CN115902224A - High Sensitivity Immunochromatography Detection Kit - Google Patents

Abstract

Translated fromChinese

本发明涉及高灵敏度免疫层析检测试剂盒，包括抗体检测液盛放装置和试纸条，其中所述抗体检测液盛放装置中含有相应的抗体检测液；所述由反应垫、样品垫、吸水垫在底板上依次交错地组装而成，反应垫一端连接样品垫，另一端与吸水垫相连，反应垫预包被有相互分离的检测线和质控线，其特征在于：在试纸条和标记物上均结合有生物桥。本发明进一步涉及利用所述试剂盒进行免疫层析检测的方法。通过本发明的试剂盒进行免疫层析测试，提高了检测灵敏度，降低了抗体用量。

The invention relates to a high-sensitivity immunochromatography detection kit, comprising an antibody detection liquid storage device and a test strip, wherein the antibody detection liquid storage device contains corresponding antibody detection liquid; the reaction pad, sample pad, The water-absorbing pads are assembled on the bottom plate in a staggered order. One end of the reaction pad is connected to the sample pad, and the other end is connected to the water-absorbing pad. The reaction pad is pre-coated with a detection line and a quality control line that are separated from each other. It is characterized by: Biological bridges are combined with the markers. The present invention further relates to a method for immunochromatographic detection using the kit. The immunochromatographic test is carried out through the kit of the invention, which improves the detection sensitivity and reduces the antibody dosage.

Description

Translated fromChinese

高灵敏度免疫层析检测试剂盒High Sensitivity Immunochromatography Detection Kit

技术领域technical field

本发明属于免疫学检测领域，具体而言，本发明涉及一种高灵敏度免疫层析检测试剂盒。The invention belongs to the field of immunological detection, in particular, the invention relates to a high-sensitivity immunochromatographic detection kit.

背景技术Background technique

免疫层析技术，或称侧向流层析技术，因其检测快速、操作简便、成本低廉的优点而广泛应用于食品安全、临床医学检验等领域。根据待测物质分子量大小的差异，免疫层析技术的检测模式分为适用于小分子物质的竞争法和适用于分子量大的物质的双抗夹心法。Immunochromatography, or lateral flow chromatography, is widely used in food safety, clinical medical testing and other fields due to its advantages of rapid detection, easy operation and low cost. According to the difference in molecular weight of the substances to be tested, the detection mode of immunochromatography technology is divided into the competition method suitable for small molecular substances and the double antibody sandwich method suitable for substances with large molecular weight.

现有的免疫层析技术存在检测灵敏度不高，抗体利用率低的问题，主要原因如下：1、标记物与抗体的连接有关，标记物与抗体的连接存在无序性，即标记物既可以连接抗体的恒定区，使可变区(与抗原结合的局域)充分暴露；也可以连接抗体的可变区，使抗体失效或效果降低；2、标记物与抗体直接连接，由于二者之间的距离过近，形成空间位阻，降低抗体与抗原的结合效率；3、在层析过程中，由于流速较快，在检测线位置抗体抗原结合的不够充分。The existing immunochromatography technology has the problems of low detection sensitivity and low antibody utilization. The main reasons are as follows: 1. The connection between the marker and the antibody is related, and the connection between the marker and the antibody is disordered, that is, the marker can be Connect the constant region of the antibody to fully expose the variable region (the region that binds to the antigen); it can also be connected to the variable region of the antibody to make the antibody invalid or reduce its effect; 2. The label is directly connected to the antibody. 3. During the chromatography process, due to the fast flow rate, the antibody-antigen binding at the detection line is not sufficient.

为了解决以上问题，本发明提供了一种高灵敏度的试剂盒，在标记物与检测线位置设有“生物桥”，利用所述试剂盒的免疫层析测试，提高了检测灵敏度，降低了抗体用量。In order to solve the above problems, the present invention provides a high-sensitivity kit, which is provided with a "biological bridge" at the position of the marker and the detection line. Using the immunochromatographic test of the kit, the detection sensitivity is improved and the antibody concentration is reduced. Dosage.

发明内容Contents of the invention

本发明的目的在于提供一种高灵敏度免疫层析检测试剂盒，包括试纸条和抗体检测液盛放装置，其中所述装置中含有相应的抗体检测液；所述试纸条由反应垫、样品垫、吸水垫在底板上依次交错地组装而成，所述反应垫预包被有相互分离的检测线和质控线，其特征在于：在试纸条和抗体检测液中均结合有生物桥。The object of the present invention is to provide a high-sensitivity immunochromatographic detection kit, including a test strip and an antibody detection solution holding device, wherein the device contains a corresponding antibody detection solution; the test strip consists of a reaction pad, The sample pad and the water-absorbing pad are sequentially assembled on the bottom plate, and the reaction pad is pre-coated with a detection line and a quality control line that are separated from each other. It is characterized in that: both the test strip and the antibody detection solution are combined with biological bridge.

所述生物桥可以有多个，例如两个、三个，其一位于试纸条的反应垫上，结合在检测线位置，其它生物桥位于抗体检测液中，与标记物结合。There may be multiple biological bridges, such as two or three, one of which is located on the reaction pad of the test strip and combined with the detection line, and the other biological bridges are located in the antibody detection solution and combined with the marker.

所述试纸条的反应垫一端连接样品垫，另一端与吸水垫相连；反应垫、样品垫、吸水垫例如可以相互交错1.5-2.5mm，例如2mm。One end of the reaction pad of the test strip is connected to the sample pad, and the other end is connected to the water-absorbing pad; the reaction pad, the sample pad, and the water-absorbing pad can be interlaced by 1.5-2.5 mm, for example, 2 mm.

所述“生物桥”为可特异结合抗体恒定区的生物制品，且具有高亲和力。所述生物桥为能与抗体特异性结合的抗抗体、蛋白A、蛋白G等分子。生物桥保证至少一个功能区是特异性结合检测抗体的恒定区段。The "biological bridge" is a biological product that can specifically bind to the constant region of an antibody with high affinity. The biological bridge is anti-antibody, protein A, protein G and other molecules that can specifically bind to the antibody. Biobridge ensures that at least one functional domain is a constant segment that specifically binds the detection antibody.

所述抗体检测液包括抗体1、抗体2和标记物偶联的生物桥1。The antibody detection solution includesantibody 1,antibody 2 and marker-coupledbiobridge 1.

所述抗体1和抗体2可以为单克隆抗体，或多克隆抗体，或单链抗体，优选为单克隆抗体；所述抗体1与抗体2为不同种属的抗体。Theantibody 1 and theantibody 2 can be monoclonal antibodies, polyclonal antibodies, or single-chain antibodies, preferably monoclonal antibodies; theantibody 1 andantibody 2 are antibodies of different species.

所述标记物可以是乳胶颗粒、也可以是胶体金、胶体碳、胶体硒、超顺磁性颗粒、荧光颗粒、上转换发光颗粒等标记物。The markers can be latex particles, colloidal gold, colloidal carbon, colloidal selenium, superparamagnetic particles, fluorescent particles, up-conversion luminescent particles and other markers.

所述生物桥1为能与抗体1的恒定区特异性结合的抗抗体、蛋白A、蛋白G等分子。Thebiobridge 1 is an anti-antibody, protein A, protein G and other molecules that can specifically bind to the constant region of theantibody 1.

在标记物和抗体1之间具有生物桥1，生物桥1可特异结合抗体1恒定区，且具有高亲和力，可提高抗体1的利用率。可使抗体1的可变区充分暴露，且使抗体1远离标记物，一定程度降低空间位阻，从而提高检测灵敏度。There is abiological bridge 1 between the marker and theantibody 1, and thebiological bridge 1 can specifically bind to the constant region of theantibody 1 with high affinity, and can improve the utilization rate of theantibody 1. The variable region of theantibody 1 can be fully exposed, and theantibody 1 can be kept away from the label to reduce steric hindrance to a certain extent, thereby improving the detection sensitivity.

所述试纸条的检测线含有生物桥2。所述生物桥2为能与抗体2的恒定区特异性结合的抗抗体、蛋白A、蛋白G等分子；所述质控线含有能与所述生物桥1特异性结合的抗体3。The detection line of the test strip contains Biobridge 2. Thebiobridge 2 is an anti-antibody, protein A, protein G and other molecules that can specifically bind to the constant region of theantibody 2; the quality control line contains theantibody 3 that can specifically bind to thebiobridge 1.

所述抗体3可以为单克隆抗体，也可以为多克隆抗体，优选为多克隆抗体。Theantibody 3 can be a monoclonal antibody or a polyclonal antibody, preferably a polyclonal antibody.

在一个实施方案中，本发明的试剂盒为双抗体夹心模式。在此模式下，共有两个生物桥，所述两个生物桥分别特异性结合双抗夹心中的两个抗体，即：一个生物桥连在标记物上，一个生物桥连接在试纸条检测线上。In one embodiment, the kit of the present invention is a double-antibody sandwich format. In this mode, there are two biological bridges, which specifically bind to the two antibodies in the double-antibody sandwich, namely: one biological bridge is connected to the marker, and the other is connected to the test strip detection on-line.

在待测物加入试纸条层析前，先与抗体1、抗体2和标记物偶联的生物桥1混合，反应充分；并且，在层析过程中，检测线位置有可以与抗体2特异性结合的生物桥，与抗体2的结合力高。Before the test substance is added to the test strip chromatography, it is mixed withantibody 1,antibody 2 and thebiobridge 1 coupled with the marker, and the reaction is sufficient; and, during the chromatography process, the position of the detection line may be specific to antibody 2 A sex-binding biobridge with high binding affinity toAntibody 2.

与直接在反应垫上包被抗体2的传统方法相比，本发明的技术方案中对于抗体2利用率更高，检测灵敏度更高。Compared with the traditional method of directly coating theantibody 2 on the reaction pad, the technical solution of the present invention has a higher utilization rate of theantibody 2 and a higher detection sensitivity.

本发明的目的还在于提供一种高灵敏度的免疫层析检测方法，其特征在于使用本发明的试剂盒进行检测。The purpose of the present invention is also to provide a high-sensitivity immunochromatographic detection method, which is characterized by using the kit of the present invention for detection.

在本发明中，在标记物与“生物桥”连接后，再通过生物桥特异性结合抗体恒定区，可使抗体的可变区充分暴露，且使抗体远离标记物，一定程度降低空间位阻。检测线的位置固定有“生物桥”，由于“生物桥”与抗体的结合亲和力更高，在层析时“生物桥”与抗体的结合效率更高，从而提高了检测灵敏度。在层析过程中，检测线位置有可以与抗体特异性结合的“生物桥”，与抗体的结合力高，可提高抗体利用率和检测灵敏度。本发明具有所述优点的原因在于，在抗体所具有的两端中，一端是结合区，即：发挥作用的区域，另一端是恒定区，不发挥作用。传统的化学偶联法将抗体和标记物偶联时，存在的问题是抗体的结合区有可能偶联到标记物上而导致该抗体浪费；也可能有的抗体的中段偶联到标记物上，由此导致抗体的结合力变弱。本发明中添加的生物桥能够定向结合抗体的恒定区，让结合区充分暴露于反应体系中，因此，生物桥的加入可以降低抗体的用量，提升测试灵敏度。In the present invention, after the marker is connected to the "bio-bridge", the antibody's variable region can be fully exposed through the specific binding of the biological bridge to the constant region of the antibody, and the antibody is kept away from the marker, reducing steric hindrance to a certain extent . The position of the detection line is fixed with a "bio-bridge". Since the "bio-bridge" has a higher binding affinity with the antibody, the "bio-bridge" has a higher binding efficiency with the antibody during chromatography, thereby improving the detection sensitivity. During the chromatography process, there is a "biological bridge" that can specifically bind to the antibody at the position of the detection line, and has a high binding force with the antibody, which can improve the utilization rate of the antibody and the detection sensitivity. The reason why the present invention has the above advantages is that, among the two ends of an antibody, one end is a binding region, that is, a functioning region, and the other end is a constant region, which does not function. When the traditional chemical coupling method couples the antibody and the marker, the problem is that the binding region of the antibody may be coupled to the marker, resulting in waste of the antibody; it is also possible that the middle part of the antibody may be coupled to the marker , resulting in weakened antibody binding. The biobridge added in the present invention can bind to the constant region of the antibody in a directional manner, allowing the binding region to be fully exposed to the reaction system. Therefore, the addition of the biobridge can reduce the amount of antibody used and improve test sensitivity.

附图说明Description of drawings

图1为根据本发明的试剂盒的试纸条。Fig. 1 is a test strip of the kit according to the present invention.

图2为本发明用于双抗体夹心模式测试的试剂盒的示意图，其包括I：抗体检测液盛放装置(其中装有相应的抗体检测液)和II：试纸条。Fig. 2 is a schematic diagram of the kit for double-antibody sandwich mode test of the present invention, which includes I: an antibody detection solution holding device (where the corresponding antibody detection solution is contained) and II: a test strip.

附图标记说明：Explanation of reference signs:

1—抗体1；2—抗体2；3—生物桥1；4—标记物；5—样品垫；6—检测线；7—生物桥2；8—质控线；9—反应垫；10—吸水垫；11—底板；I-抗体检测液盛放装置；II-试纸条。1—Antibody 1; 2—Antibody 2; 3—Biobridge 1; 4—Marker; 5—Sample pad; 6—Detection line; 7—Biobridge 2; 8—Quality control line; 9—Reaction pad; 10— Absorbent pad; 11—bottom plate; I—antibody detection solution holding device; II—test strip.

具体实施方式Detailed ways

下面结合具体的实施例对本发明做进一步详细的说明，但是本发明的范围不限制于此。本发明实施例中使用的试剂均可市场购得，并且如无特殊说明，所有操作方法均为常规操作方法。The present invention will be described in further detail below in conjunction with specific examples, but the scope of the present invention is not limited thereto. All the reagents used in the examples of the present invention are commercially available, and unless otherwise specified, all operating methods are conventional operating methods.

实施例1：Example 1:

人脑啡肽检测Human enkephalin detection

脑啡肽抗体检测液的制备：Preparation of enkephalin antibody detection solution:

(1)将2.5mg粒径为200nm的超顺磁性复合粒子(美国Quantum Design公司，产品号100001)、、0.96mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC，美国Thermo公司，产品号22980)、1.15mg N-羟基丁二酰亚胺(NHS，美国Thermo公司，产品号24500)和1mL浓度为0.1MpH＝4.7的2-(N-吗啡啉)乙磺酸(MES，美国Sigma-Aldrich公司，产品号M3671)缓冲液(称取1.066g MES，0.45g NaCl溶于50mL纯水，调pH至4.7)混匀，37℃反应30min，得到活化后超顺磁性复合粒子；(1) 2.5mg particle diameter is the superparamagnetic composite particle of 200nm (U.S. Quantum Design company, product number 100001), 0.96mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide ( EDC, U.S. Thermo Company, product number 22980), 1.15mg N-hydroxysuccinimide (NHS, U.S. Thermo Company, product No. 24500) and 1mL concentration of 0.1MpH=4.7 2-(N-morpholine) B Sulfonic acid (MES, American Sigma-Aldrich company, product number M3671) buffer solution (weigh 1.066g MES, 0.45g NaCl dissolved in 50mL pure water, adjust the pH to 4.7), mix, and react at 37°C for 30min to obtain the activated ultra Paramagnetic composite particles;

(2)用磁铁吸附磁微粒，弃上层清液，并用上述MES缓冲液洗涤两次；将步骤(1)得到的活化的超顺磁性复合粒子、0.08mg“生物桥1”(此实施例中为羊抗鼠IgG Fc段抗体)和浓度为50mM pH＝8.5的硼酸缓冲液混匀，37℃反应3h，得到偶联后标记物反应液；(2) Adsorb the magnetic microparticles with a magnet, discard the supernatant, and wash twice with the above-mentioned MES buffer solution; the activated superparamagnetic composite particles obtained in step (1), 0.08 mg "Biobridge 1" (in this embodiment Goat anti-mouse IgG Fc fragment antibody) mixed with borate buffer solution with a concentration of 50mM pH=8.5, and reacted at 37°C for 3h to obtain the conjugated marker reaction solution;

(3)向步骤(2)中的反应液加入含有BSA(美国Sigma-Aldrich公司，产品号A4612)的50mM pH＝8.5的硼酸缓冲液，使BSA终浓度为5％，37℃反应30min，得到含有封闭后的磁性粒子的反应液；用磁铁吸附磁微粒，弃上层清液，并用0.8mLpH8.5的硼酸缓冲液洗涤两次；最后加入0.5mL含有1％BSA，0.5％Tween-20，1％蔗糖的pH8.5的磷酸盐缓冲液，得到超顺磁性复合粒子标记溶液。向该溶液再加入0.01mg抗脑啡肽单克隆抗体和0.05mg抗脑啡肽兔多克隆抗体后，即为脑啡肽抗体检测液。(3) to the reaction solution in step (2), add the boric acid buffer solution containing 50mM pH=8.5 containing BSA (Sigma-Aldrich Company of the United States, product number A4612), so that the final concentration of BSA is 5%, and react for 30min at 37°C to obtain The reaction solution containing the blocked magnetic particles; adsorb the magnetic particles with a magnet, discard the supernatant, and wash twice with 0.8mL pH8.5 borate buffer solution; finally add 0.5mL containing 1% BSA, 0.5% Tween-20, 1 % sucrose in pH 8.5 phosphate buffer to obtain superparamagnetic composite particle labeling solution. After adding 0.01 mg anti-enkephalin monoclonal antibody and 0.05 mg anti-enkephalin rabbit polyclonal antibody to the solution, it becomes the enkephalin antibody detection solution.

脑啡肽试纸条的制备：Preparation of enkephalin test strips:

(1)反应垫的处理：用包被缓冲液(含2％BSA的0.01M PBS溶液，pH为7.2，过0.22μm滤膜)将“生物桥2”(羊抗兔IgG抗体)调节浓度至1mg/mL(作检测线)，鼠IgG抗体调节浓度至1mg/mL(作质控线)，按照1μL/cm的量将二者以5mm的间隔喷涂于硝酸纤维素膜上，37℃晾干后，加入干燥剂封存备用。(1) Treatment of the reaction pad: adjust the concentration of "Biobridge 2" (goat anti-rabbit IgG antibody) to Adjust the concentration of mouse IgG antibody to 1 mg/mL (as the detection line) to 1 mg/mL (as the quality control line), spray the two at 5 mm intervals on the nitrocellulose membrane according to the amount of 1 μL/cm, and dry at 37 ° C Finally, add a desiccant and store it for later use.

(2)试纸板的组装：将反应垫，吸水垫，样品垫，依次相互交错2mm粘贴在底板上，组成试纸板。(2) Assembling the test board: Paste the reaction pad, the water-absorbing pad, and the sample pad on the base plate in order to form a test board.

(3)试纸条的裁切：使用切条机将上述试纸板切成3mm宽的试纸条。(3) Cutting of test strips: the above-mentioned test strips were cut into 3 mm wide test strips using a strip cutter.

脑啡肽的检测：Detection of enkephalins:

取10μL血清与90μL脑啡肽抗体检测液于0.5mL离心管中混合后，将脑啡肽试纸条插入离心管中(样品垫在下)，室温下层析10min。After mixing 10 μL of serum and 90 μL of enkephalin antibody detection solution in a 0.5 mL centrifuge tube, insert the enkephalin test strip into the centrifuge tube (with the sample pad at the bottom), and perform chromatography at room temperature for 10 min.

经系列调试，该方法的检出限位5μg/mL。当检测线和质控线同时出现褐色时，证明样本中含有的脑啡肽高于检出限；当检测线没有颜色，而质控线出现褐色时，证明样本中含有的脑啡肽低于检出限；当检测线和质控线均没有颜色时试纸条无效。After serial debugging, the detection limit of this method is 5 μg/mL. When the test line and quality control line appear brown at the same time, it proves that the enkephalin contained in the sample is higher than the detection limit; when the test line has no color and the quality control line appears brown, it proves that the enkephalin contained in the sample is lower than Detection limit; the test strip is invalid when the test line and quality control line have no color.

Claims (8)

Translated fromChinese

1.一种高灵敏度免疫层析检测试剂盒，包括抗体检测液盛放装置(I)和试纸条(II)，其中所述抗体检测液盛放装置中含有相应的抗体检测液；所述试纸条由反应垫(9)、样品垫(5)、吸水垫(10)在底板(11)上依次交错地组装而成，反应垫(9)一端连接样品垫(5)，另一端与吸水垫(10)相连，反应垫(9)预包被有相互分离的检测线(6)和质控线(8)，其特征在于：在试纸条和抗体检测液中均结合有生物桥。1. A high-sensitivity immunochromatographic detection kit, comprising an antibody detection liquid holding device (I) and a test strip (II), wherein the antibody detection liquid holding device contains corresponding antibody detection liquid; The test strip is composed of reaction pad (9), sample pad (5), and water-absorbing pad (10) on the bottom plate (11), which are assembled alternately in sequence. One end of the reaction pad (9) is connected to the sample pad (5), and the other end is connected to the sample pad (5). The absorbent pad (10) is connected, and the reaction pad (9) is pre-coated with a detection line (6) and a quality control line (8) that are separated from each other. It is characterized in that: both the test strip and the antibody detection solution are combined with a biological bridge .2.根据权利要求1所述的试剂盒，其特征在于：所述抗体检测液包括抗体1、抗体2和标记物偶联的生物桥1；所述试纸条的检测线含有生物桥2，质控线含有能与所述生物桥1特异性结合的抗体3。2. kit according to claim 1, is characterized in that: described antibody detection liquid comprises antibody 1, antibody 2 and the biological bridge 1 of marker coupling; The detection line of described test strip contains biological bridge 2, The quality control line contains antibody 3 that can specifically bind to the biobridge 1.3.根据权利要求1所述的试剂盒，其特征在于：所述生物桥为能与抗体特异性结合的抗抗体、蛋白A、蛋白G分子。3 . The kit according to claim 1 , wherein the biobridge is an anti-antibody, protein A, or protein G molecule capable of specifically binding to an antibody.4.根据权利要求1所述的试剂盒，其特征在于：所述试剂盒为双抗体夹心模式。4. The kit according to claim 1, characterized in that: the kit is a double-antibody sandwich mode.5.根据权利要求1所述的试剂盒，其特征在于：所述抗体1与抗体2为不同种属的抗体，所述抗体1和抗体2为单克隆抗体，或多克隆抗体，或单链抗体；所述抗体3为单克隆抗体或多克隆抗体。5. The kit according to claim 1, characterized in that: said antibody 1 and antibody 2 are antibodies of different species, and said antibody 1 and antibody 2 are monoclonal antibodies, or polyclonal antibodies, or single-chain antibodies Antibody; the antibody 3 is a monoclonal antibody or a polyclonal antibody.6.根据权利要求2所述的试剂盒，其特征在于：所述标记物是乳胶颗粒、胶体金、胶体碳、胶体硒、超顺磁性颗粒、荧光颗粒或上转换发光颗粒。6 . The kit according to claim 2 , wherein the marker is latex particles, colloidal gold, colloidal carbon, colloidal selenium, superparamagnetic particles, fluorescent particles or upconversion luminescent particles.7.根据权利要求2所述的试剂盒，其特征在于：所述生物桥1为能与抗体1的恒定区特异性结合的抗抗体、蛋白A、蛋白G分子；所述生物桥2为能与抗体2的恒定区特异性结合的抗抗体、蛋白A、蛋白G分子。7. The kit according to claim 2, characterized in that: said biobridge 1 is an anti-antibody, protein A, protein G molecule that can specifically bind to the constant region of antibody 1; Anti-antibody, protein A, protein G molecule that specifically binds to the constant region of antibody 2.8.一种高灵敏度的免疫层析检测方法，其特征在于：使用前述权利要求任一项所述的试剂盒进行检测。8. A high-sensitivity immunochromatographic detection method, characterized in that: the detection kit is used according to any one of the preceding claims.

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