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CN1117587A - Membranous type enzyme linked immunoassay device and method - Google Patents

Membranous type enzyme linked immunoassay device and method Download PDF

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CN1117587A
CN1117587A CN 94115015 CN94115015A CN1117587A CN 1117587 A CN1117587 A CN 1117587A CN 94115015 CN94115015 CN 94115015 CN 94115015 A CN94115015 A CN 94115015A CN 1117587 A CN1117587 A CN 1117587A
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method
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simple
enzyme
processing
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张永新
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张永新
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The method is used for examination of whole blood and serum antibody or antigen, the reaction membrane is coated with a substance which can combine with the object to be detected. After adding of enzyme label, washing and colour-display processing, the coated spot will exhibit, colors. Advantage: simple structure, simple detection method, safety and high sensitivity.

Description

膜型酶联免疫检测装置及方法 ELISA film type device and method

本发明涉及酶联免疫检测装置及方法,特别是膜型联免疫检测装置及方法,用于检测抗原或抗体。 The present invention relates to apparatus and method for enzyme immunoassay, in particular membrane-linked immunosorbent detection device and method for detecting an antigen or antibody.

酶联免疫检测技术已广泛用于许多疾病的血清学诊断,特别是那些与微生物感染有关的抗原和抗体的检测,但是目前所采用的方法检测方法复杂,速度慢、时间长、还要有专业人员检测,而且由于放射性污染和检测样本和洗液的排放对环境污染,而且还会感染检测人员。 Enzyme-linked immunosorbent assay technique has been widely used for serological diagnosis of many diseases, especially those related to the detection of microbial infection of antigen and antibody, but the method detection methods currently used complex, slow, long time, but also a professional personnel testing, and because of radioactive contamination and the test sample and lotions pollution emissions on the environment, but also to detect infected persons.

本发明的目的在于克服上述已有技术的缺点与不足,而提供一种简便、安全、快速的检测装置及方法。 Object of the present invention is to overcome the drawbacks and disadvantages of the above prior art, and to provide a simple, safe, rapid detection apparatus and method.

本发明的目的是通过下列技术方案实现的,技术方案是根据酶联免疫测定原理结合斑点膜免疫渗滤技术,定性检测抗原或抗体,适用于全血和血清样本。 Object of the present invention is achieved by the following technical solution, the technical solution is a combination of dot immunogold filtration membrane enzyme immunoassay technique according to the measurement principle, qualitative detection of antigen or antibody, applicable to whole blood and serum samples. 当样本中含有抗原或抗体时,这些待检物分别会与反应膜包被物结合(圆点形),当加入酶结合物时,待测抗原或抗体与包被抗体结合或抗体或抗原结合,当多余的酶结合被洗掉后,加入底物显色液即可被酶催化,使阳性样本呈色,而阴性无色,但由于质控点系由可与酶结合物结合的物质包被而成,故无论阳性或阴性均可呈色,说明检测结果有效,相反无效。 When the sample containing the antigen or antibody, the analyte is bound, respectively (dot-shaped) with the reaction membrane package, when added to the enzyme conjugate, the antibody or the antigen to bind with an antibody or antibody-coated or antigen-binding when the excess enzyme conjugate is washed away, chromogenic substrate was added to catalyzed by an enzyme, the sample so that coloration positive, negative and colorless, but the quality control point of the packet-based substance may be bound by the binding of the enzyme it is made, whether it is positive or negative can coloration, indicating the effective detection result, contrary invalid.

膜型酶联免疫检测装置,其特征在于它由漏斗、反应池、反应管、反应膜组成,漏斗按装在反应池内,漏斗外壁与反应池上口配合,漏斗底口装有滤纸或滤膜,并与反应池底口装有的反应膜相接,反应膜下面装有吸水纸垫,反应池下口与反应管上口配合,反应管内装有吸水性填充物,底端面有一通气孔。 ELISA-type membrane means, characterized in that it consists of a funnel, the reaction cell, reaction tubes, film composition, according to the funnel installed in the reaction pool, pool hopper port with the outer wall of the reaction, the funnel with a filter paper or filter bottom nozzle, the reaction with the membrane and the bottom opening of the reaction phase, the reaction with absorbent paper pad under the membrane, the reaction cell with the reactor tube inlet fitting catchy reaction tube that has the water absorbing filler, has a bottom end face air vent.

膜型酶联免疫检测方法,包括检测试剂的配制,反应膜的包被和操作步骤,其特征在于:a)检测试剂由酶结合物、清洗液、底物显色液,血液处理液和终止液组成,酶结合物为酶标记抗原或抗体,亦可为其它标记物如金属标记物,即试剂1;清洗液采用吐温磷酸盐缓冲液,即为试剂2;底物显色液采用二氨基联苯胺或四甲基联苯胺或邻联甲苯胺,也可是上述两种以上的联用,即试剂3;血液处理液由1—3%聚乙二醇与2%枸椽酸钠,即试剂4;终止液为0.2N硫酸,即试剂5。 Membrane ELISA method comprising formulating detection reagents, package film is and the reaction steps, characterized in that: a) the detection of enzyme conjugate reagent, cleaning fluid, chromogenic substrate solution, the blood treatment fluid and termination solution composition, enzyme-labeled conjugate is an antigen or antibody, other markers may also be a metal such as labels, i.e., reagent 1; Tween washing solution using a phosphate buffer, is the reagent 2; chromogenic substrate solution using two diaminobenzidine or tetramethyl benzidine or o-tolidine, it may also be combined with the two or more, i.e., reagent 3; 1-3% of the blood treatment liquid polyethylene glycol and 2% sodium citrate, i.e., 4 reagent; of 0.2N sulfuric acid stop solution, i.e., reagent 5.

b)反应膜的包被,用微机或人工或微机和人工混合方法包被,反应膜中心部位包被为可与待检物结合的抗体或抗原,以圆点形状包被,边缘包被为可与酶标记物结合的物质,以小圆点形状包被,即为质控点。 b) the reaction of the coated film, or a microcomputer or microcomputers and artificial hybrid artificial coating method, the central portion of the reaction membrane to be coated in combination with antigen or antibody to be detected, the packet is a dot shape, and edges to be substance may be bound to the enzyme label was coated to form small dots, is the quality control point.

c)检测步骤:1)用吸管取未稍血在吸管内与血液处理液(试剂4)混合摇匀制成样本,加入漏斗过滤后,去除漏斗进入反应池或者采集静脉血分离血清标本,直接加入反应池,在室温静置;2)然后在反应池内加入试剂1静置;3)再加入清洗液试剂2,静置;4)待试剂2渗滤完毕立即加入显色液试剂3后,观察结果;5)当质控点显色后加入终止液试剂5保留结果;6)结果阅读。 c) detecting the step of: after 1) does not take a little blood with a pipette in the pipette blood treatment solution (Reagent 4) is made of a mixed sample shaking, addition funnel was filtered to remove the funnel into the reaction vessel, or separate venous blood serum samples, direct into the reaction vessel, allowed to stand at room temperature; 2) was then added to the reaction cell was allowed to stand reagent 1; 3) the washing liquid reagent 2 was added, allowed to stand; 4) to be completed immediately diafiltered reagent 2 was added to the color reagent 3, observation; 5) when the quality control point of the color reagent was added to terminate the reservation result 5; 6) to read the results.

对于不需要高敏感性的纯净样本检测可不用漏斗,反应膜可以是硝纤膜或含有硝酸纤维素的混纤膜,包被稀释液为磷酸盐或碳酸盐缓冲液、糖、聚乙二醇、叠氮钠组成,抗原或抗体的浓度根据所需敏感性的高低进行调节,一般在ELISA的5—200倍,但包被蛋白的含量不能大于5mg/ml。 For pure samples do not require high sensitivity without detecting funnel, the reaction may be a film or film web nitrate mixed fiber containing cellulose nitrate film, the package was diluted phosphate or carbonate buffer, sugar, polyethylene glycol alcohols, sodium azide composition, the concentration of antigen or antibody be adjusted according to the desired level of sensitivity, ELISA is generally 5-200 times, but the coat protein content is not greater than 5mg / ml.

在检测全血样本时,血液处理液由柠檬酸三钠、聚乙二醇及磷酸盐缓冲液组成。 When whole blood sample, a blood treatment liquid composed of sodium citrate, polyethylene glycol, and a phosphate buffer.

本发明与已有技术相比具有如下优点及效果:a)检测速度快,在10分钟内可完成全部检测过程;b)简化了检测步骤,可适用于家庭、医院及身体检查;c)检测过程无废物污染,防止对环境的污染和人员感染;d)根据检测项目要求,可制成系列反应膜。 Prior art and the present invention has the following advantages and effects compared to: a) detection speed, in 10 minutes to complete the entire testing process; b) simplifies the detection step, used in home, hospitals and medical examination; c) detecting process without waste pollution, environmental pollution and prevent infection of personnel; D) according to the detection project requirements, can be made into a film series of reactions.

附图说明 BRIEF DESCRIPTION

:图1为检测装置示意图;图2为结果显示图。 : FIG. 1 is a schematic view of the detection apparatus; FIG. 2 is a graph showing the results.

下面结合附图及实施侧进一步说明本发明技术:本发明的检测装置,由漏斗(1)、反应池(2)、反应管(4)、反应膜(7),组成,漏斗按装在反应池内,使待检样本集中地通过反应膜的包被点,适当延缓样本通过速度以使反应有充分时间完成,同时过滤掉样本中的颗粒物质以免影响结果,由此可使试剂敏感性提高3—10倍;漏斗外壁与反应池上的配合漏斗底口装有滤纸或滤膜(图中示出)并与反应池底口装有的反应膜相接,反应膜下面装有吸水纸垫(3),反应池下面与反应管上口配合,反应管内装有吸水性充填物(5),底端面有一通气孔(6)。 Further illustrate the techniques of this disclosure in conjunction with the accompanying drawings and the following embodiments side: detecting apparatus according to the present invention, by a hopper (1), the reaction cell (2), the reaction tube (4), the reaction membrane (7), composed by a funnel attached to the reaction pool the samples to be examined are collectively point the reaction by coating the membrane, suitably delayed by the sample rate to allow sufficient time to complete the reaction, while filtering out particulate matter so as not to affect the results of the sample, thereby allowing to improve the sensitivity of the reagent 3 -10 times; reaction membrane with an outer wall of the funnel and the reaction pool hopper bottom opening with a filter paper or filtering membrane (shown), and the reaction in contact with the bottom opening, with the following reaction the membrane pad of absorbent paper (3 ), reaction cell fitted below the upper opening of the reaction tube, the reaction tube that has the water absorbing filling (5), the bottom end face has a vent hole (6).

反应膜的中心部位包被为可与待检物结合的抗原或抗体,如检测乙肝表面抗原时,包被点为乙肝表面抗体,用圆点形状(8)表示,边缘包被为可与酶标记物结合的物质,以小圆点形状(9)表示,即为质控点。 The central portion of bag film is a reaction to an antigen or an antibody can bind to analyte, such as detection of HBsAg, hepatitis B surface coated with antibody to the point, represented by a dot shape (8), the edges to be coated with the enzyme marker substance bound to form small dots (9), is the quality control point.

检测乙肝表面抗体时,反应膜包被为乙肝抗原液。 When the detection of hepatitis B surface antibody reaction film coating hepatitis B antigen solution.

用吸管取末稍血在吸管内与试剂4混合摇匀一分钟静止,其目的是抗凝和加速免疫反应,使抗原和抗体结合更牢,提高反应敏感性,然后加入反应池内,再加试剂1四滴,使酶结合物与待测物结合,静置4分钟,加试剂2四清静置,以清洗未结合物,渗干后加1—2滴试剂3酶催化底物显色,当质控点呈色后加入试剂5,保存结果,反应膜中心圆点(B)呈色,边缘小圆点呈色,即为阳性,反应膜无反应点,为无效结果,只有边缘有呈色点,中心无显色点,即为阴性。 Peripheral blood taken with a pipette in the pipette and the reagent mixture shaken one minute rest 4, and its purpose is to accelerate the immune response anticoagulant, an antibody and antigen binding more firmly, improving the sensitivity of the reaction, was then added to the reaction tanks, plus reagent 1 four drops of the enzyme conjugate bound to the analyte, stand for 4 minutes, add 2 four quiet home agent to the washing of unbound material, after adding 1-2 drops of dry osmotic agent 3 chromogenic enzymatic substrate, when after adding reagent 5 the coloring quality control point, save the results, the reaction center of the film dot (B) coloring, coloring dots edge, is positive, the reaction no reaction point film, invalid result, only the edge coloring point, the center point of no color, that is negative.

同样,也可检测乙肝抗原。 Similarly, the detectable HBsAg.

Claims (7)

1.膜型酶联免疫检测装置,其特征在于它由漏斗、反应池、反应管、反应膜组成,漏斗按装在反应池内,漏斗外壁与反应池上口配合,漏斗底口装有滤纸或滤膜,并与反应池底口装有的反应膜相接,反应膜下面装有吸水纸垫,反应池下口与反应管上口配合,反应管内装有吸水性填充物,底端面有一通气孔。 1. ELISA type membrane means, characterized in that it consists of a funnel, the reaction cell, reaction tubes, film composition, according to the funnel installed in the reaction pool, pool hopper port with the outer wall of the reaction, the funnel with filter paper or filter bottom nozzle membrane, the membrane and the reaction with the bottom opening of the reaction phase, the reaction with absorbent paper pad under the membrane, the reaction cell with the reactor tube inlet fitting catchy reaction tube that has the water absorbing filler, has a bottom end face air vent.
2.根据权利要求1所述的检测装置,其特征在于对于不需高敏感性的纯净样本检测可不用漏斗。 2. The detection apparatus according to claim 1, characterized in that the need for high-sensitivity detection may not pure sample funnel.
3.膜型酶联免疫检测方法,包括检测试剂的配制,反应膜的包被和操作步骤,其特征在于:a)检测试剂由酶结合物、清洗液、底物显色液,血液处理液和终止液组成,酶结合物为酶标记抗原或抗体,亦可为其它标记物如金属标记物,即试剂1;清洗液采用吐温磷酸盐缓冲液,即为试剂2;底物显色液采用二氨基联苯胺或四甲基联苯胺或邻联甲苯胺,也可是上述两种以上的联用,即试剂3;血液处理液由1—3%聚乙二醇与2%枸椽酸钠,即试剂4;终止液为0.2N硫酸,即试剂5。 3. The membrane ELISA method comprising formulating detection reagents, package film is and the reaction steps, characterized in that: a) the detection of enzyme conjugate reagent, cleaning fluid, chromogenic substrate solution, the blood treatment fluid and terminating liquid composition, enzyme-labeled conjugate is an antigen or antibody, other markers may also be a metal such as labels, i.e., reagent 1; Tween washing solution using a phosphate buffer, is the reagent 2; chromogenic substrate solution using diaminobenzidine or tetramethyl benzidine or o-tolidine, it may also be combined with the two or more, i.e., reagent 3; 1-3% of the blood treatment liquid polyethylene glycol and 2% sodium citrate , i.e., 4 agent; of 0.2N sulfuric acid stop solution, i.e., reagent 5. b)反应膜的包被,用微机或人工或微机和人工混合方法包被,反应膜中心部位包被为可与待检物结合的抗体或抗原,以圆点形状包被,边缘包被为可与酶标记物结合的物质,以小圆点形状包被,即为质控点。 b) the reaction of the coated film, or a microcomputer or microcomputers and artificial hybrid artificial coating method, the central portion of the reaction membrane to be coated in combination with antigen or antibody to be detected, the packet is a dot shape, and edges to be substance may be bound to the enzyme label was coated to form small dots, is the quality control point. c)检测步骤:1)用吸管取未稍血在吸管内与血液处理液(试剂4)混合摇匀制成样本,加入漏斗过滤后,去除漏斗进入反应池或者采集静脉血分离血清标本,直接加入反应池,在室温静置;2)然后在反应池内加入试剂1,静置;3)再加入清洗液试剂2,静置;4)待试剂2渗滤完毕立即加入显色液试剂3后,观察结果;5)当质控点显色后加入终止液试剂5保留结果;6)结果阅读。 c) detecting the step of: after 1) does not take a little blood with a pipette in the pipette blood treatment solution (Reagent 4) is made of a mixed sample shaking, addition funnel was filtered to remove the funnel into the reaction vessel, or separate venous blood serum samples, direct into the reaction vessel, allowed to stand at room temperature; 2) was then added to the reaction reagent tanks 1, allowed to stand; 3) 2 was added cleaning liquid reagent, allowed to stand; 4) to be completed immediately diafiltered reagent 2 was added to the color reagent 3 , observation; 5) when the quality control point of the color reagent was added to terminate the reservation result 5; 6) to read the results.
4.根据权利要求3所述的检测方法,其特征在于反应膜可以是硝纤膜或含有硝酸纤维素的混纤膜。 4. The detection method according to claim 3, characterized in that the reaction film may be a film or fiber nitrate mixed fiber membrane containing nitrocellulose.
5.根据权利要求3所述的检测方法,其特征在于包被稀释液为磷酸盐或碳酸盐缓冲液、糖,聚乙二醇,叠氮钠组成; The detection method according to claim 3, characterized in that the package was diluted phosphate or carbonate buffer, sugars, polyethylene glycol, sodium azide composition;
6.根据权利要求3所述的检测方法,其特征在于抗原和抗体的浓度根据所需敏感性的高低进行调节,一般在ELISA的5—200倍,但包被蛋白的含量不能大于5mg/ml; 6. The detection method according to claim 3, characterized in that the concentration of the antigen and antibody is adjusted depending on the desired level of sensitivity, ELISA is generally 5-200 times, but the coat protein content is not greater than 5mg / ml ;
7.根据权利要求3所述的检测方法,其特征在于检测全血样本时血液处理液由柠檬酸三钠,聚乙二醇及磷酸盐缓冲液组成。 The detection method according to claim 3, characterized in that the whole blood sample during blood treatment fluid from the trisodium citrate, polyethylene glycol, and phosphate buffer.
CN 94115015 1994-08-24 1994-08-24 Membranous type enzyme linked immunoassay device and method CN1117587A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100510747C (en) 2001-12-12 2009-07-08 普罗托姆系统有限公司 Diagnostic testing process
CN102621300A (en) * 2011-01-26 2012-08-01 上海铭源数康生物芯片有限公司 Device and method for removing cellulose nitrate film based protein chip luminescent substrate
CN104569432A (en) * 2015-01-06 2015-04-29 通标标准技术服务有限公司 Method and device for detecting transgenic ingredients in food

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100510747C (en) 2001-12-12 2009-07-08 普罗托姆系统有限公司 Diagnostic testing process
CN102621300A (en) * 2011-01-26 2012-08-01 上海铭源数康生物芯片有限公司 Device and method for removing cellulose nitrate film based protein chip luminescent substrate
CN104569432A (en) * 2015-01-06 2015-04-29 通标标准技术服务有限公司 Method and device for detecting transgenic ingredients in food
CN104569432B (en) * 2015-01-06 2016-10-05 通标标准技术服务有限公司 Method and apparatus for detecting genetically modified organisms in food

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