CN106872693A - Antibody fragment modified micro beam preparation method and micro beam immune sensing detection system based on antibody fragment modification - Google Patents

Antibody fragment modified micro beam preparation method and micro beam immune sensing detection system based on antibody fragment modification Download PDF

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CN106872693A
CN106872693A CN 201710218984 CN201710218984A CN106872693A CN 106872693 A CN106872693 A CN 106872693A CN 201710218984 CN201710218984 CN 201710218984 CN 201710218984 A CN201710218984 A CN 201710218984A CN 106872693 A CN106872693 A CN 106872693A
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antibody
microcantilever
micro
fragments
fragment
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CN 201710218984
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张青川
吴尚犬
邬林
伍小平
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中国科学技术大学
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Abstract

The invention provides an antibody fragment modified micro cantilever. A semi-antibody fragment or an antibody Fab' fragment is directionally fixed on the gold plating surface of the micro cantilever; the sensitivity of a micro cantilever immunodetection method based on the surface stress effect is improved. The invention also discloses a preparation of the micro cantilever, an immune sensing detection system of the micro cantilever based on antibody fragment modification and a detection method.

Description

抗体片段修饰的微梁制备方法和基于抗体片段修饰的微梁免疫传感检测系统 The method of preparing the modified micro beam antibody fragments and antibody fragments based on the modified micro beam sensing the immune system

技术领域 FIELD

[0001]本发明涉及抗体片段修饰的微悬臂梁(以下也简称为微梁),及其制备方法。 [0001] The present invention relates to modified antibody fragments microcantilever (hereinafter, simply referred to as micro beam), and its preparation method. 本发明还公开了基于所述抗体片段修饰的微梁的免疫传感检测系统和检测方法,所述系统和方法可应用于食品安全、环境污染、生物医学、科学研究和生产制造等领域的监控和检测。 The present invention also discloses an immunoassay method for detecting and sensing systems based on the micro beam modified antibody fragments, the system and method can be used in food safety, environmental pollution, biomedical research and other fields of production monitoring and detection.

背景技术 Background technique

[0002]免疫传感技术的原理是基于检测抗原抗体的特异性配对反应,即针对需要检测的某种靶标分子(抗原),通过生物免疫(把抗原注入小动物体内产生的)方法,产生出相应的探针分子(抗体),提取、纯化出这种探针分子,再利用抗原抗体的特异性结合去检测样品中的靶分子。 [0002] immunological principles sensing technology is based on detecting the antigen-antibody reaction specific pairing, i.e., for certain target molecules (antigens), by immune (injecting the antigen produced in vivo small animal) to be detected methods, produce corresponding probe molecule (antibody), extracted, purified such a probe molecule, then an antigen-antibody binding specifically to the target molecule in the test sample. 已有的免疫传感技术如:酶联免疫吸附试验(ELISA,原理是利用抗原与抗体的特异性反应与酶标的催化放大来进行)和免疫荧光法(IF,原理是用荧光片段标记抗原或抗体),均需要标记物来示踪抗原抗体的反应结果,就是说仅有抗体,并不能建立相应的检测方法,还需要有酶标物或荧光标记物或蛋白复合物。 Conventional immunological techniques such as sensing: enzyme-linked immunosorbent assay (ELISA, using the principle of antigen and an antibody specifically reactive with the enzyme catalyzed amplification is performed) and immunofluorescence (IF, principle is a fluorescent labeled antigen or a fragment antibody), are required to label antigen-tracer antibody reaction results, that the antibody only, and can not establish the corresponding detection method further requires fluorescent label or enzyme or protein complex. 而一些难制备的靶标分子的标记物价格昂贵,或几乎不可能制备或购买。 And the prices of some marker target molecule expensive difficult to prepare, or nearly impossible to prepare or buy. 同时酶联免疫试剂盒和蛋白芯片的检测从原理操作上都要求每一步反应完后进行清洗,标记过程繁琐耗时,是事后的检测,不能实时,原位、在线的检测。 Simultaneous detection ELISA kits and protein chip from the principle operations require cleaning after every step of the reaction, the labeling process cumbersome time-consuming, is to detect afterthought, not real-time, in-situ, on-line detection.

[0003] 基于表面应力检测的微悬臂梁免疫传感技术是近年出现的一种新的免疫生化传感方法[1],其原理是:把探针(抗原或抗体)分子用直接或间接的方式固定(修饰)到微梁一侧的镀金层上,当被检测样品液中的靶分子与微梁金表面上的探针分子发生免疫生化反应时,会使微梁表面应力改变,从而导致微梁弯曲变形,通过光学或电学方法检测这种变形的过程,可得到免疫生化反应的实时信息。 [0003] Based on microcantilever immunosensor detection of surface stress is a new method of immunological and biochemical sensing emerged in recent years [1], which is the principle: the probe (antibody or antigen) directly or indirectly with a molecule fixing means (modified) to a gold plating layer on the side of the micro beam, when the probe molecule is detected in the sample liquid and the gold surface of the micro beam target molecule immunoreactive biochemical reactions, the stress will change the micro beam surface, resulting in micro beam bending deformation which is detected by the process of optical or electrical methods, real-time information obtained immune biochemical reactions. 基于免疫特异性识别建立的微梁免疫传感技术与传统的需要标记物的免疫传感方法相比,它无需使用任何酶标、荧光物质和放射性作为反应示踪剂,消除了标记过程的影响,灵敏度高(比酶联免疫试验高数倍[24]),还可以通过监测微梁变形来实时、定量的监测抗原抗体的反应过程,得到更丰富的免疫生化反应的信息。 Beam microprocessor-based immunosensor immunospecifically recognize established as compared with the conventional immunological method requires sensing marker, it does not require the use of any enzyme, fluorescent substance and a radioactive tracer as a reaction, to eliminate the influence marking process , high sensitivity (several times higher than the enzyme-linked immunosorbent test [24]), may also be modified in real time by monitoring the micro beam, quantitative monitoring of antigen-antibody reaction to give a more informative immune biochemical reactions. 经过这些年的发展,微梁传感被作为一种新兴技术,在生物工程和环境污染监测技术等方面与传统的方法进行对比研宄,如RNA转录因子、酶、汞排放及挥发性化合物等,优于常规的酶联免疫方法。 After years of development, micro beam sensor is used as an emerging technology, comparison study based, such as RNA transcription factors, enzymes, and mercury emissions of volatile compounds in biological engineering technology and environmental pollution monitoring and conventional methods , superior to conventional enzyme immunoassay method. 但即使如此,微悬臂梁免疫传感技术的检测灵敏度还不足以在成本方面获得相对于常规酶联免疫法的巨大优势,即如果微悬臂梁免疫传感技术的灵敏度或检测极限只比酶联免疫法高数倍,而微悬臂梁免疫传感技术的设备成本与酶联免疫相对较高,使得微悬臂梁免疫传感技术难以商业化。 Even so, the detection sensitivity of microcantilever immunosensor is not enough to have a big advantage compared to conventional ELISA method in terms of cost, i.e., if the microcantilever immunosensor or sensitivity than the detection limit of enzyme-linked only immunization several times, the microcantilever immunosensor enzyme immunoassay equipment cost is relatively high, so that the microcantilever immunosensor difficult to commercialize.

[0004]目前,国内外文献报道的方法主要是利用具有双功能基团的疏基化试剂的疏基(-SH)抓住微梁表面的金表面,和另一功能团抓住抗体,实现抗体在微梁镀金层上的固定。 [0004] Currently, the method reported in the literature is the use of mercapto (-SH) group having a hydrophobic group bifunctional reagent of the surface of the micro beam to seize the gold surface, and the other functional groups seize antibody achieve antibody is immobilized on the gold plated layer micro beams. 例如先将巯基化试剂11-羧酸硫醇结合到微梁的金表面,活化其上的羧基,使之与抗体上的氨基结合来固定抗体(图2)。 First, for example, mercapto carboxylic acids 11- thiol-binding agent to the gold surface of the micro beam, activated carboxyl groups thereon, so that the immobilized antibody (FIG. 2) in combination with an amino group on the antibody. 或用一种巯基化试剂盐酸硫醇亚胺,通过与抗体反应,使抗体连接一个带巯基的分子基团,再通过这个巯基将抗体联结到微梁的金表面上(中国专利CN101407548)(见图3)。 Or with one sulfhydryl reagents hydrochloride thiol imine by reaction with an antibody, the antibody linker molecule group with a mercapto group, and then through the mercapto group to join the antibody onto the gold surface of the micro beam (Chinese Patent No. CN101407548) (see image 3). 这些方法固定抗体,存在如下共同的问题,d)Y字形抗体(见图1)的Fc段或Fab段的顶端部,会等概率的被固定在金表面上(见图2、3),没有方向性[5]。 These methods immobilized antibody, following common problems exist, the Fc segment d) Y-shaped antibody (see FIG. 1) or the tip portion of the Fab fragment, will equal probability is fixed on the gold surface (see FIG. 3), no directivity [5]. 而当Fab 段的顶端被固定在金表面上时,结合位点被遮挡,限制了抗体的抗原结合位点与抗原的充分结合,从而降低了微梁传感灵敏度;(¾抗体与微梁之间存在不同数目C原子的单链分子, 降低了抗原抗体反应产生的应力传递到梁上的效率。由此可见,中国专利CN101407548中公开的方法的灵敏度与酶联免疫法相比仅提高数倍,还无法达到商业化或用于极微量被分析物的检测。 When the top section is Fab immobilized on a gold surface, binding sites are blocked, antigen-binding limit the full antigen binding site of antibodies, thereby reducing the sensitivity of the micro beam sensing; (¾ of the antibody to the micro-beam exists between single-stranded molecules of different numbers of C atoms, reducing the efficiency of the stress generated by antigen-antibody reaction to the transmitted beam. Thus, the method disclosed in Chinese Patent No. CN101407548 ELISA sensitivity compared to only increase several times, You can not achieve commercial or a very small amount for the detection of the analyte.

[0005]因此,在本领域中存在着改进微梁表面与抗体的结合和设计从而进一步提高微梁免疫传感灵敏度和效率的需求。 [0005] Accordingly, there is a need for improved design and the binding surface of the micro beam and further enhance the immune antibodies sensing sensitivity and efficiency of micro beams in the art.

发明内容 SUMMARY

[0006]综上所述,在已有的微梁免疫传感技术报道中,最突出的问题就是检测灵敏度。 [0006] As described above, in the conventional micro beam immunosensor reported, the most prominent problem is the detection sensitivity. 影响微梁免疫传感器灵敏度的因素主要有三个方面:①抗体的灵敏度(或抗体与抗原结合的亲和性)、②微梁上抗体的固定方法和③微梁的设计与信号读出。 Effect of micro beam sensor sensitivity factors immunized three main aspects: ① Sensitivity antibody (or antigen-binding affinity of the antibody), antibody fixing method ② and ③ micro beam micro-beam design and signal readout. 由于抗体的灵敏度和微梁的规格与信号读出方式在微梁免疫传感器系统成型之后就无法改变,唯一可变的是微梁表面抗体的固定方法。 As the sensitivity of the antibody and the specifications of the micro beam and the signal readout mode can not be changed after the immunosensor micro beam forming system, the only variable is the method of fixing the surface of the micro beam antibody. 一种合适的微梁表面抗体的修饰方法,能使固定在微梁表面的抗体与样品溶液中的抗原充分结合,并能高效地将抗原抗体结合所产生的应力变化传递到微梁表面。 A suitable method of modifying the surface of the micro beam antibody, enabling the antibody to the sample solution is fixed to the micro-beam surface antigen sufficiently binding, and can change in conjunction with stress is transferred to the surface of the micro beam efficiently antigen-antibody. 抗体在微梁表面固定的方向性、密度、活性以及抗体与微梁表面之间的联接分子的长度与刚性都有可能影响最终检测的灵敏度。 Antibody molecules of the length of the rigid coupling between the fixed directional beam micro-surface density, the active surface of the micro beam and the antibody may affect the ultimate sensitivity of detection.

[0007] 鉴于现有技术中存在的问题,本发明用于解决上述问题的技术方案是通过1)减少抗体分子结合位点以外的抗体分子质量和体积,2)取消使用连接微梁镀金表面与抗体分子的过渡分子层(巯基化试剂),3)提供一种制备镀金表面上定向固定有抗体片段的微梁的方法,达到提高微梁免疫检测方法的灵敏度目的。 [0007] In view of the problems in the prior art, the present invention is to solve the above problems of the technical solution is to reduce the mass and volume of the antibody molecule binding site of an antibody molecule other than by 1), 2) eliminate the use of the plated surface of the connecting micro beam molecular layer transition antibody molecule (sulfhydryl reagents), 3) provided with a fixed directional beam micro antibody fragments on the surface of the gold plating method for preparing one kind, to achieve the object of the sensitivity of the immunodetection methods to improve micro beam.

[0008]抗体Ig分子的结构示意图如图1,包括两个相同的抗原结合片段(Fab)和一个可结晶片段(Fc),Fab和Fc通过中间的铰链区连接,构成Y字形结构。 [0008] The schematic structure of an antibody Ig molecule 1, comprising two identical antigen binding fragment (Fab) and a crystalline fragment (Fc), Fab & Fc and hinge region through an intermediate connector constituting the Y-shaped structure. Fab的顶端为与抗原分子的结合位点。 Fab top of the antigen binding site of the molecule. 在免疫球蛋白抗体分子的“Y”字形四肽链结构(图1)中,由两条完全相同的重链和两条完全相同的轻链以二硫键连接而成。 In the "Y" shaped immunoglobulin antibody molecules tetrapeptide chain structure (FIG. 1), the two identical heavy chains and two identical light chains disulfide bonded together. 对于Fc段修饰在微梁的镀金表面上时(抗体修饰的定向性好),抗体对称(Fab段)的两个上端部抗原结合位点与抗原结合后产生的应力, 通过抗体Y字型的腿部(Fc段)传递到镀金微梁表面。 Respect to the Fc fragment modified gold plating on the surface of the micro beam (modified antibody good orientation), bound antibody produced after symmetrically (Fab segment) of the upper portion of two antigen-binding sites of the antigen stress, through the Y-shaped antibody leg portions (Fc segment) to the plated surface of the micro beam. 抗体(IgG)的分子量约150kDa,如果被检测的抗原分子分子量为数百Da,抗体抗原质量比近1000。 Molecular weight antibody (IgG) about 150kDa, if the molecular weight of the antigen molecule to be detected hundreds Da, antigen-antibody ratio of nearly 1,000 mass. 参与抗原分子结合的位点由重链和轻链的高变区(VH和VL)构成,其与抗原分子的质量比约150,而抗体分子上不参加与抗原分子结合的Fc段,其与抗原分子的质量比近400。 Involved in antigenic site molecule is composed of hypervariable regions (VH and VL) of the heavy and light chains, the quality and antigen molecule ratio of about 150, but do not participate in the Fc portion bound to the antigen molecules on the antibody molecule with the antigen molecular mass ratio of nearly 400. 因此我们设想,如果能够减少结合位点以外的抗体分子质量和体积,保持抗体的结合位点朝向固定表面的外法线方向,就能够^ 高应力的传递效率和抗体在微梁表面的有效修饰密度,从而提高微梁免疫传感的灵敏度。 Therefore, we assume that if an antibody molecule capable of reducing weight and volume than the binding site to maintain normal direction toward the binding site of the antibody fixing surface, and transmission efficiency can ^ antibodies effective modification of high stress in the surface of the micro beam density, thereby improving the sensitivity of the micro beam immune sense. [0009]为此,在第一个方面,本发明提供了一种制备微悬臂梁的方法,包括以下步骤: [0010] 1)提供抗体和带有镀金层的微悬臂梁; ' [0009] For this purpose, in a first aspect, the present invention provides a method of making the micro-cantilever, comprising the steps of: [0010] 1) providing an antibody and a gold plating layer with a micro-cantilever; '

[0011] 2)二硫键还原步骤:使用二硫键还原剂处理所述抗体,使得所述抗体较链区的二硫键还原成巯基,从而得到两个对称的带有巯基的半抗体片段; [0011] 2) reduction of disulfide bonds steps: the antibody using disulfide reducing agent, such that the more chain antibody region disulfide is reduced to mercapto group, to obtain two symmetrical half-antibody fragment having a mercapto group ;

[0012] 3)固定步骤:将步骤2)获得的半抗体片段与镀有金层的微悬臂梁结合,得到镀金表面上固定有所述半抗体片段的微悬臂梁。 [0012] 3) fixing steps: Step 2 half-antibody fragment) obtained in the microcantilever plated gold layer obtained by combining the fixed upper half-antibody fragment plated surface of the microcantilever.

[0013] 在第二个方面,本发明还提供了一种制备微悬臂梁的方法,包括以下步骤: [0013] In a second aspect, the present invention also provides a method of making the micro-cantilever, comprising the steps of:

[0014] 1)提供抗体和带有镀金层的微悬臂梁; [0014] 1) providing an antibody with the micro-cantilever and a gold plating layer;

[0015] 2)酶解步骤:用蛋白酶将所述抗体裂解成F (ab')2片段和多个小分子片段,所述蛋白酶选自胃蛋白酶、木瓜蛋白酶或其组合; [0015] 2) hydrolysis step: the antibody with a protease cleavage of F (ab ') 2 fragments and multiple small molecule fragment, the protease is selected from pepsin, papain or a combination thereof;

[0016] 3)二硫键还原步骤:使用一硫键还原剂处理所述F (ab')2片段,使得所述抗体较链区的二硫键还原成巯基,从而得到两个对称的带有巯基的Fab'片段; [0016] 3) reduction of disulfide bonds steps of: using a sulfide bond reducing agent of the F (ab ') 2 fragments, disulfide antibody such that the region is reduced to more chain mercapto group, whereby two symmetrical band mercapto group Fab 'fragment;

[0017] 4)固定步骤:将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金表面上固定有所述Fab'片段的微悬臂梁。 [0017] 4) fixing steps: Step. 3) obtained Fab 'fragment microcantilever plated gold layer obtained by combining fixed on the plated surface of the Fab' fragment of the microcantilever.

[0018] 在第三个方面,本发明还提供了一种制备微悬臂梁的方法,包括以下步骤: [0018] In a third aspect, the present invention also provides a method of making the micro-cantilever, comprising the steps of:

[0019] 1)提供抗体和带有镀金层的微悬臂梁; [0019] 1) providing an antibody with the micro-cantilever and a gold plating layer;

[0020] 2)二硫键还原步骤:使用二硫键还原剂处理所抗体,使得所述抗体铰链区的二硫键还原成巯基,从而得到两个对称的带有巯基的半抗体片段; [0020] 2) the step of reduction of disulfide bonds: The antibody using disulfide reducing agent, such that the antibody hinge region disulfide is reduced to the thiol group, to obtain two symmetrical half-antibody fragment with a thiol group;

[0021] 3)酶解步骤:用蛋白酶将所述半抗体片段裂解成Fab'片段和多个小分子片段,所述蛋白酶选自胃蛋白酶、木瓜蛋白酶或其组合; [0021] 3) digestion step of: the half-antibody with a protease is cleaved into fragments' fragments and a plurality of small molecule fragments Fab, the protease is selected from pepsin, papain or a combination thereof;

[0022] 4)固定步骤:将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述Fab'片段的微悬臂梁。 [0022] 4) fixing steps: Step. 3) obtained Fab 'fragment microcantilever plated gold layer obtained by combining fixed on the gold plated layer Fab' fragments of the microcantilever.

[0023]在第四个方面,本发明提供了一种应力传感元件,其包括基于应力的微悬臂梁,所述微悬臂梁的镀金层上固定有抗体的半抗体片段或Fab'片段。 [0023] In a fourth aspect, the present invention provides a stress sensing element, which comprises a micro-cantilever-based stress fixed half antibody fragments or Fab 'fragments of the gold plating layer on the micro-cantilever. 所述微悬臂梁优选通过本发明的方法制备而成。 The micro-cantilever is preferably prepared by the process of the present invention is formed.

[0024]在第五个方面,本发明提供了一种基于应力的微悬臂梁免疫传感检测系统,其包括本发明的应力传感元件。 [0024] In a fifth aspect, the present invention provides a stress based on microcantilever sensing the immune system, which comprises a strain sensing element of the present invention.

[0025]在第六个方面,本发明提供了一种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: [0025] In a sixth aspect, the present invention provides a method of using the immune system of the sensing microcantilever sensing test sample, comprising the steps of:

[0026] 1)提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; [0026] 1) providing an antibody specific for the target antigen and microcantilever sensing system, the sensing microcantilever system includes a reaction tank and a stress-based micro-cantilever with a gold plating layer;

[0027] 2)使用二硫键还原剂处理所述抗体,使得所述抗体铰链区的二硫键还原成巯基, 从而得到两个对称的带有巯基的半抗体片段; [0027] 2) the antibody using disulfide reducing agent, such that the antibody hinge region disulfide bonds reduced to the mercapto group, to obtain two symmetrical half-antibody fragment with a thiol group;

[0028] 3)将步骤2)获得的半抗体片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述半抗体片段的微悬臂梁; [0028] 3) Step 2 The half-antibody fragment) obtained in the microcantilever plated gold layer obtained by combining the fixed upper half-antibody fragment microcantilever gold plated layer;

[0029] 4)将步骤3)获得的固定有所述半抗体片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; [0029] 4) The Step 3) obtained in the fixed half of the micro-cantilever antibody fragment is fixed to a well microcantilever sensing system;

[0030] 5)将待测样品加入至步骤4)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原。 [0030] 5) The sample to be tested is added to step 4) microcantilever sensing system obtained, the test sample is detected in presence or absence of the target antigen.

[0031]在第七个方面,本发明还提供了一种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: [0031] In a seventh aspect, the present invention also provides a method of using the immune system of the sensing microcantilever sensing test sample, comprising the steps of:

[0032] 1)提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; [0032] 1) providing an antibody specific for the target antigen and microcantilever sensing system, the sensing microcantilever system includes a reaction tank and a stress-based micro-cantilever with a gold plating layer;

[0033] 2)用蛋白酶将所述抗体裂解成F(ab')2片段和多个小分子片段,所述蛋白酶选自胃蛋白酶、木瓜蛋白酶或其组合; [0033] 2) the antibody with a protease cleavage of F (ab ') 2 fragments and multiple small molecule fragment, the protease is selected from pepsin, papain or a combination thereof;

[0034] 3)使用二硫键还原剂处理所述F (ab')2片段,使得所述抗体铰链区的二硫键还原成巯基,从而得到两个对称的带有巯基的Fab'片段; [0034] 3) the use of a disulfide bond reducing agent F (ab ') 2 fragments, such that the antibody hinge region disulfide bonds reduced to the thiol group, whereby two symmetrical Fab having a mercapto group' fragment;

[0035] 4)将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述Fab'片段的微悬臂梁; Fab [0035] 4) The Step 3) obtained in 'fragment microcantilever plated gold layer obtained by combining fixed on the gold plated layer Fab' fragments of the microcantilever;

[0036] 5)将步骤4)获得的固定有所述Fab'片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; [0036] 5) The Step 4) obtained in micro cantilever is fixed to a well secured microcantilever sensing system of the Fab 'fragment;

[0037] 6)将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原。 [0037] 6) The sample to be tested is added to the step 5) microcantilever sensing system obtained, the test sample is detected in presence or absence of the target antigen.

[0038]在第八个方面,本发明还提供了一种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: [0038] In an eighth aspect, the present invention also provides a method of using the immune system of the sensing microcantilever sensing test sample, comprising the steps of:

[0039] 1)提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; [0039] 1) providing an antibody specific for the target antigen and microcantilever sensing system, the sensing microcantilever system includes a reaction tank and a stress-based micro-cantilever with a gold plating layer;

[0040] 2)使用二硫键还原剂处理所述抗体,使得所述抗体铰链区的二硫键还原成巯基, 从而得到两个对称的带有巯基的半抗体片段; [0040] 2) the antibody using disulfide reducing agent, such that the antibody hinge region disulfide bonds reduced to the mercapto group, to obtain two symmetrical half-antibody fragment with a thiol group;

[0041] 3)用蛋白酶将所述半抗体片段裂解成Fab'片段和多个小分子片段,所述蛋白酶选自胃蛋白酶、木瓜蛋白酶或其组合; [0041] 3) the half-antibody with a protease is cleaved into fragments' fragments and a plurality of small molecule fragments Fab, the protease is selected from pepsin, papain or a combination thereof;

[0042] 4)将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述Fab'片段的微悬臂梁; Fab [0042] 4) The Step 3) obtained in 'fragment microcantilever plated gold layer obtained by combining fixed on the gold plated layer Fab' fragments of the microcantilever;

[0043] 5)将步骤4)获得的固定有所述Fab'片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; [0043] 5) The Step 4) obtained in micro cantilever is fixed to a well secured microcantilever sensing system of the Fab 'fragment;

[0044] 6)将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原。 [0044] 6) The sample to be tested is added to the step 5) microcantilever sensing system obtained, the test sample is detected in presence or absence of the target antigen.

[GG45] 本发明的有益效果: [GG45] Advantageous effects of the invention:

[0046]鉴于微梁免疫传感检测技术中抗体固定的重要性及巯基化试剂参与抗体固定的复杂性,本发明用二硫键还原剂将抗体拆分成带有巯基的对称的半抗体片段,或在拆分之前或之后通过蛋白酶消化从而获得Fab'片段,然后将获得的半抗体片段或Fab,片段通过自带的双巯基固定到微梁的金表面上。 [0046] In view of the half-antibody fragment immunospecifically sensing technique antibody is immobilized in micro beam importance and involvement sulfhydryl reagents immobilized antibody complex, the present invention is an antibody with a disulfide bond reducing agent split symmetrically with a mercapto group , or before or after the split to obtain a Fab 'fragment was digested by a protease, and then half-antibody or fragment obtained Fab, fragments carrying the bis-sulfhydryl affixed to the gold surface of the micro beam. 结果表明,本发明的方法与现有的微梁免疫方法相比较具有以下优点: The results show that the method of the present invention and the conventional method of immunization as compared to the micro beam has the following advantages:

[0047] D半抗体片段或Fab'片段通过自带的双巯基,直接固定到微梁镀金表面,一步完成,操作简单; [0047] D semi antibody fragment or Fab 'fragment by carrying double mercapto, secured directly to the plated surface of the micro beam, one step, simple operation;

[0048] 2)半抗体片段或Fab'片段与微梁表面之间没有附加的其它的分子,有利于应力传递; [0048] 2) semi antibody fragment or Fab 'without additional molecules between the fragment and the other surface of the micro beam, is conducive to stress transfer;

[0049] 3)减少了抗体抗原结合位点以外的抗体分子质量和体积; [0049] 3) reducing the volume and mass of antibody molecules other than the antigen-binding site;

[0050] 4)减小了抗体的抗原结合位点到梁表面距离,提高应力的传递效率; [0050] 4) reduce the antigen-binding site to the distance from the surface of the beam, improve the transmission efficiency of stress;

[0051] 5)半抗体片段或Mb'片段与微梁表面之间的双巯基固定的较单巯基分子链联结的刚性要大,更有益于抗原抗体反应的作用力传递到梁上; [0051] 5) an antibody fragment, or semi-Mb 'between the segments and the surface of the micro beam fixed bis mercapto than single chain molecule coupled mercapto rigidity larger, more useful antigen-antibody reaction force is transmitted to the beam;

[0052] 6)通过自身双巯基一步完成固定,固定的抗原结合位点密度高,稳定性好; [0052] 6) through its own fixed bis mercapto one step, high immobilized antigen binding site density, good stability;

[0053] 7)镀金表面上固定的抗体的方向性好,易于与抗原结合。 [0053] 7) fixed on the plated surface is good directionality antibody, antigen binding easily.

附图说明 BRIEF DESCRIPTION

[0054] 图1为典型的抗体分子结构示意图。 [0054] FIG. 1 is a schematic view of a typical structure of an antibody molecule.

[0055] 图2为羧酸硫醇类化合物和双功能交联剂法固定抗体示意图。 [0055] FIG. 2 is a carboxylic acid compound and the immobilized antibody thiol schematic cross-linker method.

[0056] 图3为盐酸硫醇亚胺法固定抗体示意图。 [0056] FIG. 3 is a schematic diagram of an immobilized antibody thiol imine hydrochloride method. t〇〇57]图4为基于自身巯基(-SH)定向固定半抗体片段示意图。 t〇〇57] FIG. 4 is based on its own thiol group (-SH) oriented semi-schematic fixed antibody fragments.

[0058]图5胃蛋白酶水解抗体形成抗体F (ab')2片段示意图。 [0058] FIG. 5 pepsin hydrolysis of the antibody to form an antibody F (ab ') 2 fragments FIG.

[0059] 图6为通过自身疏基(-SH)定向固定抗体Fab'片段示意图。 [0059] FIG. 6 is a 'schematic fragment by itself mercapto (-SH) oriented immobilized antibody Fab.

[0060]图7为先用巯基乙胺将抗体拆分成半抗体,再用胃蛋白酶水解半抗体形成抗体Fab'片段,最后通过自身疏基(-SH)定向固定抗体Fab'片段示意图。 [0060] FIG. 7 is a first with mercaptoethylamine split into half-antibody antibody, pepsin hydrolysis half-antibodies then form an antibody Fab 'fragment, by itself finally mercapto (-SH) oriented immobilized antibody Fab' fragment. FIG.

[0061]图8为抗人参皂苷GS-Re巯基化抗体修饰微梁,检测不同浓度抗原时微梁尖端的时间位移曲线。 [0061] FIG. 8 is an anti-ginsenoside GS-Re time displacement curve when a micro tip of the beam thiolated antibody modified micro-beams, with different concentrations of antigen.

[0062]图9为抗人参皂苷GS-Re半抗体片段修饰微梁,检测不同浓度抗原时微梁尖端的时间位移曲线。 Time displacement curve when a micro tip of the beam [0062] FIG. 9 is an anti-GS-Re ginsenoside half antibody fragments modified micro beam, different concentration of antigen.

具体实施方式[0063] 定义 DETAILED DESCRIPTION [0063] defined

[0064] 抗体:抗体(antibody)指机体的免疫系统在抗原刺激下,由B淋巴细胞或记忆细胞增殖分化成的浆细胞所产生的、可与相应抗原发生特异性结合的免疫球蛋白。 [0064] Antibody: an antibody (Antibody) refers to the body's immune system in an antigen stimulation, the B lymphocytes or plasma cell proliferation and differentiation into memory cells produced, specifically bound immunoglobulins with the corresponding antigen. 典型的抗体分子具有4条多肽链的对称结构,包括2条较长、相对分子量较大的相同的重链(H链);2条较短、相对分子量较小的相同的轻链(L链)。 A typical antibody molecule has a symmetrical structure of four polypeptide chains, including two long, relatively larger molecular weight identical heavy chains (H chain); two shorter, relatively small molecular weight identical light chains (L chain ). 链间由二硫键和非共价键联结形成一个由4条多肽链构成的单体分子。 Inter-chain disulfide bond and non-covalent coupling forming a monomer molecules consisting of four polypeptide chains. 轻链有k和A两种,重链有u、S、Y、£和(1五种。整个抗体分子可分为恒定区和可变区两部分。在给定的物种中,不同抗体分子的恒定区都具有相同的或几乎相同的氨基酸序列。可变区位于〃Y 〃的两臂末端。在可变区内有一小部分氨基酸残基变化特别强烈,这些氨基酸的残基组成和排列顺序更易发生变异区域称高变区。高变区位于分子表面, 最多由I7个氨基酸残基构成,少则只有2〜3个。高变区氨基酸序列决定了该抗体结合抗原的特异性。一个抗体分子上的两个抗原结合部位是相同的,位于两臂末端称抗原结合片段(antigen-binding fragment JatO/Y"的柄部称结晶片段(crystalline fragment,Fc), 糖结合在Fc上。 A and k light chain has two kinds of heavy chains have u, S, Y, £ and (1 five kinds of whole antibody molecules can be divided into two parts, the constant and variable regions. In a given species, different antibody molecules constant region have the same or nearly identical amino acid sequence of the variable region is located 〃Y 〃 end of arms in the variable region amino acid residue changes a fraction of particularly strong, residues and sequences of these amino acids said mutation region more hypervariable regions. hypervariable region of the molecule surface, composed of up to I7 amino acid residues, at least 2 to 3 months only. the amino acid sequence hypervariable regions determines the specificity of antigen binding of the antibody. antibodies a two antigen binding sites on the same molecule, located at the end of said shank arms antigen binding fragment (antigen-binding fragment JatO / Y "said crystalline fragments (crystalline fragment, Fc), the sugar bound to Fc.

[0065] 从结构上来说,在本发明中,提及抗体时均指全抗体,即包括4条链和Fc段的抗体结构(见图1)。 [0065] Structurally, in the present invention, reference to an antibody refer to the whole antibody, i.e. an antibody chain and includes four structural Fc region (see FIG. 1). 此外,从功能上来说,本发明中的“抗体”或“抗体片段”是指对靶抗原特异的抗体或抗体片段,即与抗原具有特异结合能力的抗体或抗体片段,如未特别说明,本发明中的抗体一般指单克隆抗体,抗体片段包括半抗体片段、F(ab')2片段、Fab'片段和Fab等。 In addition, terms of functionality, in the present invention, "antibody" or "antibody fragment" refers to an antibody or antibody fragment specific for a target antigen, i.e. the antigen-specific antibody or antibody fragment having binding ability, unless otherwise indicated, the antibodies of the invention generally refers to a monoclonal antibody, an antibody fragment comprises a half-antibody fragments, F (ab ') 2 fragments, Fab' and Fab fragments and the like. [0066]半抗体片段:如图4中所示,通过二硫键还原剂将全抗体拆分成各自带有巯基的对称的片段,每个半抗体片段包含一条完整的轻链和一条完整重链以及Fc片段。 [0066] Semi antibody fragment: As shown in Figure 4, split into whole antibodies each with symmetrical segments of the mercapto group by a disulfide bond reducing agent, each half of a complete antibody fragment comprises a complete heavy and light chain chains and the Fc fragment.

[_7] F (at)')2:如图5中所示,Ig (immunoglobul in,免疫球蛋白)被胃蛋白酶水解在铰链区重链间二硫键近C处切断,形成一个双价抗原结合片段简称F (ab,)2片段和一些小片段pFc/。 [_7] F (at) ') 2: As shown in FIG. 5, Ig (immunoglobul in, immunoglobulins) by pepsin hydrolysis of disulfide bonds near the cut between the C heavy chain hinge region to form a bivalent antigen Acronym binding fragment F (ab,) 2 fragments, and small fragment pFc /. 由于F (ab')2片段保留了结合相应抗原的生物学活性,又避免了Fc片段的抗原性可能引起的副作用,因而被广泛用作生物制品。 Since the F (ab ') 2 fragments retain the biological activity corresponding antigen binding, but also avoid the side effects of antigenic Fc fragment may arise, which is widely used as a biological products. 如白喉抗霉素和破伤风抗霉素,经胃蛋白酶水解后,因去掉了Fc片段的抗原性而减少了超敏反应的发生。 Such as diphtheria and tetanus antimycin antimycin, pepsin after hydrolysis, by removing the antigenic fragment and the Fc reduced the incidence of hypersensitivity. PFC/片段最终被降解,无生物学活性。 PFC / fragments are ultimately degraded biologically inactive.

[0068] Fab (fragment of antigen binding):木瓜蛋白酶使Ig在铰链区重链间二硫键近N端处切断,形成两个相同的单价抗原结合片段简称Fab段(如图1中所示),一个可结晶的片段简称Fc (fragment crystallizable)段。 [0068] Fab (fragment of antigen binding): papain make disulfide bonds near the N-terminus of the Ig cut between the heavy chain hinge region, form two identical monovalent antigen binding Fab fragment referred to in paragraph (shown in FIG. 1) , a crystallizable fragment referred Fc (fragment crystallizable) segment.

[0069] Fab' :是带巯基的单价抗原结合片段(如图6中所示,F(ab')2被二硫键还原剂拆分的各自带有巯基的片段)。 [0069] Fab ': with a mercapto group is a monovalent antigen-binding fragment (shown in FIG. 6, F (ab') 2 fragments are each disulfide reducing agent having a mercapto group resolved).

[0070] 抗原结合位点:如图1中所示,抗体分子与抗原相结合的部位,由Ig轻、重链的CDR1、CDR2 和CDR3 组成。 [0070] The antigen binding site: As shown, the antibody molecule of the antigen binding sites relative to FIG. 1, the Ig light, the heavy chain CDRl, CDR2 and CDR3 composition.

[0071] 抗原:是一类能诱导免疫系统发生免疫应答,并能与免疫应答的产物(抗体或效应细胞)发生特异性结合的物质。 [0071] Antigen: The product is a class of immune system can induce an immune response, the immune response and to (effector cells or antibodies) that specifically binds to the substance to occur. 抗原具有免疫原性和反应原性两种性质。 Antigens having immunogenicity and reactogenicity both properties. 根据抗原性质分为两类:完全抗原和不完全抗原。 Divided into two categories according to the nature of the antigen: complete and incomplete antigen antigen. 完全抗原(complete antigen)是一类既有免疫原性,又有免疫反应性的物质。 Complete antigen (complete antigen) is a class of both the immunogenicity, but also immunoreactive substance. 如大多数蛋白质、细菌、病毒、细菌外毒素等都是完全抗原。 As most proteins, bacteria, viruses, bacterial toxins are completely foreign antigen. 不完全抗原, 即半抗原(hapten)是只具有免疫反应性,而无免疫原性的物质,故又称不完全抗原。 Incomplete antigen, i.e., (hapten) the hapten is immunoreactive only, without immunogenic substance, so called antigen incomplete.

[0072] 二硫键还原剂:二硫键又称SS键,是2个SH基被氧化而形成的-SS-形式的硫原子间的键。 [0072] The disulfide reducing agent: SS disulfide bond also known, is a bond between the two SH groups formed by oxidation of a sulfur atom in the form -SS-. 在巯基乙胺(2-MEA)、2_巯基乙醇、二硫苏糖醇等的硫化合物存在下能与之发生作用,还原成巯基(-SH)。 Sulfur compounds mercaptoethylamine (2-MEA), 2_ mercaptoethanol, dithiothreitol or the like with the presence of energy takes place, is reduced to a mercapto group (-SH). 这些硫化物就是本发明中所说的二硫键还原剂。 These sulfides present invention means that a disulfide bond reducing agent. 在微量的二硫键还原剂存在的情况下抗体的重链间的二硫键被还原而其它的二硫键不被破坏。 Disulfide bond between the heavy chains of the antibody in the presence of trace amounts of disulfide bond reducing agent is not destroyed by reduction of disulfide bonds other.

[0073] 微悬臂梁(微梁):典型的微悬臂梁由氮化硅制成,如商品化的三角形微悬臂梁(Veeco Instruments)(尺寸:长200um,腿宽20um,厚0.6um),单侧镀有60nm的金;抗体通常通过巯基化试剂的巯基(-SH)与金的共价结合以及巯基化试剂的另外一端(含有-〇)011或-NH2等活性基团)与抗体结合来固定到微梁表面。 [0073] microcantilever (micro beam): A typical micro-cantilever made of silicon nitride, as commercialization of triangular micro-cantilever (Veeco Instruments) (size: length 200um, 20um leg width, thickness 0.6um), unilateral 60nm plated with gold; antibody typically via sulfhydryl reagent mercapto group (-SH) and covalently binding gold sulfhydryl reagent and the other end (containing -〇) 011 or the like reactive -NH2 groups) bound to the antibody fixed to the surface of the micro beam.

[0074] 基于表面应力检测的微悬臂梁免疫传感系统:微悬臂梁免疫传感系统主要由激光器、分光镜、微悬臂梁、光电位置敏感器(PSD)、温度控制系统、蠕动栗、以及数据分析处理装置组成。 [0074] microcantilever surface of immune stress detection sensor system based on: sensing microcantilever immune system consists of a laser, a beam splitter, the micro-cantilever, the position of the photoelectric sensors (the PSD), the temperature control system, a peristaltic Li, and analysis of the data processing apparatus composition. 典型的微悬臂梁免疫检测方法的步骤如下:将微悬臂梁固定到反应池中,以蠕动栗控制流动缓冲液通过反应池,待反应池中气泡排净后以0. lmL/min的速度流动缓冲液通过反应池。 Typical microcantilever step immunoassay method is as follows: The micro-cantilever is fixed to a well, a peristaltic buffer Li control the flow through the reaction cell, the reaction cell to be drained at a rate of bubble 0. lmL / min flow buffer was prepared by the reaction cell. 反应池的温度控制在37 ±0 • 01 °C,室温控制在27 ±0 • 01°C。 Controlling the temperature of the reaction cell 37 ± 0 • 01 ° C, temperature control at 27 ± 0 • 01 ° C. 激光器发出一束激光经分光镜后照射在微悬臂梁的尖端,经微悬臂梁反射后再经分光镜反射后照在PSD的靶面上。 The laser emits a laser beam irradiated by the beam splitter at the tip of the micro-cantilever, micro-cantilevers after reflection by the dichroic mirror and then reflected on the target surface as the PSD. 当微悬臂梁的位移信号稳定后,加入缓冲液稀释的样品溶液,PSD实时记录微悬臂梁的尖端位移。 When the signal is stable displacement of the microcantilever, dilution buffer was added to the sample solution, PSD real time recording microcantilever tip displacement.

[0075] 本发明的优选实施方案 [0075] Preferred embodiments of the present invention

[0076] 在本发明中,抗体类型可以包括1811、186、18六、18〇、1§£。 [0076] In the present invention, the six types of antibodies may include 1811,186,18, 18〇, 1§ £. 此外,抗体可以通过本领域中已知的任何方法制备,包括但不限于免疫法、杂交瘤法、化学合成法、基因工程法等。 Furthermore, antibodies can be prepared by any method known in the art, including, but not limited to, immunoassay, hybridoma, chemical synthesis, genetic engineering method or the like. 所述抗体还可以是基因工程修饰的杂合抗体,诸如人源化抗体,骆驼源化抗体等针对某种哺乳动物改造的抗体,例如人-鼠杂合抗体等。 The antibody may also be modified genetically engineered hybrid antibodies, such as humanized antibodies, antibodies and other antibodies against camelized transformation of some mammals, such as human - mouse hybrid antibodies.

[0077] 本发明中提到的待测样品可以是生物样品,例如来自于哺乳动物尤其是人的样品,包括组织样品(如病理组织切片、活检、毛发、拭子等)、细胞样品(如细胞涂片、血液涂片等)、体液样品(如血液、尿液、脑脊液、唾液等)、排泄物(例如呕吐物、汗液、粪便等)。 Test sample [0077] The present invention may be mentioned in a biological sample, such as from a mammal, especially a human sample, including tissue samples (e.g., pathological tissue sections, biopsy, hair, swab, etc.), cell sample (e.g. smear, blood smears, etc.), a body fluid sample (such as blood, urine, cerebrospinal fluid, saliva, etc.), excreta (e.g. vomit, sweat, feces, etc.). 所述待测样品还可以是环境样品,诸如土壤样品、水样、浮尘等;其他生产领域中获得的样品,诸如污水样品、食品样品等。 The test sample may also be an environmental sample, such as a soil sample, a water sample, dust and the like; sample obtained in other production areas, such as water and food samples and the like.

[0078] 本发明中所提及的抗原包括但不限于完全抗原和半抗原,其可以是本领域中所知晓的在本发明中所提到的待测样品中可能存在的需要检测的任何类型的抗原。 [0078] The present invention is mentioned antigens include but are not limited to a complete antigen and a hapten, which may be any type of sample to be tested in the present invention, mentioned as known in the art may be present to be detected antigen.

[0079] 本发明中提到的二硫键还原剂可以是巯基乙胺(2-MEA)、2_疏基乙醇、二硫苏糖醇等。 [0079] The disulfide-reducing agent of the present invention, it may be mentioned mercaptoethylamine (2-MEA), 2_ mercaptoethanol, dithiothreitol and the like. 使用二硫键还原剂还原二硫键从而拆分抗体的方法和条件是本领域中公知的,例如,可以根据靶抗原的类型、大小和性质等,遵照本领域中的已知方法或市售的相关试剂盒的说明书来选择具体的二硫键还原剂和浓度、抗体浓度,两者的用量和质量比,温育条件和温育时间等。 The method and conditions employed disulfide bond reducing agent so as to split the disulfide bond antibodies are known in the art, for example, the target antigen according to the type, size and nature, in accordance with methods known in the art or commercially available kit instructions related to selecting specific concentrations of disulfide reducing agent and the antibody concentration, and the mass ratio of the two amounts, incubation conditions and time of incubation. 在本发明的优选实施方案中,在用二硫键还原剂处理抗体或抗体片段之后,可以进行透析去除未反应的二硫键还原剂,以免影响带巯基的抗体片段与微梁镀金层的结合和固定。 In a preferred embodiment of the present invention, after treatment with the antibody or antibody fragment disulfide bond reducing agent, a disulfide bond reducing agent may be removed by dialysis unreacted, so as not to affect binding antibody fragment with a thiol group and the gold plating layer of micro beam and fixed.

[0080] 在本发明中,在酶解步骤中使用的蛋白酶可以根据要被水解的抗体的种类或其他因素任意地选择,只要其水解产物中包括带有巯基的抗原抗体结合片段从而可以直接固定在微梁金表面上,所述抗原抗体结合片段包括不限于单价或二价的抗原抗体结合片段,诸如F (ab')2、Fab'等。 [0080] In the present invention, the protease digestion step may be according to the kind or other factors of the hydrolyzed antibodies arbitrarily selected, as long as it comprises a hydrolyzate having a mercapto group antigen-antibody binding fragments can be directly fixed on the surface of the gold micro-beams, the antigen-binding antibody fragments include monovalent or divalent not limited to antigen-antibody binding fragments such as F (ab ') 2, Fab' and the like. 可选的蛋白酶诸如胃蛋白酶,木瓜蛋白酶等,也可以使用两者的组合。 Optional protease pepsin, papain, such as may be used in combination of both.

[0081] 在本发明中,采用蛋白酶水解抗体Ig的方法和反应条件是本领域公知的,本领域普通技术人员可以根据要水解的抗体的种类来确定合适的蛋白酶和相应的水解条件。 The method and the reaction conditions [0081] In the present invention, a proteolytic Ig antibodies are well known in the art, those of ordinary skill in the art can determine the appropriate and suitable protease hydrolysis conditions depending on the type of antibody to be hydrolysed.

[0082] 例如,当选择胃蛋白酶时,冑蛋白酶可以发挥其活性的环境,一般是指pH 2〜5和在25〜6(TC下的环境。胃蛋白酶的最适PH和最适温度根据其来源而定,一般为pH 2左右和37r左右(可以根据市售产品的要求来确定)。在本发明中可以使用的胃蛋白酶的来源不受限制,只要其可以将抗体分子裂解为F (ab')2片段或将半抗体片段裂解为Fab'片段。在本发明中,抗体或抗体片段与胃蛋白酶的质量比不受限制,只要足以将抗体或抗体片段充分地水解为Fab'片段。两者的质量比可以根据常规方法来选择,也可以同时参考所选择胃蛋白酶的活性、所选择的抗体类型等因素,一般可以在10:1〜200:1的范围内选择。 [0082] For example, when the selected pepsin, protease helmet can exert its activity in the environment, and generally refers to the pH 2~5 25~6 environment (at TC. The optimum temperature and the optimum PH pepsin according to its source may be, generally about 2 and about pH 37r (may be determined according to the requirements of the commercial product). pepsin source is not limited in the present invention may be used as long as it can be an antibody molecule is cleaved F (ab ') 2 fragments or antibody fragments semi-cleaved Fab' fragments. in the present invention, the quality of the antibody or antibody fragment with pepsin ratio is not limited, as long as the antibody or antibody fragment sufficient to fully hydrolyze Fab 'fragments. two by mass ratio may be selected according to conventional methods, may be simultaneously active pepsin, the selected reference antibody selected type and other factors, generally in the 10: 1 range selection: 1~200.

[0083] 另外,还可以选择木瓜蛋白酶。 [0083] Further, papain may be selected. 在合适的条件下木瓜蛋白酶也可以将抗体水解为F (ab')2片段,例如,在文献[6]中在偏酸性(PH5.5)的环境中在37°C下1S小时木瓜蛋白酶可以将小鼠IgGi水解成F (ab,)2片段。 Under suitable conditions papain may be hydrolyzed to an antibody F (ab ') 2 fragments, for example, in [6] may be at 37 ° C 1S hours papain acidic (PH5.5) environment the mouse IgGi hydrolyzed to the F (ab,) 2 fragments. 在本发明中,抗体或抗体片段与木瓜蛋白酶的质量比不受限制,只要足以将抗体或抗体片段充分地水解SFab'片段。 In the present invention, the quality of the antibody or antibody fragment papain ratio is not limited, as long as sufficient to fully hydrolyze antibody or antibody fragment SFab 'fragment. 两者的质量比可以根据常规方法来选择,也可以同时参考所选择木瓜蛋白酶的活性、所选择的抗体类型等因素,一般可以在10:1〜200:1的范围内选择。 Mass ratio of them may be selected according to conventional methods, may be simultaneously active papain, antibodies selected with reference to the type of selected factors, may be generally 10: 1 range selection: 1~200.

[0084] 当使用胃蛋白酶和木瓜蛋白酶的混合物时,由于两者的活性环境均为酸性,因此根据具体的应用适当地调整两者的质量比,从而可以根据需要调整所需的水解活性和水解时间。 [0084] When a mixture of the stomach and proteases papain, since both are active acidic environment, thus appropriately adjusting the mass ratio of the two depending on the application, can be adjusted so that the desired hydrolysis activity and hydrolysis as needed time. 在本发明中,酶解步骤可以在拆分抗体之前或之后进行。 In the present invention, enzymatic resolution step may be performed before or after the antibody. 当在拆分抗体之前用蛋白酶水解时,即用蛋白酶水解抗体时,得到一个带有两个Fab'片段的F (ab')2片段和多个小分子片段。 When the hydrolysis antibody with a protease prior to splitting, i.e. hydrolysis of the antibody with a protease, to give a two Fab 'fragments F (ab') 2 fragments and multiple small fragments with molecules. 当在拆分抗体之后用蛋白酶水解时,即用蛋白酶水解半抗体片段时,得到了一个Fab ' 片段和多个小分子片段。 When the hydrolysis with proteinase antibodies after the split, i.e. an antibody fragment with a protease to hydrolyze hemicellulose, to give a Fab 'fragment and a plurality of small molecule fragments. 两种处理方式最终均可以得到带有双巯基的Fab'片段。 Two approaches can be finally obtained Fab 'fragment with a double mercapto group.

[0085] 在本发明中,抗体或抗体片段与微梁的镀金层的结合可以使用本领域公知的方法和条件,例如在适于两者结合的条件下温育一段时间来结合(参见下面的实施例或市售产品的说明书)。 [0085] In the present invention, the antibody or antibody fragment in combination with the gold plating layer may be micro-beams using the methods and conditions known in the art, for example under conditions suitable for binding of both the period of incubation to bound (see below Example commercially available products or instructions) embodiment. 在本发明中使用的微梁可以根据本领域中的公知方法自行制备,也可以购买市售商品,对此在本发明中并无任何限制。 Micro beam used in the present invention may themselves be prepared according to methods well known in the art, may be purchased commercially available, this is not any limitation in the present invention.

[0086]下面结合具体实施例对本发明做进一步说明,但需要说明的是下面的实施例不限制本发明的范围。 [0086] in conjunction with the following specific examples further illustrate the invention, but it should be noted that the following embodiments do not limit the scope of the invention.

[0087] 实施例 [0087] Example

[0088]实施例1固定半抗体片段的微梁 [0088] The semi-fixed beam micro Example antibody fragments

[0089]实验1利用巯基乙胺将抗体拆分成带巯基的半抗体片段 [0089] Experiment 1 using mercaptoethylamine split into half-antibody antibody fragment with a thiol group

[0090] 1 •准确称取人参阜苷GS-Re单克隆抗体(购自美国USBiological公司)7 . Omg溶于l.OmL PBS(含0.15M NaCl和5.0mM EDTA,pH 6.5)中得到浓度为7.0g/L的抗体溶液; [0090] 1 • Accurately weigh Fu ginseng glycoside GS-Re monoclonal antibody (purchased from USBiological Corporation) 7. Omg dissolved in l.OmL PBS (containing 0.15M NaCl and 5.0mM EDTA, pH 6.5) to give a concentration of 7.0g / L of the antibody solution;

[0091] 2.称取lmg疏基乙胺(购自美国Sigma公司)溶于l.OmL水中得到浓度为1.0g/L的抗体拆分试剂; [0091] 2. Weigh lmg mercapto ethylamine (commercially available from Sigma, USA) was dissolved in l.OmL water to give a concentration of 1.0g / L antibody resolving agent;

[0092] 3 •将1 • OmL上述步骤1的抗体溶液和291.2UL上述步骤2的抗体拆分试剂混合,室温下反应1.5小时。 [0092] 3 • 1 • OmL the above step 1 and the antibody solution 291.2UL antibody reagent mixing the above step 2 to split the reaction at room temperature for 1.5 hours.

[0093] 4•用2〇mM的PBS缓冲液(含0.15M NaCl和5.0mM EDTA,pH 6.5)对上述步骤3的反应液透析48小时,期间每3小时更换一次透析液,得到带巯基的半抗体片段。 [0093] 4 • 2〇mM with PBS buffer (with 0.15M NaCl and 5.0mM EDTA, pH 6.5) dialyzed for 48 hours the reaction step 3 above liquid, once every 3 hours to replace the dialysate during a mercapto group to give half-antibody fragment. 用甘油等体积稀释后-20°C保存。 Save -20 ° C was diluted with an equal volume of glycerol.

[0094] 实验2半抗体片段与微梁金表面的结合 2 semi-binding antibody fragment gold surface [0094] micro beam experiments

[0095] 将洗净并用氮气吹千的镀有金层的微梁(购自美国Veeco公司)放入酶标板小孔中,加入150uL浓度为5 • 0mg/mL的实验1步骤1获得的抗人参皂苷GS-Re单克隆抗体片段,37 °C温育1小时,即得到固定有半抗体片段的微梁。 [0095] The cleaned and purged with nitrogen for one thousand gold plated layer micro beam (commercially available from Veeco Instruments Inc. USA) were placed in microtiter plate wells, 150uL was added at a concentration of 5 • 0mg / mL Experiment 1 obtained in Step 1 anti ginsenoside GS-Re monoclonal antibody fragment, 37 ° C incubation for 1 hour, to obtain half-fixed antibody fragments micro beam.

[00%] 实验3直接竞争ELISA方法评价半抗体片段与微梁金表面的结合情况具体步骤如下: [00%] 3 experimental evaluation direct competitive ELISA binding of antibody fragment half microbeam gold surface the following steps:

[0097] U青洗:用镊子小心取出上述固定了半抗体片段的微梁,用洗涤液(每1L洗涤液中含有8_0g NaCl、0_2g KH2P〇4、2_96g Na2HP〇4. 12H20、1.0mL Tween-20,其余为水)冲洗数次,氮气吹干; [0097] U cyan wash: carefully removed with forceps micro beam above the fixed half-antibody fragment with a washing solution (washing solution containing per 1L 8_0g NaCl, 0_2g KH2P〇4,2_96g Na2HP〇4 12H20,1.0mL Tween-. 20, the remaining water is washed several times), dried by nitrogen;

[0098] 2•封闭:将氮气吹干后的微梁分别放入酶标板中,加入1〇〇此质量百分含量为5% 的BSA溶液(用PBS溶解)封闭,37°C温育30分钟; [0098] 2 • closed: the micro beam after nitrogen blow were placed in microtiter plates, this added 1〇〇 mass percentage of 5% BSA solution (dissolved in PBS) is closed, 37 ° C incubation 30 minutes;

[0099] 3 •清洗:用镊子小心取出微梁,用洗涤液冲洗数次,氮气吹干; [0099] 3 • Cleaning: micro beam carefully removed with forceps, rinsed several times with a washing solution, dried by nitrogen;

[0100] 4•竞争:将氮气吹干后的微梁分别放入酶标板的两个小孔中(做好标记,抑制孔与非抑制孔),抑制孔加入50yL经样品稀释液(含有终浓度为0. lmol/L pH7.5的ros、0.1%体积百分含量的吐温-20和0.1 %质量百分含量的明胶)稀释的浓度为l〇〇mg/L的人参阜苷GS-Re溶液(购自美国Sigma公司),非抑制孔加入50此样品稀释液作为对照;然后再分别加入浓度为1 • 0mg/L的人参皂苷GS-Re过氧化物酶联结物(购自美国Sigma公司),37°C温育30分钟; [0101] 5.清洗:用镊子小心取出上述抑制孔与非抑制孔中的微梁,用洗涤液冲洗数次,氮气吹干。 [0100] 4 • Competition: micro beam after nitrogen blow were placed in microtiter plate two holes (marked to be suppressed and non-suppressed bore hole), suppressing 50yL well of diluted sample solution (containing final concentration of 0. lmol / L pH7.5 the ros, the volume percentage of 0.1% gelatin, Tween-20 and 0.1% by mass percentage of) the concentration of the diluted l〇〇mg / L Fu ginseng glycoside GS -Re solution (purchased from Sigma), a non-restraining hole 50 this sample diluent was added as a control; were then added at a concentration of 1 • 0mg / L ginsenoside GS-re coupling peroxidase (purchased from U.S. Sigma Co.), 37 ° C for 30 minutes; [0101] 5. wash: carefully removed with forceps hole and said suppressing micro beam non-inhibited wells, washed several times with a washing solution, blown dry with nitrogen.

[0102] 6.显色:将氮气吹干后的两根微梁分别放入酶标板的两个小孔中,每孔加入i〇〇uL 显色溶液(TMB底物使用液与底物缓冲液按1:9的体积比混匀,每10mL上述混合液中加入4.0mg过氧化脲),室温显色; [0102] 6. color: the two micro beam after nitrogen blow holes were placed in two microtiter plates, each well was added i〇〇uL developing solution (TMB substrate is used with a substrate solution buffer 1: 9 volume ratio of mixing, the mixed solution was added per 10mL 4.0mg carbamide peroxide) at room temperature for color development;

[0103] 7.终止:15分钟后,每孔加入50UL终止液(2 • 0M的硫酸溶液)终止反应,取出微梁, 450nm波长处分别读取㈤值。 [0103] 7. Termination: After 15 minutes, was added to each well to terminate 50UL (2 • 0M solution of sulfuric acid) to terminate the reaction, remove the micro beam, at a wavelength of 450nm, respectively, (v) the value read.

[0104]实验设三次重复,结果表明,抑制孔和非抑制孔的450nm波长下的吸光度值分别为0.121和0.727,说明抗人参皂苷GS-Re半抗体片段能有效结合微梁的金表面上并具有活性。 [0104] Experimental set up in triplicate, the results suggest that inhibition of the absorbance value at a wavelength of 450nm holes and non-suppressor holes were 0.121 and 0.727, indicating that on the gold surface of the anti-ginsenoside GS-Re semi antibody fragments can effectively combine the micro-beams and active. [0105] 实验4微梁传感器效果监测 [0105] Experiment 4 micro beam sensors to monitor the effect of

[0106] 1 •清洗:无水乙醇、丙酮和PBS分别流经微梁传感系统,流速0.5mL/min; [0106] 1 • Cleaning: ethanol, acetone and PBS respectively, flows through the micro beam sensing systems, flow rate 0.5mL / min;

[0107] 2•固定微梁:将步骤2中固定有抗人参皂苷GS-Re抗体片段的微梁固定到微梁传感系统的反应池中,流动PBS通过反应池子。 [0107] 2 • fixing micro beam: The Step 2 is fixed in an anti-ginsenoside GS-Re antibody fragment micro beam fixed to a well of micro-beam sensor system, the flow through the reactor pool PBS. 排除反应池中的气泡后以〇. imL/min流动PBS。 After the reaction vessel to remove air bubbles billion. ImL / min flow of PBS. [0108] 3.调节光路:调节激光器与PSD (光电位置敏感器)的位置使激光器发出的激光照在微梁的尖端反射后被PSD接收。 [0108] 3. Adjust the optical path: the laser and adjusting the position of PSD (photoelectric position sensor) in the laser beam emitted by the laser according to the reception after reflection in the tip of the PSD micro beam.

[0109] 4.样品测定:当PSD接收的微梁偏转信号稳定后,加入2mL PBS稀释的人参皂苷GS- Re 标准样品。 [0109] 4. Determination of sample: when the micro-beam deflection received signal PSD stable, ginsenoside GS- Re added 2mL PBS diluted standard samples. 通过PSD 实时记录微梁尖端位移信息。 PSD real-time recording by the micro beam tip displacement information.

[0110]图9为抗人参皂苷GS-Re半抗体片段修饰微梁,检测不同浓度抗原时微梁尖端的时间位移曲线。 Time displacement curve when a micro tip of the beam [0110] FIG. 9 is an anti-GS-Re ginsenoside half antibody fragments modified micro beam, different concentration of antigen. 检测了三个浓度1 • 0ng/mL、0. lng/mL、0.02ng/mL的样品得到的微梁尖端位移幅值分别为77、58和32nm。 Detecting a concentration of the three 1 • 0ng / mL, 0. Lng / mL, micro beam tip displacement amplitude samples 0.02ng / mL were obtained 77,58 and 32nm. 不同浓度的人参皂苷GS-Re标准样品可以产生不同程度的微梁位移,说明抗人参皂苷GS-Re半抗体片段能有效结合到微梁的金表面上并能对人参皂苷GS-Re 进行定量检测。 Ginsenoside GS-Re standard samples with different concentrations may be generated micro beam displacement varying degrees, indicating anti ginsenoside GS-Re semi antibody fragment can be effectively bound to the gold surface of the micro beam on and can be quantitative detection of ginsenoside GS-Re . 并且具有很高的检测灵敏度(图8为利用盐酸硫醇亚胺做为巯基化试剂来巯基化抗体后将抗体固定到微梁上(中国专利CN101407548)对人参皂苷进行检测的结果)。 And has high sensitivity (FIG. 8 utilizing imine hydrochloride as a thiol reagent after thiolated antibody thiolated antibody is immobilized to the micro beam (Chinese Patent No. CN101407548) on the result of detecting ginsenoside).

[0111] 结论: [0111] Conclusion:

[0112] 用本发明的半抗体片段修饰的微梁和完整抗体修饰的微梁进行实验测试对比,检测不同浓度的人参皂苷GS-Re抗原时,微梁尖端的时间位移响应曲线(图9和图8)显示,在微梁位移响应为20纳米左右时,半抗体片段修饰和巯基化抗体修饰的检测人参皂苷GS-Re抗原浓度分别为〇• 〇2ng/mL和0 • 5ng/mL,灵敏度提高了约20倍。 When the [0112] Semi antibody fragment of the invention modified micro beam and intact antibody modified micro beams experimental tests comparing different concentrations of ginsenoside GS-Re antigen, tip timeout micro beam response curves (FIG. 9 and FIG. 8) show, when the micro beam displacement response of about 20 nm, a half-antibody fragments modification and thiolated antibody modified detection ginsenoside GS-Re antigen concentration were square • 〇2ng / mL and 0 • 5ng / mL, sensitivity increased by about 20 times.

[0113] 实施例2抗体Fab'片段修饰的微梁 [0113] Example 2 antibody Fab fragments modified micro beam "embodiment

[0114] 实验1抗体F (ab')2片段的制备 Preparation of [0114] Experiment 1 antibody F (ab ') 2 fragments

[0115] 1 •用0 • lmol/L柠檬酸缓冲液(pH3 •5)溶解人参皂苷GS-Re单克隆抗体(IgG)(购自美国USBiological公司)至10mg/ml; [0115] 1 • ginsenoside was dissolved with GS-Re 0 • lmol / L citric acid buffer (pH3 • 5) monoclonal antibody (IgG) (purchased from USBiological Corporation) to 10mg / ml;

[0116] 2•用0• lmol/L梓檬酸缓冲液(pH3_ f5)溶解胃蛋白酶(pepsin)(购自美国Sigma公司)至lmg/ml; [0116] 2 • dissolved pepsin (Pepsin) (commercially available from Sigma, USA) with 0 • lmol / L citric acid buffer Zi (pH3_ f5) to lmg / ml;

[0117] 3.将IgG:胃蛋白酶按质量比100:1混合,放在37°C水浴箱中2小时,然后加入3mol/ L Tris使pH在7左右以终止反应。 [0117] 3. IgG: pepsin mass ratio of 100: 1 were mixed in 37 ° C water bath for 2 hours, followed by addition of 3mol / L Tris at a pH of about 7 to terminate the reaction.

[0118] 4•用20mM的PBS缓冲液(含0.15M NaCl和5.0mM EDTA,pH 6.5)对上述步骤3的反应混合液透析48小时,期间每3小时更换一次透析液,得到抗体F(ab')2片段。 [0118] 4 • in 20mM PBS buffer (containing 0.15M NaCl and 5.0mM EDTA, pH 6.5) to the reaction mixture of Step 3 above dialyzed for 48 hours once every 3 hours during the replacement of the dialysis fluid, to obtain antibody F (ab ') 2 fragments.

[0119] 实验2抗体片段Fab'的制备 Preparation of [0119] Experiment 2 antibody Fab 'fragment of

[0120] 1.用去离子水溶解巯基乙胺(2-MEA)(购自美国Sigma公司)至lmg/ml; [0120] 1 was dissolved in deionized water mercaptoethylamine (2-MEA) (purchased from Sigma) to lmg / ml;

[0121] 2.将步骤1中制备的F (ab')2: 2-MEA按质量比20:1进行混合,放在37°C水浴箱中2 小时; [0121] 2. Step 1 Preparation of F (ab ') 2: 2-MEA mass ratio of 20: 1 were mixed, placed in 37 ° C water bath for 2 hours;

[0122] 3•用20mM的PBS缓冲液(含〇_1洲NaCl和5.0mM EDTA,pH 6.5)对上述步骤2的反应混合液透析48小时,期间每3小时更换一次透析液,得到抗体Fab'片段。 [0122] 3 • with 20mM PBS buffer (with NaCl and 〇_1 Island 5.0mM EDTA, pH 6.5) to the reaction mixture of step 2 above dialyzed for 48 hours once every 3 hours during the replacement of the dialysis fluid, to obtain antibody Fab 'fragment. 用甘油等体积稀释后保存在-20°C环境下。 After dilution volume and the like with glycerol and stored at -20 ° C environment.

[0123] 实验3将Fab'抗体片段修饰到微梁金表面上 [0123] Experiment 3 The Fab 'antibody fragment to a modified surface of the gold micro-beam

[0124] 将洗净并用氮气吹干的镀有金膜的微梁(购自美国Veeco公司)放入酶标板小孔中,加入15〇uL浓度为5.0mg/mL的上述步骤2制得的Fab'抗体片段溶液,放在37°C水浴箱中2 小时,即得到固定有抗体Fab'片段的微梁。 The above step [0124] The cleaned and blown dry with nitrogen plated with gold film microbeam (available from Veeco Instruments Inc. USA) were placed in microtiter plate wells, 15〇uL was added at a concentration of 5.0mg / mL of the prepared 2 the Fab 'antibody fragment solution, placed in 37 ° C water bath for 2 hours, to obtain antibodies are immobilized Fab' fragments of the micro beam.

[0125] 实验4 ELISA直接竞争法效果检测 [0125] Experiment 4 ELISA direct competitive effect detection method

[0126] 1.清洗:用镊子小心取出上述固定了抗体Fab'片段的微悬臂梁,用洗涤液侮1L洗潘液中含有8.0g NaCl、0.2g KH2P〇4、2_%g NasHPO4* l2H2〇、1.0mL Tween-20,其余为水)冲洗数次,氮气吹干; [0126] 1. Wash: carefully taken out with tweezers immobilized antibody Fab 'fragment microcantilever, insult with a washing solution containing 8.0g NaCl solution washed Pan 1L, 0.2g KH2P〇4,2_% g NasHPO4 * l2H2〇 , 1.0mL Tween-20, the remaining water is washed several times), dried by nitrogen;

[0127] 2•封闭:将氮气吹干后的微悬臂梁分别放入酶标板中,加入100uL质量百分含量为5%的BSA溶液(用PBS溶解)封闭,37°C温育30分钟; [0127] 2 • closed: the microcantilever dry nitrogen were placed in the microtiter plate, add 100uL mass percentage of blocked 5% BSA solution (dissolved in PBS), 37 ° C for 30 minutes ;

[0128] 3 •清洗:用镊子小心取出微悬臂梁,用洗潘液冲洗数次,氮气吹干; [0128] 3 • Cleaning: microcantilever carefully removed with forceps, washed several times with a washing solution Pan, blown dry with nitrogen;

[0129] 4•竞争:将氮气吹干后的微悬臂梁分别放入酶标板的两个小孔中(做好标记,抑制孔与非抑制孔),抑制孔加入50yL经样品稀释液(含有终浓度为〇• im〇i/L PH7.5的PBS、 0• 1%体积百分含量的吐温-20和0.1%质量百分含量的明胶)稀释的浓度为100mg/L的人参皂苷GS-Re溶液(购自美国Sigma公司),非抑制孔加入50uL样品稀释液作为对照;然后再分另IJ加入浓度为l_〇mg/L的人参皂苷GS-Re过氧化物酶联结物(购自美国Sigma公司),,匚温育30分钟; [0129] 4 • competition: the microcantilever after nitrogen blow were placed in microtiter plate two holes (marked to be suppressed and non-suppressed bore hole), suppressing 50yL well of diluted sample solution ( • square containing a final concentration im〇i / L PH7.5 in PBS, Tween-20 and 0.1% gelatin mass percentage of 0 • 1% of the volume percentage) was diluted to a concentration of 100mg / L ginsenoside GS-re solution (purchased from Sigma), non-inhibitory well was added 50uL sample dilution as a control; the other points then added at a concentration l_〇mg IJ / L ginsenoside GS-re peroxidase was coupled to ( was purchased from Sigma, USA) ,, contraband incubated for 30 minutes;

[0130] 5 •清洗:用镊子小心取出上述抑制孔与非抑制孔中的微悬臂梁,用洗涤液冲洗数次,氮气吹干; [0130] 5 • Wash: carefully removed with forceps hole and the micro-cantilever said suppressing non-inhibited wells, washed several times with a washing solution, dried by nitrogen;

[0131] 6.显色:将氮气吹干后的两根微悬臂梁分别放入酶标板的两个小孔中,每孔加入100UL显色溶液(TMB底物使用液与底物缓冲液按1:9的体积比混匀,每i〇mL上述混合液中加入4.0mg过氧化脲),室温显色; [0131] 6. color: two microcantilever after the nitrogen blow holes were placed in two microtiter plates, 100UL added per well developing solution (TMB Substrate working solution with substrate buffer 1: 9 volume ratio of mixing, the mixed solution was added per i〇mL 4.0mg carbamide peroxide) at room temperature for color development;

[0132] 7 •终止:15分钟后,每孔加入50此终止液(2.0M的硫酸溶液)终止反应,取出微悬臂梁,45〇nm波长处分别读取㈤值。 [0132] 7 • Termination: After 15 minutes, added to each well 50 of this stop solution (2.0M solution in sulfuric acid) to terminate the reaction, remove the microcantilever, were read at a wavelength 45〇nm v value.

[0133]实验设三次重复,结果表明,抑制孔和非抑制孔的450nm波长下的吸光度值分别为0 • 178和0 •853,说明抗人参皂苷GS-Re抗体Fab'片段能有效结合微悬臂梁的金表面上。 [0133] Experimental set up in triplicate, the results suggest that inhibition of the absorbance value at a wavelength of 450nm holes and non-suppressor holes were 0 • 178 and 0 • 853, described anti ginsenoside GS-Re antibody Fab 'fragment can effectively combine microcantilever gold on the surface of the beam.

[0134] 实验5微梁传感器效果检测 [0134] Experiment 5 Effect sensor detecting micro beam

[0135] 1 •清洗:无水乙醇、丙酮和PBS分别流经微梁传感系统,流速0.5mL/min; [0135] 1 • Cleaning: ethanol, acetone and PBS respectively, flows through the micro beam sensing systems, flow rate 0.5mL / min;

[0136] 2•固定微梁:将实验3中修饰有抗人参皂苷GS-Re抗体片段Fab'的微悬臂梁固定到微悬臂梁传感系统的反应池中,流动PBS通过反应池子。 [0136] 2 • fixing micro beam: the modified anti Experiment 3 ginsenoside GS-Re antibody Fab 'fragment microcantilever is fixed to a well microcantilever sensing system, the flow through the reactor pool PBS. 排除反应池中的气泡后以〇.lmL/ min 流动PBS。 After the reaction vessel to remove air bubbles 〇.lmL / min flow of PBS.

[0137] 3 •调节光路:调节激光器与PSD (光电位置敏感器)的位置使的激光器发出的激光照在微梁的尖端并被PSD接收。 [0137] 3 • adjusting the optical path: the laser and adjusting the laser position as the PSD (photoelectric position sensor) so that the emitted laser beam is received in the tip of the micro and PSD.

[0138] 4•样品测定:当PSD接收的微梁偏转信号稳定后,加入2mL PBS稀释的人参皂苷GS-Re 标准样品。 [0138] 4 • Sample measurement: when the micro-beam deflection signal received PSD stable, diluted 2mL PBS ginsenoside GS-Re standard sample. 通过PSD 实时记录微梁尖端位移信息。 PSD real-time recording by the micro beam tip displacement information.

[0139] 检测了二个浓度1 • 〇ng/mL、0 • 1 ng/mL、0 • 01 ng/mL的样品得到的微梁尖端位移幅值分别为79、61和21nm;不同浓度的人参皂苷GS—Re标准样品可以产生不同程度的微悬臂梁位移,说明抗人参皂苷GS-Re抗体Fab'片段能有效结合到微悬臂梁的金表面上并能对人参皂苷GS-Re进行检测。 [0139] detecting a concentration of 1 • two 〇ng / mL, 0 • 1 ng / mL, the sample micro beam tip displacement amplitude 0 • 01 ng / mL were obtained for the 79,61 and 21nm; different concentrations of ginseng GS-Re saponin standard samples can produce different degrees of displacement of the microcantilever described anti ginsenoside GS-Re antibody Fab 'fragment can be effectively bound to the gold surface of the micro-cantilever and can detect ginsenoside GS-Re.

[0140] 结论: [0140] Conclusion:

[0141]用本发明的抗体Fab'片段修饰的微梁和完整抗体修饰的微梁进行实验测试对比, 检测不同浓度的人参皂苷GS-Re抗原时,在微梁位移响应为20纳米左右时,抗体Fab'片段修饰和巯基化抗体修饰检测人参皂苷GS-Re抗原浓度分别为0.01ng/mL和0 • 5ng/mL,灵敏度提高了近40倍。 When the [0141] experimental test comparison with the antibody Fab of the present invention, 'fragments modified micro beam and intact antibody modified micro beam, different concentrations of ginsenoside GS-Re antigen, in the micro beam displacement response is about 20 nanometers, antibody Fab 'fragments modified and mercapto-modified antibody detection ginsenoside GS-Re antigen concentrations of 0.01ng / mL and 0 • 5ng / mL, nearly 40-fold increase in sensitivity.

[0142] 实施例3抗体Fab'片段修饰的微梁 [0142] EXAMPLE 3 Antibody Fab 'fragments modified embodiment micro beam

[0143] 实验1利用巯基乙胺将抗体拆分成带巯基的半抗体片段 [0143] Experiment 1 using mercaptoethylamine split into half-antibody antibody fragment with a thiol group

[0144] 1.准确称取人参皂苷GS-Re单克隆抗体(购自美国USBiological公司)7.0mg溶于l.OmL PBS (含0.15M NaCl和5.0mM EDTA,pH 6.5)中得到浓度为7.0g/L的抗体溶液; [0144] 1. Weigh accurately ginsenoside GS-Re monoclonal antibody (purchased from USBiological Corporation) 7.0 mg was dissolved in l.OmL PBS (containing 0.15M NaCl and 5.0mM EDTA, pH 6.5) to give a concentration of 7.0g / L of antibody solution;

[0145] 2•称取lmg巯基乙胺(购自美国Sigma公司)溶于l.OmL水中得到浓度为l.〇g/L的抗体拆分试剂; [0145] 2 • weighed lmg mercaptoethylamine (purchased from Sigma) was dissolved in water to give a concentration of l.〇g l.OmL / L antibody resolving agent;

[0146] 3•将1 .OmL上述步骤1的抗体溶液和291.2此上述步骤2的抗体拆分试剂混合,室温下反应1.5小时。 [0146] 3 • 1 .oml the above step antibody solution 1 and 291.2 of this antibody reagent mixing resolved the above step 2, the reaction at room temperature for 1.5 hours.

[0147] 4•用20mM的PBS缓冲液(含0.15M NaCl和5.0mM EDTA,pH 6.5)对上述步骤3的反应液透析48小时,期间每3小时更换一次透析液,得到带巯基的半抗体片段。 [0147] 4 • in 20mM PBS buffer (containing 0.15M NaCl and 5.0mM EDTA, pH 6.5) reaction of the above step 3 was dialyzed for 48 hours half-antibody once every 3 hours during the replacement of the dialysis fluid, to obtain a mercapto group fragments. 用甘油等体积稀释后-20 °C保存。 Save -20 ° C was diluted with an equal volume of glycerol.

[0148]实验2抗体Fab'片段的制备 Preparation of [0148] Experiment 2 antibody Fab 'fragments

[0149] 1.用0_lm〇l/L柠檬酸缓冲液(pH3.5)溶解胃蛋白酶(pepsin)(购自美国Sigma公司)至lmg/ml; [0149] 1. dissolved with pepsin 0_lm〇l / L citrate buffer (pH3.5) (pepsin) (purchased from Sigma) to lmg / ml;

[0150] 2 •将步骤1中制备的半抗体片段与胃蛋白酶按质量比100:1混合反应,放在37°C水浴箱中2小时,然后加入3mol/L Tris使pH在7左右以终止反应。 [0150] 2 • Semi pepsin antibody fragment prepared in Step 1 in a mass ratio of 100: 1, mixed and reacted in 37 ° C water bath for 2 hours, followed by addition of 3mol / L Tris at a pH of about 7 to terminate reaction.

[0151] 3.用20禮的? [0151] 3. 20 ceremony? 83缓冲液(含0.151恥(:1和5.(^1\^0了4,?116.5)对上述步骤2的反应混合液透析48小时,期间每3小时更换一次透析液,得到抗体Fab'片段。用甘油等体积稀释后保存在-20°C环境下。 83 buffer (0.151 shame (:? 1 and 5 (^ 1 \ 4 ^ 0, 116.5) 2 the reaction mixture was dialyzed against the above-described steps 48 hours, once every 3 hours during the replacement of the dialysis fluid, to obtain the antibody Fab ' fragments diluted with a volume such as glycerin and stored at -20 ° C environment.

[0152] 实验3将抗体Fab'片段修饰到微梁金表面上 [0152] Experiment 3 antibody Fab 'fragment to a modified surface of the gold micro-beam

[0153]将洗净并用氮气吹干的镀有金膜的微梁(购自美国Veeco公司)放入酶标板小孔中,加入150此浓度为5 • 0mg/mL的上述步骤2制得的抗体Fab'片段溶液,放在37 °C水浴箱中2 小时,即得到固定有抗体Fab'片段的微梁。 [0153] The cleaned and blown dry with nitrogen plated with gold film microbeam (available from Veeco Instruments Inc. USA) were placed in microtiter plate wells, 150 of this concentration 5 • 0mg / mL prepared in the above Step 2 antibody Fab 'fragment was placed in 37 ° C water bath for 2 hours, to obtain antibodies are immobilized Fab' fragments of the micro beam.

[0154] 实验4 ELISA直接竞争法效果检测 [0154] Experiment 4 ELISA direct competitive effect detection method

[0155] 1.清洗:用镊子小心取出上述固定了抗体Fab'片段的微悬臂梁,用洗涤液(每1L洗涤液中含有8.0g NaCl、0.2g KH2P〇4、2.96g Na2HP〇4* 12H20、1.0mL Tween-20,其余为水)冲洗数次,氮气吹千; [0155] 1. Wash: carefully taken out with tweezers immobilized antibody Fab 'fragments of the micro-cantilever, with a washing solution (1L per wash solution containing 8.0g NaCl, 0.2g KH2P〇4,2.96g Na2HP〇4 * 12H20 , 1.0mL Tween-20, the remainder is washed several times water), nitrogen purge one thousand;

[0156] 2 •封闭:将氮气吹干后的微悬臂梁分别放入酶标板中,加入lOOyL质量百分含量为5%的BSA溶液(用PBS溶解)封闭,37°C温育30分钟; [0156] 2 • closed: the microcantilever after nitrogen blow were placed in microtiter plates, was added lOOyL mass percentage of blocked 5% BSA solution (dissolved in PBS), 37 ° C for 30 minutes ;

[0157] 3 •清洗:用镊子小心取出微悬臂梁,用洗涤液冲洗数次,氮气吹干; [0157] 3 • Cleaning: microcantilever carefully removed with forceps, rinsed several times with a washing solution, dried by nitrogen;

[0158] 4.竞争:将氮气吹干后的微悬臂梁分别放入酶标板的两个小孔中(做好标记,抑制孔与非抑制孔),抑制孔加入50uL经样品稀释液(含有终浓度为〇.lm〇l/L pH 7.5的PBS、 0 • 1 %体积百分含量的吐温-20和0 • 1 %质量百分含量的明胶)稀释的浓度为i〇〇mg/L的人参皂苷GS-Re溶液(购自美国Sigma公司),非抑制孔加入50此样品稀释液作为对照;然后再分别加入浓度为1.0mg/L的人参皂苷GS-Re过氧化物酶联结物(购自美国Sigma公司),37°C温育30分钟; 10159] 5 •浪洗:用镊子小心取出上述抑制孔与非抑制孔中的微悬臂梁,用洗涤液冲洗数次,氮气吹干; [0158] 4. Competition: the microcantilever after nitrogen blow were placed in microtiter plate two holes (marked to be suppressed and non-suppressed bore hole), suppressing 50uL well of diluted sample solution ( PBS containing a final concentration 〇.lm〇l / L pH 7.5, the gelatin concentration of 0 • 1% volume percentage of Tween-20 and 0 • 1% by mass percentage content) was diluted i〇〇mg / L ginsenoside GS-re solution (purchased from Sigma), a non-restraining hole 50 this sample diluent was added as a control; were then added at a concentration of 1.0mg / L ginsenoside GS-re was coupled to peroxidase (commercially available from Sigma, USA), 37 ° C for 30 minutes; 10159] 5 • waves wash: microcantilever carefully removed with forceps hole and to suppress the above-described non-inhibited wells, washed several times with a washing solution, blown dry with nitrogen ;

[0160] 6 •显色:将氮气吹干后的两根微悬臂梁分别放入酶标板的两个小孔中,每孔加入100UL显色溶液(TMB底物使用液与底物缓冲液按1:9的体积比混匀,每l〇mL上述混合液中加入4.0mg过氧化脲),室温显色; [0160] 6 • color: the two micro-cantilevers after the nitrogen blow holes were placed in two microtiter plates, 100UL added per well developing solution (TMB Substrate working solution with substrate buffer 1: 9 volume ratio of mixing, the mixed solution was added per l〇mL 4.0mg carbamide peroxide) at room temperature for color development;

[0161] 7 •终止:15分钟后,每孔加入50uL终止液(2.0M的硫酸溶液)终止反应,取出微悬臂梁,450nm波长处分别读取0D值。 [0161] 7 • Termination: After 15 minutes, added to 50uL stop solution per well (in 2.0M sulfuric acid solution) to terminate the reaction, remove the microcantilever, at a wavelength of 450nm are read 0D value.

[0162]实验设三次重复,结果表明,抑制孔和非抑制孔的450nm波长下的吸光度值分别为0 • 152和0 • 763,说明抗人参皂苷GS-Re抗体Fab'片段能有效结合微悬臂梁的金表面上。 [0162] Experimental set up in triplicate, the results suggest that inhibition of the absorbance value at a wavelength of 450nm holes and non-suppressor holes were 0 • 152 and 0 • 763, described anti ginsenoside GS-Re antibody Fab 'fragment can effectively combine microcantilever gold on the surface of the beam.

[0163]实验5微梁传感器效果检测 [0163] Experiment 5 Effect sensor detecting micro beam

[0164] 1 •清洗:无水乙醇、丙酮和PBS分别流经微梁传感系统,流速〇.5mL/min; [0164] 1 • Cleaning: ethanol, acetone and PBS respectively, flows through the micro beam sensing systems, flow 〇.5mL / min;

[0165] 2.固定微梁:将实验3中修饰有抗人参皂苷GS-Re抗体Fab'片段的微悬臂梁固定到微悬臂梁传感系统的反应池中,流动PBS通过反应池子。 [0165] 2. Fixed micro beam: Experiment 3 The modified anti microcantilever ginsenoside GS-Re antibody Fab 'fragment is fixed to a well microcantilever sensing system, the flow through the reactor pool PBS. 排除反应池中的气泡后以〇. lmL/ min 流动PBS。 After the reaction vessel to remove air bubbles billion. LmL / min flow of PBS.

[0166] 3.调节光路:调节激光器与PSD (光电位置敏感器)的位置使的激光器发出的激光照在微梁的尖端并被PSD接收。 [0166] 3. Adjust the optical path: the laser and adjusting the laser position as the PSD (photoelectric position sensor) so that the emitted laser beam is received in the tip of the micro and PSD.

[°167^ 4•样品测定:当PSD接收的微梁偏转信号稳定后,加入2mL PBS稀释的人参皂苷GS- Re标准样品。 ° 167 ^ 4 • Sample measurement [: when the micro-beam deflection signal received PSD stable, diluted 2mL PBS ginsenoside GS- Re standard sample. 通过PSD实时记录微梁尖端位移信息。 PSD real-time recording by the micro beam tip displacement information.

[0168] 检测二个浓度1.0ng/mL、0. lng/mL、0.015ng/mL的样品得到的微梁尖端位移幅值分别为68、42和22nm;不同浓度的人参皂苷GS-Re标准样品可以产生不同程度的微悬臂梁位移,说明抗人参皂苷GS-Re抗体Fab'片段能有效结合到微悬臂梁的金表面上并能对人参皂苷GS-Re进行检测。 [0168] detecting two concentration 1.0ng / mL, 0 lng / mL, micro beam tip displacement amplitude samples 0.015ng / mL were obtained for the 22nm and 68,42;. Ginsenoside GS-Re standard samples with different concentrations of can produce different degrees of displacement of the microcantilever described anti ginsenoside GS-Re antibody Fab 'fragment can be effectively bound to the gold surface of the micro-cantilever and can detect ginsenoside GS-Re.

[0169]结论: [0169] Conclusion:

[0170]通过先拆分后酶解的方法制备的本发明的抗体Fab'片段修饰的微梁和完整抗体修饰的微梁进行实验测试对比,检测不同浓度的人参皂苷GS-Re抗原时,在微梁位移响应为20纳米左右时,抗体Fab'片段修饰和巯基化抗体修饰检测人参皂苷GS-Re抗原浓度分别为0 • 015ng/mL和0 • 5ng/mL,灵敏度提高了近33倍。 When the [0170] Fab antibody of the present invention prepared by the process of digestion after the first split 'fragments modified micro beam and a modified intact antibody test comparison micro beam experiments, different concentrations of ginsenoside GS-Re antigen, in when the micro-beam displacement response of about 20 nanometers, an antibody Fab 'fragments modified and mercapto groups of the modified antibody detection ginsenoside GS-Re antigen concentrations of 0 • 015ng / mL and 0 • 5ng / mL, the sensitivity increased nearly 33 fold.

[0171] 通过上述非限制性的实施例,可以看出,本发明的方法和通过本发明的方法制备的微悬臂梁在免疫传感检测技术中与现有技术的微梁法相比可以提高20-40倍,相当于常规酶联免疫法的200-400倍,实现了本发明的目的,即在食品安全、环境污染、生物医学、科学研宄和生产制造等领域中监控和检测极微量被分析物的可能性和实用性,完全可以实现实际的商业化应用。 [0171] By the above-described non-limiting embodiments, it can be seen, the method of the present invention and prepared by the process of the micro-cantilever of the present invention in an immunoassay sensing techniques and prior art micro-beam method can be improved as compared to 20 -40-fold, 200-400-fold equivalent to a conventional ELISA method, to achieve the object of the present invention, i.e., food safety, environmental pollution, biomedical, and scientific study based on manufacturing and other areas are to monitor and detect a trace amount of the possibility and usefulness of the analyte, can achieve real business applications.

[0172] 参考文献 [0172] Reference

[0173] [l]Koutilya R.Buchapudi ,Xin Huang,Xin Yang,HaiFeng Ji and Thomas Thundat?Microcantilever biosensors for chemicals and bioorganisms.Analyst, 2011,136,1539-1556 [0173] [l] Koutilya R.Buchapudi, Xin Huang, Xin Yang, HaiFeng Ji and Thomas Thundat? Microcantilever biosensors for chemicals and bioorganisms.Analyst, 2011,136,1539-1556

[0174] [2]Weiming Tan,Yuan Huang,Tiegui Nan,Changguo Xue?Zhaohu Li?Qingchuan Zhang?and Baominffang,Development of Protein A Functionalized Microcantilever Immunosensors for the Analyses of Small Molecules at part per trillion Levels.Analysis Chemistry?2010?82 (2) ,615-620 [0174] [2] Weiming Tan, Yuan Huang, Tiegui Nan, Changguo Xue? Zhaohu Li? Qingchuan Zhang? And Baominffang, Development of Protein A Functionalized Microcantilever Immunosensors for the Analyses of Small Molecules at part per trillion Levels.Analysis Chemistry? 2010 ? 82 (2), 615-620

[0175] [3] Hongwei Zhao ;Changguo Xue; Tiegui Nan;Guiyu Tan; Zhaohu Li ; Qing X. Li;Qingchuan Zhang;Baomin Wang ? Detection of Copper Ions Using Microcantilever Immunosensors and Enzyme-linked Immunosorbent Assay?Analytica Chimica Acta,2010,676,8卜86, [0175] [3] Hongwei Zhao; Changguo Xue; Tiegui Nan; Guiyu Tan; Zhaohu Li; Qing X. Li; Qingchuan Zhang; Baomin Wang Detection of Copper Ions Using Microcantilever Immunosensors and Enzyme-linked Immunosorbent Assay Analytica Chimica Acta,?? 2010,676,8 BU 86,

[0176] [4] Changguo Xue,Hongwei Zhao,Yanyun Chen?Baomin Wang,Qingchuan Zhang? Xiaoping Wu, Development of sulfhydrylated antibody functionalized microcantilever immunosensor for taxol,Sensors and Actuators B,2011,156,863-866 [0176] [4] Changguo Xue, Hongwei Zhao, Yanyun Chen? Baomin Wang, Qingchuan Zhang? Xiaoping Wu, Development of sulfhydrylated antibody functionalized microcantilever immunosensor for taxol, Sensors and Actuators B, 2011,156,863-866

[0177] [5]A.Kausaite-Minkstimiene?A. Ramanavi c iene?J.Kirlyte?and A . Ramanav icius.Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor.Analysis Chemistry,2010,82,6401-6408 [0177] [5] A.Kausaite-Minkstimiene? A. Ramanavi c iene? J.Kirlyte? And A. Ramanav icius.Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor.Analysis Chemistry, 2010,82 , 6401-6408

[0178] [6] [J]仇凯等,木瓜蛋白酶制备抗人肝癌抗F(ab')2及Fab片段。 [0178] [6] [J] Qiu Kay et al., Papain preparation of anti-anti-human hepatoma F (ab ') 2 and Fab fragments. 第四军医大学学报,1995;16®^^*^。 Fourth Military Medical University, 1995; 16® ^^ * ^.

Claims (10)

  1. 1. 一种制备微悬臂梁的方法,包括以下步骤: 1) 提供抗体和带有镀金层的微悬臂梁; 2) 二硫键还原步骤:使用二硫键还原剂处理所述抗体,使得所述抗体铰链区的二硫键还原成巯基,从而得到两个对称的带有巯基的半抗体片段; 3) 固定步骤:将步骤2)获得的半抗体片段与镀有金层的微悬臂梁结合,得到镀金表面上固定有所述半抗体片段的微悬臂梁。 1. A process for preparing micro-cantilever, comprising the following steps: 1) providing an antibody with the micro-cantilever and the gold plating layer; 2) disulfide bond reduction steps: the antibody using disulfide reducing agent, such as said antibody hinge region disulfide bonds reduced to the mercapto group, to give a half-antibody fragment with two symmetrical thiol group; 3) a fixing step of: half-antibody fragments step 2) is obtained with a plating layer of gold binding microcantilever to afford the semi-fixed antibody fragments on the plated surface of the microcantilever.
  2. 2. —种制备微悬臂梁的方法,包括以下步骤: 1) 提供抗体和带有镀金层的微悬臂梁; 2) 酶解步骤:用蛋白酶将所述抗体裂解成F(ab')2片段和多个小分子片段,所述蛋白酶选自胃蛋白酶、木瓜蛋白酶或其组合; 3) 二硫键还原步骤:使用二硫键还原剂处理所述F (ab')2片段,使得所述抗体铰链区的二硫键还原成巯基,从而得到两个对称的带有巯基的Fab'片段; 4) 固定步骤:将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金表面上固定有所述Fab'片段的微悬臂梁。 2. - Preparation of the microcantilever species, comprising the steps of: a) providing an antibody with the micro-cantilever and the gold plating layer; 2) hydrolysis step: the antibody with a protease cleavage of F (ab ') 2 fragments and a plurality of small molecule fragments, the protease is selected from pepsin, papain, or a combination thereof; 3) the step of reduction of disulfide bonds: reducing agent of the disulfide using F (ab ') 2 fragments, such that the antibody hinge region disulfide bonds reduced to the thiol group, whereby two symmetrical Fab having a mercapto group 'fragment; 4) fixing step: step 3 Fab)' obtained fragment microcantilever plated gold layer obtained by combining said fixed microcantilever Fab 'fragments of the plated surface.
  3. 3. —种制备微悬臂梁的方法,包括以下步骤: 1) 提供抗体和带有镀金层的微悬臂梁; 2) 二硫键还原步骤:使用二硫键还原剂处理所抗体,使得所述抗体较链区的二硫键还原成巯基,从而得到两个对称的带有巯基的半抗体片段; 3) 酶解步骤:用蛋白酶将所述半抗体片段裂解成Fab'片段和多个小分子片段,所述蛋白酶选自胃蛋白酶、木瓜蛋白酶或其组合; 4) 固定步骤:将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述Fab'片段的微悬臂梁。 3. - Preparation of the microcantilever species, comprising the steps of: a) providing an antibody with the micro-cantilever and the gold plating layer; 2) a disulfide bond reduction step: The antibodies used were disulfide bond reducing agent, such that the chain antibody region more disulfide is reduced to mercapto group, to obtain two symmetrical half-antibody fragment with a thiol group; 3) digestion step of: using a protease is cleaved into the half-antibody fragment Fab 'fragment and a plurality of small molecules fragment, the protease selected from pepsin, papain, or a combination thereof; 4) the step of fixing: Fab 'fragment in step 3) is obtained with a plating layer of gold has a microcantilever bound, Fab obtained fixed on the gold plated layer 'microcantilever fragments.
  4. 4. 一种应力传感元件,其包括基于应力的微悬臂梁,所述微悬臂梁的镀金层上固定有抗体的半抗体片段或Fab'片段,所述应力改变,从而导致微梁弯曲变形,通过光学或电学方法检测这种变形的过程,可得到免疫生化反应的实时信息。 A strain sensing element comprises a stress-based micro-cantilever, a fixed half antibody fragments or Fab 'fragments, a gold plating layer on the stress changes of the micro-cantilever, micro beam resulting in bending detecting this deformation process by optical or electrical methods, real-time information can be obtained immune biochemical reactions.
  5. 5.—种基于应力的微悬臂梁免疫传感检测系统,其包括权利要求4的应力传感元件,所述应力改变,从而导致微梁弯曲变形,通过光学或电学方法检测这种变形的过程,可得到免疫生化反应的实时信息。 5.- Process species microcantilever immune stress-based sensing system, in which the stress sensing element 4, including the claims, the stress change, thereby causing micro beam bending deformation which is detected by optical or electrical methods , real-time information can be obtained immune biochemical reactions.
  6. 6. —种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: 1) 提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; 2) 使用二硫键还原剂处理所述抗体,使得所述抗体铰链区的二硫键还原成巯基,从而得到两个对称的带有巯基的半抗体片段; 3) 将步骤2)获得的半抗体片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述半抗体片段的微悬臂梁; 4) 将步骤3)获得的固定有所述半抗体片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; 5) 将待测样品加入至步骤4)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原; 所述应力改变,从而导致微梁弯曲变形,通过光学或电学方法检测这种变形的过程,可得到免疫生化反应的实时 6. - kinds sensing microcantilever sensing system immunoassay test sample, comprising the steps of: a) providing a target antibody specific for the antigen and microcantilever sensing system, said transmission microcantilever sensing detection system comprises a reaction cell and a stress-based micro-cantilever with a gold plating layer; 2) a disulfide bond reducing agent using the antibody so that the antibody hinge region disulfide bonds reduced to the thiol group, whereby two symmetrical half-antibody fragment having a mercapto group; half antibody fragment 3) step 2) obtained and the plated gold layer microcantilever binding, fixed on the gold plating layer to give the microcantilever the half-antibody fragment; 4) in step 3) are fixed to said semi-obtained antibody fragments micro-cantilever is fixed to a well microcantilever sensing system; 5) the sample to be tested is added to step 4) obtained by sensing microcantilever system, detecting whether the test sample in the presence of the target antigen; real-time change of the stress, leading to micro beam bending deformation which is detected by an optical or electrical process method, to obtain an immune response and biochemical 息。 Information.
  7. 7.—种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: 1) 提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; 2) 用蛋白酶将所述抗体裂解成F(ab')2片段和多个小分子片段,所述蛋白酶选自冑蛋白酶、木瓜蛋白酶或其组合; 3) 使用二硫键还原剂处理所述F (ab')2片段,使得所述抗体铰链区的二硫键还原成巯基,从而得到两个对称的带有巯基的Fab'片段; 4) 将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述Fab'片段的微悬臂梁; 5) 将步骤4)获得的固定有所述Fab'片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; 6) 将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原; 所述应 7.- kinds sensing microcantilever sensing system immunoassay test sample, comprising the steps of: a) providing a target antibody specific for the antigen and microcantilever sensing system, said transmission microcantilever sensing detection system comprises a reaction cell and a stress-based micro-cantilever with a gold plating layer; 2) the antibody with a protease cleavage of F (ab ') 2 fragments and multiple small molecule fragment, the protease is a protease selected helmet , papain or a combination thereof; 3) the use of a disulfide bond reducing agent F (ab ') 2 fragments, such that the antibody hinge region disulfide bonds reduced to the thiol group, whereby two symmetrical having a mercapto group Fab 'fragments; Fab & 4) in step 3)' obtained fragment microcantilever plated gold layer obtained by combining the micro-cantilever is fixed to Fab 'fragments of the plated layer; 5) in step 4) is obtained fixed to the Fab 'fragment of the micro-cantilever is fixed to a well microcantilever sensing system; 6) the sample to be tested is added to the step 5) obtained microcantilever sensing system, the detection test the presence or absence of the target antigen in the sample; should the 改变,从而导致微梁弯曲变形,通过光学或电学方法检测这种变形的过程,可得到免疫生化反应的实时信息。 Change, thereby causing micro beam bending deformation, this deformation is detected by an optical or electrical process methods, real-time information obtained immune biochemical reactions.
  8. 8. —种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: 1) 提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; 2) 使用二硫键还原剂处理所述抗体,使得所述抗体铰链区的二硫键还原成巯基,从而得到两个对称的带有巯基的半抗体片段; 3) 用蛋白酶将所述半抗体片段裂解成Fab'片段和多个小分子片段,所述蛋白酶选自胃蛋白酶、木瓜蛋白酶或其组合; 4) 将步骤3)获得的Fab'片段与镀有金层的微悬臂梁结合,得到镀金层上固定有所述Fab'片段的微悬臂梁; 5) 将步骤4)获得的固定有所述Fab'片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; 6) 将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原; 所述应 8. - kinds sensing microcantilever sensing system immunoassay test sample, comprising the steps of: a) providing a target antibody specific for the antigen and microcantilever sensing system, said transmission microcantilever sensing detection system comprises a reaction cell and a stress-based micro-cantilever with a gold plating layer; 2) a disulfide bond reducing agent using the antibody so that the antibody hinge region disulfide bonds reduced to the thiol group, whereby two symmetrical half-antibody fragment having a mercapto group; and 3) the half-antibody with a protease is cleaved into fragments' fragments and a plurality of small molecule fragments Fab, the protease is selected from pepsin, papain, or a combination thereof; 4) the step of . 3) obtained Fab 'fragment microcantilever plated gold layer obtained by combining fixed on the gold plated layer Fab' fragments of the microcantilever; 5) in step 4) are fixed to obtain the Fab 'fragments is fixed to a well microcantilever sensing microcantilever system; 6) the sample to be tested is added to the step 5) microcantilever sensing system obtained, the presence or absence of the target antigen in the sample to be tested ; should the 改变,从而导致微梁弯曲变形,通过光学或电学方法检测这种变形的过程,可得到免疫生化反应的实时信息。 Change, thereby causing micro beam bending deformation, this deformation is detected by an optical or electrical process methods, real-time information obtained immune biochemical reactions.
  9. 9. 根据权利要求1-8中任一项所述的方法、应力传感元件或免疫传感检测系统,其特征在于所述二硫键还原剂选自疏基乙胺、2-疏基乙醇、二硫苏糖醇中的一种或多种。 9. The method as claimed in any one of claims immune stress sensing element or sensing system, wherein said disulfide reducing agent is selected mercapto ethylamine, 2-mercaptoethanol , dithiothreitol one or more.
  10. 10. 根据权利要求1-9中任一项所述的方法、应力传感元件或免疫传感检测系统,其特征在于所述抗体为IgM、IgG、IgA、IgD、IgE中的任一种。 10. The method claimed in any one of claims immune stress sensing element or sensing system, wherein said antibody is an IgM, IgG, IgA, IgD, IgE any one of.
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