multivariate analysis of time-resolved immunoassay based on chemiluminescence, comprising the steps of:

(I)用化学发光反应动力学性质不同的化学发光标记物分别标记不同的抗原或抗体,然后与待测物同时加入包被有相应抗体或抗原的酶标板反应孔中进行免疫反应; (2)向所述反应孔中加入引发化学发光的共反应剂,然后在不同时间窗口分别检测不同的化学发光信号。 (I) by the chemiluminescent reaction kinetics properties of different chemiluminescent labels are different labeled antigen or antibody, and then added simultaneously to the analyte coated immunoreact wells microtiter plates corresponding antibodies or antigens; and ( 2) co-initiators chemiluminescent reactant is added to the reaction well, and then detect different chemiluminescent signal at different time windows.

[0007] 本发明中,由于不同的化学发光标记物具有不同的化学发光反应动力学性质,因此加入共反应剂后在不同的时间窗口呈现出不同组分的光信号。 [0007] In the present invention, because of different chemiluminescent labels having different kinetic properties chemiluminescent reaction, after addition of the co-reactant components exhibit different optical signals at different time windows.

[0008] 优选的,所述步骤(I)是用闪光型化学发光标记物和辉光型化学发光标记物分别标记不同的抗原或抗体,然后与待测物同时加入包被有相应抗体或抗原的酶标板反应孔中进行免疫反应。 [0008] Preferably, the step (I) is labeled with a chemiluminescent label flash type and a glow-type chemiluminescent labels are different antigen or antibody, and then added simultaneously with a test was coated with the corresponding antibody or antigen for immune response microtiter plates wells.

[0009] 优选的,所述步骤(I)是用辣根过氧化物酶和碱性磷酸酶分别标记克伦特罗和莱克多巴胺,然后与克伦特罗和莱克多巴胺同时加入到包被有克伦特罗抗体和莱克多巴胺抗体的酶标板反应孔中进行免疫反应。 [0009] Preferably, the step (I) is a marker clenbuterol and ractopamine are horseradish peroxidase and alkaline phosphatase, and then mixed with clenbuterol added simultaneously to ractopamine coated with immunoreact microplate wells ractopamine clenbuterol antibody and antibody.

[0010] 优选的,所述包被的具体方法为:将克伦特罗抗体和莱克多巴胺抗体溶解于O. 10M pH 8.0 Tris-HCl缓冲液中至克伦特罗抗体浓度为8. O Pg/mL,莱克多巴胺抗体的浓度为10.0 Pg/mL,混匀,得包被液;然后将包被液加入到聚苯乙烯酶标板,然后置于4 °C条件下包被过夜,倒去孔中未结合的抗体溶液,然后每孔用洗液清洗,再向每个孔中加入封闭缓冲液,在37 °C下封闭1. 5小时,将酶标板用洗液清洗3次。 [0010] Preferably, the packet of the specific method is as follows: Clenbuterol antibody and the antibody was dissolved in ractopamine O. 10M pH 8.0 Tris-HCl buffer to a concentration of clenbuterol antibody 8. O Pg / mL, a concentration of antibody is ractopamine 10.0 Pg / mL, mix to yield coating liquid; coating solution was then added to polystyrene microtiter plates are coated overnight and then placed in 4 ° C for conditions, tossed hole unbound antibody solution, and then washed with a wash solution per well, again added to each well blocking buffer, blocked at 37 ° C 1. 5 hours, the plate was washed three times with the washing solution.

[0011] 优选的,所述共反应剂含有鲁米诺,对碘苯酚,H2O2和碱性磷酸酶底物液。 [0011] Preferably, the co-reactant contains luminol, phenol, iodine, H2O2, and alkaline phosphatase substrate solution.

[0012] 优选的,所述共反应剂含有1. OXlO-4 M的鲁米诺、5. OXlO-6 M的对碘苯酚、IOmM的H2O2和25 mM的碱性磷酸酶底物液。 [0012] Preferably, the co-reactant containing 1. OXlO-4 M luminol, iodophenol for, IOmM H2O2 and 25 mM of alkaline phosphatase substrate solution 5. OXlO-6 M's.

[0013] 更优选的,所述共反应剂的加入方式为先加入鲁米诺和对碘苯酚,再加入H2O2与ALP底物液的混合物,所述H2O2与碱性磷酸酶底物液体积比为1:1。 [0013] More preferably, the co-reactant is added for the first embodiment and p-iodophenol 加入鲁米诺, H2O2 was added and the mixture ALP substrate solution, the alkaline phosphatase substrate solution H2O2 by volume 1: 1.

[0014] 优选的,所述共反应剂的pH值为9. O。 [0014] Preferably, pH, the co-reactant is 9. O.

[0015] 本发明中闪光型化学发光标记物的发光时间很短,只有零点几秒到几秒;辉光型又称持续型,发光时间从几分钟到几十分钟,或几小时至更久。 [0015] In the present invention, the light emitting time of the flash type chemiluminescent labels is very short, only a few tenths to a few seconds; glow-type, also known as persistent, light emission time from several minutes to several tens of minutes to several hours or longer .

[0016] 本发明的有益效果:本发明公开的基于时间分辨化学发光的多元免疫分析方法,与传统的多标记物模式的分光光度法和常规荧光法相比,无需采用分光系统以分辨不同组分所发出不同波长的光信号;与时间分辨荧光法相比,化学发光法的时间窗口更宽:前者的时间窗口在毫秒级,而后者的时间窗口从秒级到小时级不等,因此为发光信号的时间分辨提供了更为便利的条件,无需采用复杂的荧光寿命测量系统,如基于时间相关单光子计数技术的精密测量系统,只需用秒表、时钟等最为常见的计时工具分辨信号;与采用阵列模式的多组分免疫分析法相比,仪器化程度低,操作简便,只需常规的化学发光分析仪即可,无需其常用的复杂仪器设备以及昂贵的阵列型检测器,因而成本较低。 [0016] Advantageous effects of the present invention are: polyhydric time resolved immunoassay method based on chemiluminescence, as compared with conventional multi-mode marker conventional fluorescent spectrophotometry method and disclosed in the present invention need not be employed to distinguish between different spectral components of the system optical signals having different wavelengths emitted; compared with time-resolved fluorescence, chemiluminescence time window wider: the former in the millisecond time window, which window of time ranging from seconds to hours grade level, thus emitting a signal to time resolved to provide a more convenient conditions, without the use of complex fluorescence lifetime measurement system, such as precision measurement system time correlated single photon counting technique based on only the most common resolution timing signal tool stopwatch, a clock and the like; and using low compared multicomponent array mode degree immunoassay instruments, simple, just the conventional chemiluminescence analyzer without the need of sophisticated equipment which is conventional and expensive array detector, and thus a lower cost.

附图说明 BRIEF DESCRIPTION

[0017] 图1为本发明的基于时间分辨的化学发光免疫分析方法的示意图。 Schematic [0017] FIG. 1 of the present invention based on chemiluminescence immunoassay method of time-resolved.

[0018] 图2为本发明检测克伦特罗和莱克多巴胺的工作曲线。 [0018] FIG detection curve and ractopamine clenbuterol present invention.

具体实施方式 Detailed ways

[0019] 为了使本发明的目的、技术方案和优点更加清楚,下面对本发明的优选实施例进行详细的描述。 [0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the following preferred embodiments of the present invention will be described in detail. [0020] 实施例1 [0020] Example 1

结合附图1对基于时间分辨化学发光的多元免疫分析法做进一步详细的说明。 Further detailed description in conjunction with the accompanying drawings one pair of polyol based on time-resolved immunoassay chemiluminescence. 在实施例中,以克伦特罗(CLE)和莱克多巴胺(RAC)作为模型分析物,辣根过氧化物酶(HRP)和碱性磷酸酶(ALP)作为化学发光标记物分别标记CLE和RAC,采用基于时间分辨化学发光的多元免疫分析法来检测不同的“瘦肉精”类物质。 In an embodiment, to clenbuterol (CLE) and ractopamine (RAC) as a model analyte, horseradish peroxidase (HRP) and alkaline phosphatase (ALP) as chemiluminescent labels and labeled CLE RAC, employed to detect different "lean" substances based on time-resolved chemiluminescence immunoassay polyol. 本发明采用竞争免疫模式,具体免疫分析步骤如下: The present invention is a competitive immunoassay mode, immunoassays specific steps are as follows:

首先将CLE抗体和RAC抗体溶解于O. 10 M pH 8. O Tris-HCl缓冲液中混合后作为包被液,至CLE抗体浓度为8. O Pg/mL,RAC抗体的浓度为10. O Pg/mL,然后将包被液加入到高亲和力的聚苯乙烯96孔酶标板,每孔加入100 UL包被液,然后置于4 °C条件下包被过夜,倒去孔中未结合的抗体溶液,然后每孔用300 μ L的洗液(含体积分数为O. 05%吐温20的Tris-HCl缓冲溶液)清洗3次,再向每个孔中加入150 μ L的封闭缓冲液(SuperBlOck®T20, Thermo Fisher Scientific Inc.)在37 °C下封闭1. 5小时,将酶标板用洗液清洗3次;然后,在每个孔中依次加入80 μ L含有不同浓度的CLE和RAC的样品溶液,同时加入含有HRP标记的CLE抗原(10 Pg/mL)和ALP标记的RAC抗原(20 Pg/mL)各10 μ L,继续在37 °〇下孵育1. 5 h,使其充分地发生竞争性免疫结合。 The first antibody and RAC CLE antibodies were dissolved in O. 10 M pH 8. O Tris-HCl solution was used as coating buffer After mixing, the concentration of the antibody to CLE 8. O Pg / mL, a concentration of antibody is RAC 10. O Pg / mL, and then the coating solution was added to the high affinity 96 well microtiter polystyrene plate, each well was added 100 UL coating buffer, then placed the package 4 ° C for overnight conditions, unbound decanted hole antibody solution, then L per well of wash solution 300 μ (volume fraction O. 05% Tween-20 Tris-HCl buffer solution) is washed 3 times, each well was added again 150 μ L of blocking buffer solution (SuperBlOck®T20, Thermo Fisher Scientific Inc.) is closed at 37 ° C 1. 5 hours, the plate was washed three times with the washing solution; then, each well were added 80 μ L containing different concentrations of CLE and RAC sample solution while adding CLE antigen (10 Pg / mL) containing HRP-labeled antigen and the ALP-labeled RAC (20 Pg / mL) for each 10 μ L, and incubation continued for 1. 5 h at 37 ° square, to make it fully competitive immunoassay binding occurs. 孵育结束后清洗酶标板,待进一步检测。 After incubation washed ELISA plate, until further testing.

[0021] 在化学发光检测过程中,通过注入共反应剂同时触发两个化学发光反应。 [0021] In the chemiluminescence detection process, by injecting two co-reactants simultaneously triggering the chemiluminescent reaction. 共反应剂包括鲁米诺,对碘苯酚,H2O2和ALP底物液。 Co-reactants include luminol, phenol, iodine, H2O2 and ALP substrate solution. 加入方式为先每孔分别加入40 μ L浓度为1. OX 10_4 M的鲁米诺和20 μ L浓度为5. OX 10_6 M的对碘苯酚,然后将触发两个化学发光反应的触发剂10 mM的H2O2与25 mM的ALP底物液按1:1的比例混合,取30 μί立即注入到待检测的微孔中触发反应,通过光电倍增管收集化学发光信号,光电倍增管的工作电压设置为-800 V,分别在O. 6 s和25 min的两个时间窗口处检测CLE和RAC的化学发光信号。 Mode is added to 40 μ L were added to each well at concentrations of 1. OX 10_4 M of luminol and 20 μ L is the concentration of 5. OX 10_6 M-iodophenol, and then triggers the chemiluminescent reaction of two triggers 10 mM, 25 mM H2O2 and ALP substrate solution of 1: 1 ratio, 30 μί taken immediately injected into the pores to be detected to trigger the reaction, the operating voltage is provided by a photomultiplier tube to collect a chemiluminescent signal, PMT is -800 V, respectively detect chemiluminescent signal CLE in the RAC and O. 6 s and at two time windows of 25 min.

[0022] 本发明所述的检测过程中,共反应剂的pH值最佳为9.0。 [0022] The detection process according to the present invention, pH, co-reactant optimum of 9.0. 对于共反应剂以Tris-HCl为共同的缓冲液体系更为兼容。 For co-reactants in Tris-HCl is more compatible for the common buffer system. 对碘苯酚作为增强剂,其浓度过大会影响HRP催化的鲁米诺-H2O2的动力学特征,使反应时间延长,故优选为5. OX 10_6 M。 Iodophenol as the enhancer to a concentration too much effect on the dynamics of HRP-catalyzed luminol -H2O2 the reaction time, it is preferable to 5. OX 10_6 M.

[0023] 在上述实验方法和条件下,测定了浓度为I ng/mL、10 ng/mL、100 ng/mL、200 ng/mL、500 ng/mL的CLE标准溶液和RAC标准溶液,结果如图2所示。 [0023] In the above test method and conditions, measured at a concentration of I ng / mL, 10 ng / mL, 100 ng / mL, 200 ng / mL, CLE standard solution 500 ng / mL and RAC standard solutions, the results 2 shown in FIG. 由检测结果可知,CLE和RAC在1. 0-500 ng/mL范围内化学发光峰高呈良好的线性关系,其相关系数(R2)分别为O. 9985 和O. 9913,检测限(S/N=2)都为O. 50 ng/mL。 We understood from the detection results, and the RAC CLE 1. 0-500 ng / mL range chemiluminescence peak height good linear relationship, the correlation coefficient (R2), respectively, and O. 9985 O. 9913, the detection limit (S / N = 2) are both O. 50 ng / mL.

[0024] 本实施例中利用了HRP催化的鲁米诺-H2O2反应在一定浓度的增强剂对碘苯酚的作用下在O. 6s即达到发光峰值,之后迅速衰减,呈现出闪光型化学发光信号;而ALP催化的金刚烷二氧丁环磷酸盐水解反应初始发光极低,随着时间延长发光信号增强,整个发光过程可持续增强数小时,呈现辉光型化学发光信号。 [0024] The present embodiment utilizes the HRP-catalyzed luminol reaction -H2O2 certain concentrations of enhancer in the action-iodophenol O. 6s i.e. emission peak reached, followed by rapid decay, showing a flash chemiluminescent signal ; ALP and adamantane-dioxo-catalyzed hydrolysis of phosphate butoxy ring initial low emission, light emission signal enhancement with time, enhance the entire process may last several hours emission, exhibits a glow type chemiluminescence signal. 由于两者的发光动力学特征差异较大,这就为以时间分辨法识别不同组分提供了可能途径。 Since both the kinetics of light emission difference is large, this provides a possible way to time-resolved method to identify the different components.

[0025] 在本实施例闪光型-辉光型化学发光组合研究中,将两个体系进行配对组合,结果显示,呈现出时间分辨化学发光的现象,两者兼容性较好,相互干扰性较小。 [0025] In the present embodiment, a flash type - Combination of a glow type chemiluminescence, the combination of pairing two systems, the results show, showing time-resolved chemiluminescence phenomenon, both good compatibility, mutual interference than small.

[0026] 为了进一步评估本发明所述方法的可行性,对其交叉干扰性进行了研究。 [0026] To further evaluate the feasibility of the method of the present invention, its cross-interference was studied. 检测时,在待检测样品中只加入一种分析物CLE (100 ng/mL)或RAC (100 ng/mL),然后进行检测。 When detected, the sample to be tested is added only one analyte CLE (100 ng / mL) or RAC (100 ng / mL), and then detected. 同时,对空白样品进行检测作为对照实验。 Meanwhile, the detection of the blank sample as a control experiment. 检测结果:当检测只含有CLE的样品时,由于CLE与其抗体的竞争免疫反应,与对照实验相比,窗口I的信号明显降低,窗口2的信号基本不变。 Test Results: When a sample containing only the detection of CLE, due to competing antibodies immunoreactive therewith CLE, compared with control experiments, the window signal I is decreased, the window signal 2 is substantially unchanged. 同样的,当检测只含有RAC的样品时,由于RAC与其抗体的竞争免疫反应,与对照实验相比,窗口2的信号明显降低,而窗口I的信号基本不变。 Similarly, when detecting the sample containing only the RAC, RAC due to competition and an antibody immune response, compared with control experiments, the window signal 2 is decreased, and the I signal window substantially unchanged. 由此表明,其交叉干扰性可有效地避免。 This indicates that the cross interference may be effectively avoided.

[0027] 实施例2 [0027] Example 2

以猪尿为样品,将猪尿用Tris-HCl缓冲液(pH7. O)稀释5倍,分别加入CLE和RAC,至CLE和RAC的浓度都为10 ng/mL、50 ng/mL和100 ng/mL。 In pig urine sample, the pig urine was diluted 5-fold with Tris-HCl buffer (pH7. O), were added CLE and RAC, to CLE and concentration of the RAC are 10 ng / mL, 50 ng / mL and 100 ng / mL. 在包被CLE抗体和RAC抗体的酶标板上封闭并清洗,然后每孔分别加入80 μ L含CLE和RAC的猪尿稀释液,同时加入含有HRP标记的CLE抗原(10 Pg/mL)和ALP标记的RAC抗原(20 Pg/mL)各10 μ L,在37 V下孵育1. 5 h,使其发生竞争性免疫反应。 The package is closed and washed RAC CLE antibodies and antibody ELISA plate, and then were added to each well containing 80 μ L of RAC CLE and pig urine dilution, while adding CLE antigen (10 Pg / mL) and containing HRP-labeled RAC ALP-labeled antigen (20 Pg / mL) for each 10 μ L, were incubated for 1. 5 h at 37 V, so a competitive immunoassay reaction. 孵育结束后再清洗酶标板。 After the end of the incubation washed ELISA plate. 在上述优化的反应条件下触发化学发光反应,分别在O. 6 s和25 min的两个时间窗口处检测CLE和RAC的化学发光信号,检测结果如表I所示: 表1.空白猪尿样品中克伦特罗和莱克多巴胺的加标回收率 In the above-described reaction conditions triggering the optimization of a chemiluminescent reaction, were used to detect chemiluminescent signal CLE in the RAC and O. 6 s and 25 min at the two time windows, the detection results are shown in Table I: Table 1. The blank pig urine sample recoveries clenbuterol and ractopamine

I i¥i I 克伦特罗 I 莱克多巴胺 I i ¥ i I I ractopamine clenbuterol

I 样品I ϊ 2 II ί I 3 I Sample I ϊ 2 II ί I 3

I 加_重(ng.mL) ' 10 一I» ^100 + 10 ' 50 100 — _ I plus weight (ng.mL) '10 a I »^ 100 + 10' 50 100 -

I I j : I I j:

+检出重(ng.mL) . 8.6 —— 54.7 104.4 —「11—2 技1 . 88.7 — + Detected weight (ng.mL) 8.6 -. 54.7 104.4 -. "Techniques 11-2 1 88.7 -

RSD (%) ' 7.6 —— 6J 5J ' 7.2 . 6.5 3J RSD (%) '7.6 - 6J 5J'. 7.2 6.5 3J

■回收率—(¾) ' 86 109 104 j 112 90 —「38 — ■ Recovery - (¾) '86 109 104 j 112 90 -' 38 -

I_1. .1 I_ I_1. .1 I_

由表I可知,利用本发明的方法测得CLE和RAC的加标回收率分别为86-104%和88-112%,两者的标准偏差均低于7. 6%。 It is seen from Table I, the use of the present invention is measured as CLE RAC and recoveries of 86-104% and 88-112%, respectively, of both the standard deviations were less than 7.6%.

[0028] 本发明中,也可以用吖啶酯-H2O2与ALP-金刚烷二氧丁环磷酸盐体系组合进行检测,吖啶酯-H2O2化学发光反应为典型的闪光型发光,在O. 4秒时达到峰值,反应条件较为简单,在H2O2的稀碱性溶液中即能发光。 [0028] In the present invention, may be detected with acridinium ester system in combination with ALP- -H2O2 adamantane ring-dioxo-butoxy phosphate, -H2O2 acridinium ester chemiluminescent reaction is a typical flash type light emission, in O. 4 when the second peak, the reaction conditions are relatively simple, i.e., it can emit light in a dilute alkaline solution of H2O2. 因此,能够与ALP-金刚烷二氧丁环磷酸盐体系组合并呈现出良好的化学发光时间分辨现象。 Thus, the system can be a combination of ALP- adamantane ring-dioxo-butoxy phosphate salt and exhibited good time-resolved chemiluminescence phenomenon.

[0029] 将吖啶酯和ALP各10 Ul混合于待检测的反应孔中,其浓度分别为5 X 10_9 M和O. 05 Pg/mL,将含有O. 06%H202的碱性缓冲液20 ul与25 mM的ALP底物液20 ul混合之后,立即加入到待检测的反应孔中触发反应,通过光电倍增管检测化学发光信号,在第一个时间窗口O. 4秒处出现第一个信号值,随着时间延长,第二个信号的化学发光强度逐渐增强,可选择15 min作为第二个时间窗口读取信号。 [0029] The reaction wells each ALP acridinium ester and mixing to be detected 10 Ul in which concentrations of 5 X 10_9 M and O. 05 Pg / mL, O. basic buffer containing 06% H202 20 after ul ALP substrate solution was mixed with 20 ul of 25 mM, was added immediately to the wells to be detected to trigger the reaction, detection by a photomultiplier tube chemiluminescent signal occurs at a first time a first window O. 4 seconds signal value, with time, chemiluminescence intensity of the second signal gradually increased, alternatively 15 min as a second time window of the read signal.

[0030] 应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。 [0030] It should be appreciated that the present invention is applied is not limited to the above-described example, those of ordinary skill in the art, can be modified or converted according to the above description, all such modifications and variations shall fall within the appended claims of the invention protected range.

Claims (8)

1.基于时间分辨化学发光的多元免疫分析方法,其特征在于,包括如下步骤: (1)用化学发光反应动力学性质不同的化学发光标记物分别标记不同的抗原或抗体,然后与待测物同时加入包被有相应抗体或抗原的酶标板反应孔中进行免疫反应; (2)向所述反应孔中加入引发化学发光的共反应剂,然后在不同时间窗口分别检测不同的化学发光信号。 1. Multivariate time-resolved immunoassay based on chemiluminescence, characterized in that it comprises the steps of: different (1) with a chemiluminescent reaction kinetics properties of chemiluminescent labels are different labeled antigen or antibody, and then with the analyte simultaneously added to the coated ELISA plate immunoreact wells corresponding antibodies or antigens; and (2) a chemiluminescent initiator coreactant is added to the reaction well, and then detect different chemiluminescent signal at different time windows .
2.根据权利要求1所述基于时间分辨化学发光的多元免疫分析方法,其特征在于:所述步骤(I)是用闪光型化学发光标记物和辉光型化学发光标记物分别标记不同的抗原或抗体,然后与待测物同时加入包被有相应抗体或抗原的酶标板反应孔中进行免疫反应。 The multivariate analysis based on time-resolved immunoassay chemiluminescence of claim 1, wherein: said step (I) is a flash chemiluminescent markers and glow-type chemiluminescent label different antigens were labeled or an antibody, the analyte and then simultaneously added to the coated ELISA plate immunoreact wells corresponding antibody or antigen.
3.根据权利要求1所述基于时间分辨化学发光的多元免疫分析方法,其特征在于:所述步骤(I)是用辣根过氧化物酶和碱性磷酸酶分别标记克伦特罗和莱克多巴胺,然后与克伦特罗和莱克多巴胺同时加入到包被有克伦特罗抗体和莱克多巴胺抗体的酶标板反应孔中进行免疫反应。 The multivariate analysis based on time-resolved immunoassay chemiluminescence of claim 1, wherein: said step (I) is labeled with clenbuterol Lake and horseradish peroxidase and alkaline phosphatase, respectively dopamine, then with clenbuterol and ractopamine added simultaneously to immunoreact with a coated microtiter plates wells ractopamine clenbuterol antibody and antibody.
4.根据权利要求3所述基于时间分辨化学发光的多元免疫分析方法,其特征在于:所述包被的具体方法为:将克伦特罗抗体和莱克多巴胺抗体溶解于O. 10 M pH 8.0 Tris-HCl缓冲液中至克伦特罗抗体浓度为8. O Pg/mL,莱克多巴胺抗体的浓度为10.0 Pg/mL,混匀,得包被液;然后将包被液加入到聚苯乙烯酶标板,然后置于4°C条件下包被过夜,倒去孔中未结合的抗体溶液,然后每孔用洗液清洗,再向每个孔中加入封闭缓冲液,在37 °C下封闭1. 5小时,将酶标板用洗液清洗3次。 The multivariate analysis based on time-resolved immunoassay chemiluminescence of claim 3, wherein: the packet is a specific method to: Clenbuterol antibody and antibody ractopamine was dissolved in O. 10 M pH 8.0 Tris-HCl buffer to a concentration of clenbuterol antibody 8. O Pg / mL, a concentration of antibody is ractopamine 10.0 Pg / mL, mix to yield coating liquid; coating solution was then added to the polystyrene microtiter plates, and then placed in 4 ° C for overnight coating conditions, unbound antibody was decanted wells, each well was then washed with a washing solution, again added to each well blocking buffer at 37 ° C for blocking 1.5 hours, the plate was washed three times with the washing solution.
5.根据权利要求1-4任一项所述基于时间分辨化学发光的多元免疫分析方法,其特征在于:所述共反应剂含有鲁米诺,对碘苯酚,H2O2和ALP底物液。 According to any of claims 1-4 over one yuan time resolved immunoassay method based on the chemiluminescence, wherein: the co-reactant contains luminol, phenol, iodine, H2O2 and ALP substrate solution.
6.根据权利要求5所述基于时间分辨化学发光的多兀免疫分析方法,其特征在于:所述共反应剂含有1. 0Χ1(Γ4 M的鲁米诺、5. 0Χ1(Γ6 M的对碘苯酚、IOmM的H2O2和25 mM的碱性磷酸酶底物液。 The multi Wu time-resolved immunoassay based on the chemiluminescence claimed in claim 5, wherein: said co-reactant comprising 1. 0Χ1 (Γ4 M luminol, 5 0Χ1 (Γ6 M of iodine. phenol, IOmM H2O2 and 25 mM of alkaline phosphatase substrate solution.
7.根据权利要求6所述基于时间分辨化学发光的多兀免疫分析方法,其特征在于:所述共反应剂的加入方式为先加入鲁米诺和对碘苯酚,再加入H2O2与碱性磷酸酶底物液的混合物,所述H2O2与碱性磷酸酶底物液体积比为1:1。 The multi Wu immunoassay method based on time-resolved chemiluminescence of claim 6, wherein: the addition method for the first co-reactant and iodine 加入鲁米诺 phenol, then with alkaline phosphatase was added H2O2 the mixture of enzyme substrate solution, H2O2 and the alkaline phosphatase substrate solution volume ratio of 1: 1.
8.根据权利要求5所述基于时间分辨化学发光的多兀免疫分析方法,其特征在于:所述共反应剂的pH值为9.0。 The multi Wu time-resolved immunoassay based on the chemiluminescence according to claim 5, wherein: pH of the co-reactant is 9.0.
CN2012105891569A 2012-12-31 2012-12-31 Multicomponent immunoassay method based on time-resolved chemiluminescence CN103018441A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2012105891569A CN103018441A (en) 2012-12-31 2012-12-31 Multicomponent immunoassay method based on time-resolved chemiluminescence

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN2012105891569A CN103018441A (en) 2012-12-31 2012-12-31 Multicomponent immunoassay method based on time-resolved chemiluminescence

Publications (1)

Publication Number Publication Date
CN103018441A true CN103018441A (en) 2013-04-03

Family

ID=47967280

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2012105891569A CN103018441A (en) 2012-12-31 2012-12-31 Multicomponent immunoassay method based on time-resolved chemiluminescence

Country Status (1)

Country Link
CN (1) CN103018441A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106796229A (en) * 2014-07-10 2017-05-31 Fio公司 Lateral flow / immuno-chromatographic strip service and cassette analysis device, system, method and computer readable medium
CN107907684A (en) * 2017-09-29 2018-04-13 何皓 Prostate cancer early-diagnosis system based on multiple fluorescence lifetime resolution detection technology

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1186513A (en) * 1995-03-10 1998-07-01 梅索磅秤技术有限公司 Multi-array, multi-specific electrochemiluminescence testing
US6015667A (en) * 1996-06-03 2000-01-18 The Perkin-Emer Corporation Multicomponent analysis method including the determination of a statistical confidence interval
CN1908663A (en) * 2006-08-16 2007-02-07 南京大学 Substrate regional band resolution chemical luminescent multi-component immunity analytical method and detection system thereof
CN101021530A (en) * 2007-03-26 2007-08-22 南京大学 Automatic channel resolution chemiluminescent multicomponent immunodetection system and analytical method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1186513A (en) * 1995-03-10 1998-07-01 梅索磅秤技术有限公司 Multi-array, multi-specific electrochemiluminescence testing
US6015667A (en) * 1996-06-03 2000-01-18 The Perkin-Emer Corporation Multicomponent analysis method including the determination of a statistical confidence interval
CN1908663A (en) * 2006-08-16 2007-02-07 南京大学 Substrate regional band resolution chemical luminescent multi-component immunity analytical method and detection system thereof
CN101021530A (en) * 2007-03-26 2007-08-22 南京大学 Automatic channel resolution chemiluminescent multicomponent immunodetection system and analytical method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOSEA.MURILLO PULGARIN: "Simultaneous stopped-flow determination of morphine and naloxone by time-resolved chemiluminescence", 《TALANTA》 *
JOSEA.MURILLO PULGARıN: "Simultaneous stopped-flow determination of morphine and naloxone by time-resolved chemiluminescence", 《TALANTA》, 2 October 2007 (2007-10-02) *
ZHENG PING LI: "Time-resolved Chemiluminescence Technique for the Microdetermination of Protein Based on their Complexation with Copper(II)", 《ANALYTICAL SCIENCES SEPTEMBER》 *
张新荣: "时间分辨化学发光分析的研究I.金和银的同时化学发光测定", 《化学学报》 *
武学成: "时间分辨荧光免疫分析技术及临床应用", 《医学综述》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106796229A (en) * 2014-07-10 2017-05-31 Fio公司 Lateral flow / immuno-chromatographic strip service and cassette analysis device, system, method and computer readable medium
CN107907684A (en) * 2017-09-29 2018-04-13 何皓 Prostate cancer early-diagnosis system based on multiple fluorescence lifetime resolution detection technology

Similar Documents

Publication Publication Date Title
Diamandis Immunoassays with time-resolved fluorescence spectroscopy: principles and applications
EP0517516B1 (en) Multiple output referencing system for evanescent wave sensor
US5756011A (en) Detecting or quantifying multiple analytes using labelling techniques
CA1284947C (en) Indirect colorimetric detection of an analyte in a sample using ratio of light signals
Dasgupta et al. Luminescence detection with a liquid core waveguide
US4582809A (en) Apparatus including optical fiber for fluorescence immunoassay
CA1113392A (en) Luminescent immunoassay
JP2690011B2 (en) Measurement of the analyte concentration to use two kinds of labeling marker
CN100464189C (en) Magnetic separating direct chemical illuminating reagent and testing method using the same reagent
Kuningas et al. Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol
EP0617286A2 (en) Biospecific solid phase carrier
Wilson et al. Electrochemiluminescence enzyme immunoassays for TNT and pentaerythritol tetranitrate
EP0478626B1 (en) Detecting or quantifying multiple analytes using labelling techniques
Kakabakos et al. Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solid-phase time-resolved fluorometry.
Mastichiadis et al. Simultaneous determination of pesticides using a four-band disposable optical capillary immunosensor
Zaman et al. Fluorescence assays for high-throughput screening of protein kinases
EP0222341B1 (en) A method for immunoassay and reagents therefor
US5945344A (en) Electrochemiluminescence method
JP3594609B2 (en) Luciferase labeling method
CN101180530B (en) Method of analyzing biochemical substance, analyzing reagent kit and analyzer
US7214539B2 (en) Scintillation proximity test
US20120270207A1 (en) Analyte detection
US5672475A (en) Mixed luminescent conjugate test
US20130034863A1 (en) Apparatus and Methods for Detecting Inflammation Using Quantum Dots
US5736320A (en) Method of detecting substances by chemiluminescence

Legal Events

Date Code Title Description
C06 Publication
C10 Entry into substantive examination
C12 Rejection of a patent application after its publication