CN1029574C - Immune percolation checking method and device - Google Patents

Immune percolation checking method and device Download PDF

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CN1029574C
CN1029574C CN 92101890 CN92101890A CN1029574C CN 1029574 C CN1029574 C CN 1029574C CN 92101890 CN92101890 CN 92101890 CN 92101890 A CN92101890 A CN 92101890A CN 1029574 C CN1029574 C CN 1029574C
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immune
percolation
checking
method
device
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CN1064550A (en )
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祝加贝
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祝加贝
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Abstract

一种一检多项、新型模式、快速斑点免疫渗滤检测方法及装置,属生化领域该方法通过一次加试样同时即能得到多项检测结果即同时得到数种定性结论或得到包括数种量程在内的宽量程定量标示,本方法应用的反应膜根据不同检测目的可在不同部位包被不同图形标示,同时对试剂成份进行不同调整此外本发明采取辣根过氧化物酶联免疫显色系统以取代碱性磷酸酶显示,在反应装置中,位于反应膜之上,设置过滤膜,以去除大颗粒杂质及胶状物。 One of a number of inspection, new patterns, dot immunogold infiltration detection method and apparatus, which belongs to the field of biochemistry method by adding one more sample can be obtained at the same time i.e. detection result obtained i.e. simultaneously or several qualitative conclusions obtained comprise several wide range including the indicated quantitative range, the reaction of the present method of application of the film may be coated in different parts of different pattern depending on the labeled detection purposes, while the different components of the reagent of the present invention is furthermore adjusted to take chromogenic horseradish peroxidase enzyme-linked immunoassay the system displays to replace the alkaline phosphatase in the reaction apparatus, located above the reaction membrane, a filtration membrane is provided, to remove large particulate impurities and gum. 本方法的装置操作简便,检测快速,装置小巧,成本低廉适于医院及家庭个人使用。 The device of the present method is simple, rapid detection, device size, low cost suitable for hospital and home for personal use.

Description

本发明属于生物化学领域快速斑点免疫渗滤检测方法(DIBA)以其快速、简便灵敏度高而正在逐渐得到广泛应用,有在许多方面取代以前的酶联固相免疫吸附检测(ELISA)的趋势,并在不断完善中。 The present invention belongs to the field of biochemistry dot immunogold detection infiltration (DIBA) with its fast, simple sensitivity is gradually widely used, there is trend to replace the previous solid phase enzyme-linked immunosorbent assay (ELISA), in many respects, and continuous improvement. 如作为固相载体的膜已从最早的非化学键结合的硝酸纤维膜部分改为以化学键的商品膜。 Chemically changed such as the commercial membranes nitrocellulose membrane from the first portion of the non-chemically bound to a solid support membrane. 该技术的显示系统也从单一的碱性磷酸酶发展为胶体金。 The display system technology from a single alkaline phosphatase development of colloidal gold. 此两种显示系统,其灵敏度皆低而成本较高,这就限制了DIBA技术的应用,此外由于技术本身的限制,还不能用以检测大颗粒抗源。 This two display systems, which are low and sensitivity is high cost, which limits the application of DIBA technology, due to restrictions in addition to the technology itself, not for detecting large particulate source of resistance.

现在的免疫检测方法都是一次加样只能测试一种结果,这样在实际应用中不仅浪费试样,效率也不能提高。 Immunoassay methods are now loaded only once testing a result, in practical applications this is not only a waste of the sample, the efficiency can not be improved. ELISA方法在定量检测时则需做许多诸如稀释等烦锁操作,使用起来不甚方便,其它方法也有此弊病。 When the ELISA method quantitatively detects the like need to do a lot of trouble, such as diluted lock operation, not convenient to use, this method also has other shortcomings.

另外,某些特殊指标由于本身固有特性的原因在单一检测中不能完成。 Further, because of some special indicators inherent characteristics can not be completed in a single assay. 以前这只有靠多次加样的多项检测才能完成,比如尿液中的LH(促黄体酮生长激素)的半定量分析即为典型一例。 Previously this only by repeatedly loading a number of testing to complete, such as urine LH (stimulating hormone progesterone) semi-quantitative analysis that is a typical example.

本发明的目的是提供一种新型免疫渗滤检测方法和装置,不仅具有高的灵敏度而且通过一次加样可同时获得几种定性结果和一个宽量程的多个分段定量(或半定性)的测试结果。 Object of the present invention is to provide a novel method and apparatus for detecting immunological diafiltration, not only high sensitivity but also through a loading several qualitative results can be obtained with a wide-range multiple segments quantitative (or semi-qualitative) simultaneously Test Results.

本发明是通过以下技术方案来实现的:一.基于在膜载体上斑点显示是相对独立的原理,我们对膜载体的不同部位进行不同处理(包被),当然也要同时对试剂成份进行调整以适应这种多个包被。 The present invention is achieved by the following technical solutions: a spot on the membrane carrier is displayed on a principle of relatively independent, we different treatment (coating) to different parts of the membrane carrier, of course, also be adjusted while the reagent composition this is to accommodate a plurality of packets. (包被的含义是将相应抗体或抗原溶于PBS中,浓度适需要而调整,然后点加在反应膜上的相应部位。)在一尽量小的圆形膜面积(为降低商业成本可选φ=12mm)中心以某一定量待测试样成份包被对照杠(即一横杠形图形),使其显色强度分别等于某两种(或几种)待测试样在某一浓度时的显色强度,这就是定性(半定量)检测对照杠,然后在横杠上下方位分别用抗该两种(或几种)试样的高亲和性抗体包被该两种(或几种)待测试样点,这就制得了定性(半定量)反应膜。 (For the meaning of the corresponding antibody or antigen dissolved in PBS, and the concentration of aptamer required to adjust, and then added to the corresponding parts of the reaction film.) In a circular membrane area as small as possible (to reduce business costs optional φ = 12mm) center of the test sample component is a control package to the bar a certain amount (i.e., a bar-shaped pattern), so that each color intensity equal to a two (or several) of the test sample at a concentration when the color intensity, which is qualitative (semi-quantitative) detection of the control bar, then using the anti-two (or several) samples in the vertical bar orientation of the high-affinity antibody-coated both (or several species) samples to be tested, which was prepared qualitative (semi-quantitative) reaction film.

在定量检测时可以某一定量待测成份包被一个中心圆点使其显色强度等于该样品在第一量程时的显色强度,此后在中心点周围按测量强度递增顺序所划分的量程用依次降低浓度的相同抗体或依次降低亲合力的不同抗体(依自身实验室抗体特征而定)来处理,使之在其相应量程的样品浓度下得到与中央对照点一样的显色强度。 When quantitative detection of a certain amount can be measured by a central dot package component to develop a color intensity equal to the intensity of the color samples in the first range, after ascending order according to the measured intensity of the divided range around a center point with successively lower concentrations of the same antibody or different successively lower affinity antibodies (antibodies characterized by their laboratories set) to process, so the central control point to obtain the same color intensity at the corresponding sample concentration range.

二.在显色系统上本发明采用辣根过氧化物显色系统,该系统与碱性磷酸酶系统相比具经济,易于制作的优点,但将其用于DIBA技术时有两个难题。 2. On the color system of the present invention is horseradish peroxidase chromogenic system compared to systems with alkaline phosphatase economic advantages of easy production, but there are two problems when it is used DIBA techniques.

(一)辣根过氧化物酶反应体系在ELISA技术中有高灵敏度,而DIBA确需要不溶性底物的酶反应体系才能有好的显色效果;而现有的辣根过氧化物酶的不溶性底物显色系统的灵敏度较低。 (A) Horseradish peroxidase reaction system has a high sensitivity ELISA technique, the enzyme reaction system DIBA really necessary either insoluble substrates in order to have a good color effect; and the conventional horseradish peroxidase insoluble less sensitive chromogenic substrate system.

(二)当前采用的辣根过氧化物酶体系底物显色溶液由两种溶液组成,第一种如OPD、α-四氯萘酚、DAB等,另一种为含有过氧化氢的缓冲液,而这两种溶液混合一定时间即告失效,由于这两个原因限制,以致现有技术无法将辣根过氧化物酶系统用于DIBA技术。 (Ii) the current system using horseradish peroxidase chromogenic substrate solution composed of two solutions, a first such OPD, α- tetrachloro-naphthol, DAB, etc., to another buffer containing hydrogen peroxide solution, which two solutions are mixed a certain lapse of time, both of these reasons limited so that the prior art systems can not be used with horseradish peroxidase DIBA techniques.

本发明经大量试验发现选用辣酶体系中的半可溶性显色底物TMB(四甲基联笨胺)并配以含有过氧化氢的柠檬酸磷酸缓冲液可解决以上难题。 It was found that the present invention by a large number of semi-soluble chromogenic substrate TMB (tetramethylbenzidine stupid amine) selected enzyme system in hot and with a citrate phosphate buffer solution containing hydrogen peroxide can solve the above problems.

底物显色溶液的配制:TMB 5mg/100ml(试剂2)Hci-Na2HPO40.2M pH5.0H2O20.004%制作辣根过氧化物酶联抗体复合物可采用常规实验室技术,但要注意获得的复合物最好进行一次凝胶过滤分离,选取分子量为20~30万的组分以避免在渗滤反应中产生过高本底。 Chromogenic substrate solution was prepared: TMB 5mg / 100ml (reagent 2) Hci-Na2HPO40.2M pH5.0H2O20.004% production horseradish peroxidase-linked antibody complex using conventional laboratory techniques, but note obtained complex is preferably carried out once separated by gel filtration, a molecular weight of 20 to select components 300,000 to avoid excessive background in the diafiltration reaction.

三.试剂1的配制要点:0.5%BSA(牛血清白蛋白)-PBS(磷酸盐缓冲液)内含0.05%TMS(硫抑汞)作为腐蚀剂,用此溶液将酶联抗体复合物稀释到适当比例清洗液为0.05%Tween20~PBS四.为测试装置(图1)设计了经济实用的滤膜(3)以取代现行DIBA技术中的特制过滤器或笨重的离心处理,滤膜仅由两层黄色的质地柔韧的卫生纸再加一层新华2号滤纸构成。 Reagent 1 prepared in three points:. 0.5% BSA (bovine serum albumin) -PBS (phosphate buffered saline) containing 0.05% TMS (sulfur suppression mercury) as an etchant, the solution was diluted with an appropriate enzyme-linked antibody complex the ratio of washing liquid is 0.05% Tween20 ~ PBS IV. test apparatus (FIG. 1) designed economical and practical filter (3) to replace the current filter DIBA special techniques or cumbersome centrifugation, membrane of only two layers flexible texture of yellow toilet paper plus a layer constituting the Xinhua No. 2 filter paper.

以下结合附图说明实施例:(一)定性、半定量检测:检测尿样(或血样)中的LH(黄本酮生长激素)和HCG(人绒毛膜促性腺激素),显示符号如图2。 BRIEF DESCRIPTION Example embodiments less :( a) qualitative and semi-quantitative detection: The urine sample (or blood) in LH (Huang ketone GH) and of HCG (human chorionic gonadotropin), the symbol 2 in FIG. .

反应膜处理:选用与LH和HCG同时反应的高亲合性抗体包被杠上方点,以对HCG特异的抗体包被杠下方点,以与40mlu的LH50mlu的HCG的显色强度相同的HCG包被对照杠,包被完的膜即用BSA-PBS溶液封闭并风干,此膜即用做为装置中的反应膜。 The reaction membrane treatment: selection of high affinity antibody-coated reaction simultaneously with LH and HCG are bar above the point to the antibody coated on the HCG-specific are bar point below to the color intensity LH50mlu of HCG 40mlu the same HCG package is a control bar, i.e. a film coated finished blocked with BSA-PBS solution and air-dried, the film that is used in the reaction as a membrane device.

操作:以晨尿液滴入装置顶端之小池(7)充盈之,待其完全渗入加3滴试剂1于过滤膜(3)上完全渗入后静置1分钟,再加4滴清洗剂,待完全渗入后用滴管(一头带尖的塑料管,此管兼做取尿样用)剥去滤膜,加3滴底物显色溶液于反应膜(4)上待完全吸收后再加2滴,显示结果如图2,图2中各小图表示:1为LH和HCG皆为阴性2为LH弱阳性但低于40mlu3为LH阳性大于或等于40mlu4为HCG弱阳性但低于50mlu5为HCG阳性大于或等于50mlu6为检测失误图中O点表示点的颜色深度浅于横杠图中·点表示点的颜色深度等于或强于杠图1中(1)为检测装置盖,(2)为装置本体,(5)为隔离膜,(6)为吸水材料(二)定量检测:检测尿样(或血样)中的HCG或HBsAg(乙型肝炎表面抗源)显示符号如图3。 Operation: In a cuvette was added dropwise to the top of the morning urine means (7) of the filling, until completely infiltrated after standing 3 drops Reagent 1 was added to the filtration membrane (3) completely infiltrated 1 minutes, add 4 drops of cleaning agent to be after completely infiltrated with a dropper (a pointed plastic tube, this tube also serves as a urine sample) filter strip, add 3 drops of chromogenic substrate solution to be completely absorbed in the reaction membrane (4) plus 2 drops, FIG. 2 shows the results, in each panel represented in FIG. 2: 1 to 2 LH and HCG were negative to weakly positive, but lower than the LH to LH 40mlu3 40mlu4 is equal to or greater than positive weakly positive but below HCG HCG is 50mlu5 positive color depth is greater than or equal to the detection error 50mlu6 FIG color point represented by point O in FIG shallower * represents a point equal to or stronger than the bars in (1) to cover detection apparatus of FIG. 1 bar, (2) device body (5) is a separator, (6) of water absorbing material (B) quantitative detection: detection of urine (or blood) of the HCG or of HBsAg (hepatitis B surface antigen source) display symbols 3 shown in FIG.

反应膜处理:首先用一定量的HCG或HBsAg包被中心圆点使其显色强度等于样品在等一量程浓度时在外围第一量程敏感点的显色强度,外围第一量程敏感点用高浓度高亲和力的抗HCG或HBsAg抗体来包被,而第二至第六量程敏感点则依次用降低浓度的相同抗体或依次用降低亲和力的不同抗体来包被,使之在其相应量程的样品浓度中得到与中央对照点一样的显色强度,而后的处理制备与一般DIBA相同,操作与上面的定性,半定量检测实施例相同,设定的六个量程及与图3对应的图号如下:1.为5mlu 2.为20mlu 3.为80mlu4.为320mlu 5.为1.2lu 6.为5lu附图说明图1为该发明装置剖视图图2为定性及半定量检测结果之显示符号图3为定量检测结果之显示符号 The reaction membrane treatment: First, with an amount of HCG or HBsAg coated center dot color development intensity sensitive points periphery of the first range when a color intensity equal to the concentration range in the sample and the like, with the periphery of the first high range Sensitive concentration of the high affinity anti-HCG antibody to HBsAg or coating, and the second to sixth range is sensitive points sequentially by the same or a reduced concentration of antibodies in turn be coated with a different affinity antibody reduces, so that the sample in its respective range concentration obtained in the central control point of the same color intensity, and then processing was prepared in the same general DIBA, operation of the above qualitative, semi-quantitative detection of the same embodiment, six set range and corresponding to FIG. 3 and FIG numbers are as follows : 1 to 20mlu 3. 5mlu 2. 80mlu4 as to 320mlu 5. 5lu is 1.2lu 6. Figure 1 is a device of the invention for the sectional view of FIG. 2 is a symbolic qualitative and semi-quantitative detection result of 3 results show the quantitative detection of symbols

Claims (2)

  1. 1.一种免疫渗滤检测方法,包括如下步骤:A.包被定性反应膜,在一圆形膜中心以其中一种待测试样成分包被对照杠,使其显色强度分别等于一定量两种待测试样成分的显色强度:B.在对照杠的上下方位分别用抗该两种试样成分的高亲合性抗体包被该两种待测试样点:其特征在于:以辣根过氧化物酶联免疫显示系统取代碱性磷酸酶显示系统或胶体金显示系统,采用斑点一横杠图形显示检测的定性结果。 An immunoassay method for detecting infiltration, comprising the steps of:. A qualitative reaction film coating, a circular center of the film in one of the components of the test sample coated with the control lever, so that a certain color intensity equal to color intensity of the test sample amounts of the two components: a high affinity antibody-coated in a vertical orientation control bar B are both samples with anti-components are the two kinds of the samples to be tested: wherein : display horseradish peroxidase-linked immune system substituted alkaline phosphatase or colloidal gold show a display system that uses a bar graphic display speckle detection qualitative results.
  2. 2.一种免疫渗滤检测方法,包括如下步骤:A包被多个分段定量反应膜,以待测成分包被反应膜的中心圆点,使其显色强度等于该样品在第一量程时的显色强度;B.在中心点周围按测量强度递增顺序所划分的量程用依次降低浓度的相同抗体或依次降低亲合力的不同抗体包被测试样点,使之在相应量程的样品浓度下得到与中央对照点一样的显色强度。 An immunoassay method for detecting infiltration, comprising the steps of: A plurality of segments coated film quantitative reaction, the reaction to the test center dot coating composition film so color intensity range is equal to the first sample when the color intensity;. B around the center point by measuring the intensity of the ascending order of the divided range by successively reducing the concentration of the same antibody or different successively lower affinity for the antibody-coated test samples, so that the corresponding sample concentration range the central control point and to obtain the same color intensity. 其特征在于:以辣根过氧化物酶联免疫显示系统取代碱性磷酸酶显示系统或胶体金显示系统,并以多斑点图形显示检测的定量结果。 Characterized in that: the display system substituted with alkaline phosphatase or colloidal gold display system a display system, and displays the results of the quantitative detection of multiple speckle patterns to horseradish peroxidase in enzyme immunoassay.
CN 92101890 1992-03-24 1992-03-24 Immune percolation checking method and device CN1029574C (en)

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