CN103175820A - Method for performing immunoassay on biofluid sample quantified by low-value target analyte in thin film sample chamber - Google Patents
Method for performing immunoassay on biofluid sample quantified by low-value target analyte in thin film sample chamber Download PDFInfo
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- CN103175820A CN103175820A CN2013101350065A CN201310135006A CN103175820A CN 103175820 A CN103175820 A CN 103175820A CN 2013101350065 A CN2013101350065 A CN 2013101350065A CN 201310135006 A CN201310135006 A CN 201310135006A CN 103175820 A CN103175820 A CN 103175820A
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Abstract
The invention discloses a method for performing immunoassay on a biofluid sample quantified by a low-value target analyte in thin film sample chamber, and is suitable for detecting and identifying any target analyte comprising more than two antigens, such as thyrotropic hormone (TSH). A biofluid sample, most preferably blood plasma or blood serum, is put into a reaction chamber; the surface size of the reaction chamber is enough for counting molecules of the analyte to the maximum extent; the bottom or the top of the reaction chamber is made of a plastic thin film; and thyroid gland antibodies (of which the number is greater than a required number) are attached to the surface and are used for catching the target analytes to the maximum extent. The caught antibodies must be bound on a fixed substrate in an irreversible manner, so that the antibodies cannot be separated from the adhesive surface during analysis. The method can be used for quantifying the monomer-molecule target analyte in the blood plasma or the blood serum in an analysis chamber and also can be used for catching target analysis molecules on the surface of a plane thin-layer sample chamber.
Description
Technical field
The present invention relates to a kind of method and apparatus that uses imaging system to detect and quantize the low concentration target analytes, specifically a kind of method that the biologicfluid sample of the indoor low value target analytes quantification of film sample is carried out immunoassays.
Background technology
As everyone knows such as hormonal some analyte, as the TSH(thyrotropic hormone), its concentration may be low to moderate thousands of molecule/microlitres, can adopt device of the present invention to come the individual molecule amount of Measurement and analysis thing for the analyte of this extremely low concentration.Of the present invention one large advantage is that it is a kind of main quantization method, thereby need not standardization.
Summary of the invention
The present invention is intended to detect and quantize certain particular target analytes, analyte in thin film bio fluid sample as contained in 2 microns ~ 10 chambers, micron thickness plane, target analytes contains two kinds of antigens at least, can be with the individual molecule of particular target analytes and fixing matrix bonding, although the cementing agent that arrives involved in the present invention can be effective for more than one antigen.Contain capture antibody or binding part in matrix.Antibody or part be mainly for the first antigen or all antigen of target analytes, and fixing analyte, to prevent diffusion.Can with target analytes and matrix bonding, then utilize label probe that the target analytes of binding is detected.Probe comprises Multiple Antibodies or the part with the surface bonding, can be for the second epitope or the antigen of target analytes.
The first and second antigens are positioned on target analytes, and its position must guarantee that the bonding of all kinds of antigens can not prevent the bonding of other class antigen." antibody " and " part " these two terms refer to anyly can the material of effective adhesive be arranged with target antigen, comprise immunoglobulin (Ig), antimer and similar all living things cementing agent with high cohesion.
The present invention is applicable to any target analytes that contains two or more antigens of detection and Identification, as the TSH(thyrotropic hormone).With the blood plasma of a kind of biological fluid sample sample-preferably or serum-put into a reaction chamber, the surface size of reaction chamber should allow the molecular amounts of analyte is carried out maximum count, and is specific as follows described.
Reaction chamber bottom surface or end face are made by plastic sheeting, and first is urged gland antibody (to surpass required quantity) and adhered to from the teeth outwards, with maximum ground captured target analyte.Capture antibody must irreversibly be bundled in fixedly on matrix, guarantees that antibody can not leave bonding surface in analytic process, and this zone is called trapping region.
Blood plasma or serum sample are added in reaction chamber, and the short gland molecules of first all in sample bond with the fixedly matrix that contains capture antibody, thereby fix all molecules in sample.Walled thickness (usually less than 10 microns) allows molecule with the fastest speed vertical diffusion, and then guarantees to carry out rapid diffusion through thin-walled is two-layer, thereby makes all molecules of analyte and arrive the capture antibody Surface Contact.Ideally, plasma or other checked biofluids should not contain the particle such as cell, prevent from causing interfere with or compromise to the detection of analytic signal to the analyte bonding.
After while or the of short duration initial soak of process, will join (to surpass required quantity) in sample with the fluorescent nano particles (gland antibody as short in anti-β first) of antibody bonding, guarantee the molecule counting that bonds with carrying out maximum.The diameter of nano particle is preferably the 10-100 nanometer, is comprised of europium fluorescent material or any detectable nano particle, for example quantum dot or other nano particles.These fluorescent nano particles must be enough little, and concentration reaches the colloidal suspension shape, unless antibody of its surface bonding and fixedly analyte be bonded together.
To contain single fluorescent nano particles and the short gland molecule bonding of first that is bonded on matrix for the antibody/body of gland of the short gland analyte of first.The fluorescent nano particle with fixing analyte bonding will not keep the colloidal suspension shape, carry out simultaneously Brownian movement.For the nano particle of distinguishing constraint and not fettering, (when fixedly luminescent nanoparticle was luminous, signal rolled up after cultivating a period of time; And mobile nano particle can produce the existence that nano particle cause bias light due to the signal of not constraint when luminous).Time shutter is measured according to surveying instrument, but should farthest be limited, because these zones may not have bound nano particle.The photon that is fixed on matrix and must rest on a nano particle in the zone is projected in some pixels; Comparatively speaking, the photometric distribution scope of nano particle of carrying out Brownian movement is much bigger, and the detection of particle becomes easier thereby make fixedly.The reaction chamber surface that does not contain capture antibody can be used as the use of control zone.
When adopting present technique, in imaging region, the concentration of nano particle should be enough little, and is fully overlapping to guarantee can not to cause, or reduce inductor to the fixing separating capacity of particle.Therefore, can distinguish the quantity that the fixing quantity of fluorescent particles should equal the contained analyte molecule of trapping region IT antibody up and down reaction chamber.Because the Fluid Volume more than the control zone is less with respect to fixed trapped antibody or the amount more than body of gland, in order to calculate reaction chamber or the arrow path cumulative volume of (as the diffusion barrier of separately control zone and trapping region), the above volume in control zone can be ignored.In practical operation, can utilize aquiclude that capture region and control area are made a distinction.The molecule maximum quantity that can measure is determined by reaction chamber trapping region and pixel enlargement factor.The concentration of target analytes is that measured molecular amounts calculates divided by the sample size in the trapping region locular wall.The volume of reaction chamber is determined by known weight and the schedule of samples area of reaction chamber, and the schedule of samples area can be determined by pixel quantity in sample areas and zone/pixel amplification factor.Therefore, be known as reaction chamber weight and enlargement factor, sample volume also can be determined by analytical instrument so.Bound particle distance each other must be guaranteed enough large, can not overlap to avoid captive label nano particle signal.For example, if the fluorescence of the contained signal of nano particle can record on the surface of 3-10 pixel, and the imaging separating distance of nano particle is at least the twice of above-mentioned distance, or 15 pixels of being separated by respectively, and the amplification imaging size is 0.5 micron/pixel, 1/4 of the sample surface area will contain enough resolution so, so that the maximum molecule molecular weight of each reaction chamber is detected.Detectable molecular weight is lower in theory, because be subject to the restriction of counting statistics.The insider thinks, reaction chamber is thinner, and is larger without the target analytes body of gland constraint of label, but the indoor sample size that contains is also less.The reaction chamber area is larger, and dynamic range is larger, but it is longer to carry out the required time of reaction chamber imaging process.
For the reaction chamber of 2 square centimeters of high 10 microns, areas, trapping region volume 2 microlitres are the existence that can measure molecule.This is corresponding with sample source amount of substance concentration susceptibility.As can increase sample size (thereby catching most of in this volume or all molecules) to 1000 microlitres by slow inflow 10 microlitres when needing susceptibility and the method for device are carried out linearity raising.Flow rate is 1-7 microlitre/second.Detection can be carried out before this, but the analysis room must be placed between sample key-course and waste layer, until add the nano particle that needs measurement after mobile end and acquisition result again.Although use absorbent material automatically to promote to flow in collecting chamber, but still need the volume sample that increases is shared.Sample must flow on trapping region and control zone.
Therefore, one of beneficial effect of the present invention is to provide a kind of quantization method to contained individual molecule target analytes in blood plasma in the analysis room or serum; Two be to provide a kind of method that the surperficial target analysis molecule in thin layer sample chamber, plane (contain at least a transparent surface, the outstanding particle that is hunted down on optics is in order to carry out the photo measure counting) is caught.
Description of drawings
Fig. 1 uses when being the blood plasma of evaluating objects analyte or serum part film sample sensing chamber schematic diagram describes as an example of short first gland example.
Fig. 2 and Fig. 1 are similar, but what show is the sensing chamber that adds after blood plasma or blood serum sample and various fluorescence analysis element.
Fig. 3 and Fig. 2 are similar, demonstration be to carry out sensing chamber's electronic imaging for the existence of determining target analytes.
Figure 4 shows that the schematic diagram that film sample sensing chamber consists of, comprise jumbo sample source district, laminated film detection zone, and sample reception district.
Fig. 5 and Fig. 4 are similar, but demonstration is the schematic diagram that sample passes through the film detection zone.
Fig. 6 and Fig. 5 are similar, but demonstration is the imaging schematic diagram of sample circulation rear film detection zone.
Embodiment
A part that has shown as shown in Figure 1 film test sample chamber 2 is represented that by numeral the test sample of analyzing is blood plasma or serum, can analyze the short first gland of TSH() existence.The surface of reaction chamber 2 or wall can allow the fixing of various bodies of gland.Body of gland 6 only limits to the first surface antigen of analyzed short first gland molecule in this case.
Fig. 2 has shown that reaction chamber 2 adds analyzed blood plasma and fluoroscopic examination particles mixture state afterwards.Particle 8 comprises body of gland, and only limits to the second antigen of target analytes, thereby some particles can bond with the target analysis molecule before being placed into sensing chamber 2.The fluorescent particles that numeral 10 expressions and target analysis molecule 12 bond.The fluorescent particles of constraint is not by numeral 8 expressions, as shown in Figure 2.Target analytes, namely TSH, by numeral 12 expressions, as shown in Figure 2, shown some analytes that are captured to 12 and a series of not fluorescent particles 8 of constraint.The particle 8 of constraint does not move in sample 4 usually, and is as shown in arrow 14.When 2 imaging of sample chamber, as shown in Fig. 3 schematic diagram, captive particle (on target analytes) fluorescence signal is relatively bright, and as shown in Fig. 3 numeral 10, and the fluorescence signal that sends of bound particle will be relatively not dark or fuzzy, as shown in numeral 8 in Fig. 3.
Therefore, in sample 4, captive target analytes can be determined simply by sample 4 imagings.Because the volume of sample chamber 2 is controlled, and the volume of the interior sample 4 of reaction chamber 2 is known, so the quantity of target analytes can be calculated according to target analytes/sample volume unit.
As shown in Fig. 4-6, having embodied can be to the device example of analyzing than the large sample amount in the present invention.This embodiment comprises sample area 16, comprising a large amount of blood plasma or the blood serum sample of need analysis.The zone 16 can hold the sample of 1 milliliter, and its upper surface is more flexible, can compress, thereby can push sample, then extracts by sample detection chamber 2.Sensing chamber 2 comprises a control zone 20, does not wherein contain to catch body of gland 6, and separates with sample area 2'.This control zone obviously do not indicate, and is much smaller with respect to trapping region, and when needing, the control zone can be connected with the diffusion barrier of trapping region, comprises that analyte catches body of gland 6.When zone 16 was compressed, sample can move according to the direction of arrow A, passed through simultaneously sample area 2 ' and control zone 20.Behind zone 2 ' and 20, sample is seated in reception area 18, wherein may contain absorption of sample agent (as needs time).
After Fig. 6 has shown that sample puts in place, the imaging results in sample area 2'.This image will demonstrate the bright image 10 of the particle that is hunted down, and demonstrate not constraint or the not fuzzyyer or dim fluorescence signal 8 of trapped particle.As effective in sample detection, control zone 20 will only comprise fuzzy fluorescence signal 8 so. Zone 16 and 18 will allow to hold more analytic sample, therefore makes testing result have higher validity.Broken line 11 in Fig. 4-6 has shown the waterproof obstacle between sample area 2' and control zone 20, and working is to prevent that two interregional samples from intersecting.
In the description of the invention scope, can carry out some modifications to related structure of the present invention.Its analysis room's region area can not wait from 1 square millimeter to 400 square millimeters, and height is between the 2-10 micron.Preferably will bind antibody and be a kind of uniform pattern storing, near the control zone making is only contained the antibody of not constraint or is not contained any antibody.The control zone is guarantee and control the accuracy of measurement corresponding to the label analyte, preferably the sample diffusion from the control zone to the trapping region is applied restriction, so that more accurate to the stereometry that is exposed to the capture antibody sample size.Simultaneously, during as needs, namely contain the known analyte of many concentration when the analysis room, and be placed on similarly when analyzing under condition, also can carry out the Drawing of Curve of standard.The discrete signal region quantity that each imaging area in known analyte concentration trapping region is measured deducts in each imaging region the analysis of drawing of detectable discrete signal, to obtain the curve plotting of standard.Its result can be used to calculate the concentration of analyte in unknown sample, namely by analyzing under condition same as described above.
The probe signals amplifying technique can be used to replace nano particle as RCAT (rolling circulation amplify technology), because they have the effect that produces fluorescent particles.Owing to can the disclosed case study on implementation of the present invention being had more modification (as long as without prejudice to concept of the present invention), unless the separate statement requirement need not to apply restrictive factor for the present invention.
Claims (2)
1. method that the biologicfluid sample that the indoor low value target analytes of film sample is quantized carries out immunoassays, it is characterized in that comprising step as described below: provide capture antibody or body of gland for all types of target analyte, be enough to the target analytes bonding with all increases, fix or be fixed on analysis room's structure the analysis room with film sample, and described capture antibody or body of gland only limit to the first epitope or the antigen (being present in biological fluid sample) on the target analytes molecule;
With various biologicfluid samples and with antibody (selectively with the target analysis molecule on the second epitope or antigen, be present in above-mentioned biologicfluid sample) the fluorescent nano particles above-mentioned sample analysis of packing into of coupling is indoor;
Make the indoor static sample imaging of sample analysis, simultaneously the target analytes molecule is counted, these molecules can be caught by described fixed trapped antibody, and can go up to get fixedly fluorescent nano particles imaging by being coupled to antibody (with the second antigen bonding of fixed target analyte), so that detect.
2. a kind of biologicfluid sample that the indoor low value target analytes of film sample is quantized according to claim 1 method of carrying out immunoassays, it is characterized in that described fluorescent nano particles is quantum dot, because of with sample in captive target analysis molecule bonding fix, the nano particle that does not fetter in fluorescent nano particles and sample can be carried out the optics differentiation by the motion that does not fetter nano particle;
Described bound and do not fetter label and measure the differentiation of antibody by adopting image or scanning analysis to be achieved by the electronics mode;
In described method, analyzed material is not diluted;
The quantity that can detect discrete signal in described trapping region unit imaging area is more than detecting discrete signal in unit imaging area in the control zone, and the quantity that multiply by trapping region of the difference amount in unit area equals the quantity of captive target analytes molecule;
Described reaction chamber comprises a control zone, does not wherein contain capture antibody or body of gland;
Described diameter of nano particles scope is the 10-100 nanometer;
Described sample volume is greater than the volume of analysis room.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110470852A (en) * | 2018-05-09 | 2019-11-19 | 豪夫迈·罗氏有限公司 | Laboratory system and method for the interfering substance for including in separation test sample |
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| WO1995017675A1 (en) * | 1993-12-23 | 1995-06-29 | Holmes, Michael, John | Method of assay |
| CN1515909A (en) * | 2003-08-27 | 2004-07-28 | 魏景艳 | Quantum point marker sandwich immunodetection method and its diagnosis kit |
| US20050277159A1 (en) * | 2003-07-23 | 2005-12-15 | Ctl Analyzers, Llc | Nanoparticle and microparticle based detection of cellular products |
| CN101915838A (en) * | 2010-08-05 | 2010-12-15 | 中国检验检疫科学研究院 | Flow fluorescent coding microballon-based avian influenza virus multi-type simultaneous detection method and kit |
| CN102047116A (en) * | 2008-04-09 | 2011-05-04 | 艾博特健康公司 | Method of detecting very low levels of analyte within a thin film fluid sample contained in a thin thickness chamber |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995017675A1 (en) * | 1993-12-23 | 1995-06-29 | Holmes, Michael, John | Method of assay |
| US20050277159A1 (en) * | 2003-07-23 | 2005-12-15 | Ctl Analyzers, Llc | Nanoparticle and microparticle based detection of cellular products |
| CN1515909A (en) * | 2003-08-27 | 2004-07-28 | 魏景艳 | Quantum point marker sandwich immunodetection method and its diagnosis kit |
| CN102047116A (en) * | 2008-04-09 | 2011-05-04 | 艾博特健康公司 | Method of detecting very low levels of analyte within a thin film fluid sample contained in a thin thickness chamber |
| CN101915838A (en) * | 2010-08-05 | 2010-12-15 | 中国检验检疫科学研究院 | Flow fluorescent coding microballon-based avian influenza virus multi-type simultaneous detection method and kit |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110470852A (en) * | 2018-05-09 | 2019-11-19 | 豪夫迈·罗氏有限公司 | Laboratory system and method for the interfering substance for including in separation test sample |
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Application publication date: 20130626 |