CN104215757A - Biological fluid sample quantitative detection device and biological fluid sample quantitative detection method - Google Patents
Biological fluid sample quantitative detection device and biological fluid sample quantitative detection method Download PDFInfo
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Abstract
The invention discloses a biological fluid sample quantitative detection device including a testing bar which is arranged in a box body, a memory for color intensity data, and a reader for color intensity data. A substrate is arranged on a bottom layer of the testing bar and is provided with a porous testing main membrane. A second membrane is disposed in an upper stream of the testing main membrane and is provided with a sample application locus and a marker ligand locus for enable a biological fluid sample, a compound and a marker ligand thereof to be flowable through capillarity. A third membrane is arranged in a lower stream of the testing main membrane and is used for adsorbing a to-be-tested biological fluid sample which passes through the testing main membrane at the end of the lower stream of the testing main membrane. Two sample adding holes and a window are disposed on a box cover of the box body. By means of the device, a standard substance curve can be pictured through a light reflecting intensity in a standard substance of which the concentration is known, so that quickly, effectively and accurately quantitative analysis of the biological fluid sample can be carried out.
Description
The divisional application that the application is application number is 201010196737.7, the applying date is on May 28th, 2010, denomination of invention is the application for a patent for invention of " biological fluid sample quantitative test device and detection method thereof ".
Technical field
The present invention relates to a kind of system for detecting biological liquid sample, particularly relating to a kind of detection and the system of concentration of thing to be checked in quantitative measurment sample.
Background technology
The quantitative test of antigen, steroids antibody, endocrine albumen and other types albumen, usually can provide vital clinical data.At biological field, immunoassay is well-known, and its principle is after reagent-impregnated, by capillary action to containing on the film of immobilized reagent, test strip detection zone be combined with visable indicia thing, as: the determinand of latex particle, metal composite reacts.It is worth mentioning that and obtain label from metal-sol, this label dealt from metal-sol can with ligand binding, be then combined with determinand, part or antibody further.Test-strips by a large amount of for detecting body fluid, as the determinand in urine and blood.Detect human chorionic gonadotrophin as assignor's pregnancy method this may be method the most general the earliest.Above-mentioned detection is carried out all on the test strip, and the display of its result only needs a step to complete, and such as, liquid sample is by absorbing membrane, and inner determinand and corresponding ligand binding, result can clearly be presented on the detection zone that is separated with sample loading zone.Carry out above-mentioned detection, proving installation conventional is at present generally containing cutting and capsule, and user just can detect without the need to extra utensil.Known pick-up unit adopts sandwich detection and Competitive assays to detect two kinds of detection methods substantially.
Sandwich-type detection procedures, is that determinand in liquid sample and mark or indicator ligands react, generates complicated determinand or label.This reaction generation will have determinand to enter detector bar, otherwise tagged ligand will be deposited on test-strips absorbing membrane completely.On the test strip, the determinand being combined with label moves on to sample capture district by capillary action; There, determinand complex can be obtained by solidification capture ligands.When gold marks, there is colour band in pattern detection district, then show have determinand to exist.This pick-up unit also containing Article 2 solidification part band, being generally not only combined with tagged ligand as quality control band, even not having determinand to deposit in case, can showing the performance of test-strips.
Competitive assays detection method, be that determinand and tagged ligand can react with the fixed ligand in sample capture district, such determinand and tagged ligand compete the fixed ligand in sample capture district jointly.The existence of any determinand all will replace the binding site of tagged ligand.Tagged ligand is caught and is occurred signal, then represent that result is negative.
In prior art, based on device with presence or absence of test-strips determination determinand, how can not provide the correct amount of determinand in liquid sample.Even if be furnished with the standard items of quantitative test in pick-up unit, also can because of temperature, air humidity, time lapse, test-strips difference, change in signal strength and inaccurate.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of biological fluid sample quantitative test device and method, the present invention can provide biological fluid samples is carried out fast, effectively, quantitative test accurately.
In order to solve the problems of the technologies described above, the technical scheme that biological fluid sample quantitative test device of the present invention is achieved is: this pick-up unit, comprise the test-strips be arranged in a box body, also comprise the storer of color intensity data and the reader of color intensity data; The bottom of described test-strips is a substrate, described substrate is provided with the main film of the test with porous; The main film of described test has a high standard band HC, a substandard band LC and a calibration tape T, has calibrating reagent, described calibration tape T has fixed ligands in described high standard band HC and substandard band LC; The upstream of the main film of described test is provided with the second film, described second film comprises at least two-layer non-activity that arranges from bottom to top and has film and a filtering membrane of porous, described second film is provided with a sample application site and a tagged ligand site, impels tested organism fluid samples and compound thereof, the flowing of marker ligand physical efficiency by capillary action; The downstream of the main film of described test is provided with tertiary membrane, described tertiary membrane is an absorption layer, described tertiary membrane is used for contacting with tested organism fluid samples, impel tested organism fluid samples to flow through the main film of test by the capillary action of described tertiary membrane, described tertiary membrane is used for the tested organism fluid samples having passed through the main film of test in the end absorption in the main film downstream of test; Described box body comprises lid, described lid is provided with two wells and a form, and the position of described two wells aligns with sample application site on the second film in box and tagged ligand site respectively; Described form is consistent with the region that high standard band HC, substandard band LC and calibration tape T on the main film of box body build-in test limit; Described storer for storing the color intensity that in high standard band HC, substandard band LC and calibration tape T, tested organism fluid samples produces, by the correlation measured the luminous value of color intensity and tested organism fluid samples concentration and form in described storer; Described storer is electric magnetic card or rfid card; Described reader is optical card reader, and described optical card reader is used for the concentration value providing all analysis things in biological fluid samples respectively.
In the present invention, utilize the detecting step of above-mentioned biological fluid sample quantitative test device as follows:
Step one, use transfer pipet drip the tested organism fluid samples of 10 to 100 microlitres in the well on pick-up unit lid;
Step 2, described tested organism fluid samples are positioned at the second film testing main film upstream and are adsorbed, and flow to the main film of test with capillary action from the second film; In the process, tested organism fluid samples flows through second diaphragm area with tagged ligand deposition, and described tested organism fluid samples is combined with tagged ligand, described second film is formed with the height of known quantity and low calibration part;
Step 3, with test main film contact, tested organism fluid samples just acts on calibration tape T and high standard band HC and substandard band LC with tagged ligand;
Step 4, compare based on the colored intensity of tested organism fluid samples and the colored intensity of index zone alignment reagent and obtain the result that quantitatively detects; That is: in tested organism fluid samples, determinand content is higher, the analysis thing be combined with tagged ligand is more, in the main film calibration tape T of test the tagged ligand that is combined of fixed ligands or determinand compound amount higher, thus the mark colored intensity increase on index zone; Increase along with analyzing thing content in tested organism fluid samples, the colored intensity of mark just increases thereupon;
Step 5, the mark colored intensity produced by tested organism fluid samples in index zone and calibration tape are together stored in an electric magnetic card or rfid card; When being inserted in optical card reader by this electric magnetic card or rfid card, this card reader provides the concentration value of all analysis things in tested organism fluid samples respectively;
Step 6, light reflection strength drawing standard product curve according to tested organism fluid samples concentration standards band.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention's application solid phase chromatography detects, as sandwich detection, one or more tested organism fluid samples with mark bond and be combined, also can solidification calibrating reagent above test-strips or labelled reagent be combined.On index zone, described labelled reagent is captured by calibrating reagent, creates a platform for measuring tested organism fluid samples concentration in detection zone.Index zone solidification calibrating reagent and corresponding labelled reagent are combined as liquid sample and pass through detector bar.Determinand in liquid sample is just combined on sample capture band.Utilize these relative intensities being marked with complex just can obtain the actual concentrations of tested organism fluid samples.Therefore, the intensity of the label (as: gold size bond) on film in various sample capture band, the degree of depth just reflect the quantity of tested organism fluid samples, and in like manner, index zone also can reflect intensity and the concentration of calibrating reagent.So the light reflection strength of sample is converted into measurement concentration by the commercial visualization device that utilizes.Finally, with the light reflection strength drawing standard product curve of concentration known standard items band.
Accompanying drawing explanation
Fig. 1 is the formation block diagram of proving installation of the present invention;
Fig. 2 is the surface structure schematic diagram of test-strips box body in proving installation of the present invention;
Fig. 3 is test-strips STRUCTURE DECOMPOSITION schematic diagram in proving installation of the present invention;
Fig. 4 is proving installation detection method process flow diagram of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
For ease of understanding the present invention, relevant word is defined as follows:
1, the antigen in tested organism fluid samples (hereinafter referred to as determinand), refers to the material that can be combined with antibody.Antigen can be compound, polypeptide, carbohydrates, nucleic acid, lipid and its analog, as virion, subunit, parasitic animal and plant, host protein etc.
2, bond, refers to the determinand part mentioned in sandwich assay, the same with the determinand in competition detection method, label, tracer part.Bond can be chosen, as antigen-antibody complex, Antibody-antigen complex from the large molecule that can be combined with determinand or compound.
3, test section (or band), refers to that bond or determinand can adhere to the position on it, such as: a part for test-strips in pick-up unit.
4, test-strips (or detector bar), refers to and can, by capillary action, make the liquid sample containing determinand and other need to determine the perforated membrane of the antigen flowing of testing concentration.General perforated membrane has glass fibre, porous nitrocellulose, tygon.The test-strips of invention is all made up of supporting film and filtrator.
5, tracer, refers to and is marked with determinand that can survey, that be preferably marked with special raadable mark thing (as: collaurum, latex, comprise the liposome of dyestuff, carbon black and analog) or bond part.
6, sample loading zone, refers to determinand detection zone, namely makes liquid sample move in test-strips.
7, liquid sample to be tested, refers to that any one contains the liquid of determinand.The sample detected can be body fluid, comprise: the urine obtained from fetus, neonate, youth, adult, blood, sweat, lymph liquid, abdomen intraluminal fluid, crude extracts or homogenate, the liquid of inactive, the water as obtained from river, lake, test water etc.
8, label, refers to molecule greatly or the compound of the direct or indirect conditioning signal of energy (as color change) pattern.This signal is used to indicate the concentration range that has that it's too late detecting target determinand in sample.Label may contain enzyme, fluorescer, liposome, red blood cell, polymer microcapsules, colour developing polymer particles (latex), also may be colloid containing metal composite.Various patent and be applied as produce optical signal provide lot of documents.In the present invention, the application of several label types patent can be found.United States Patent (USP): 3,646,346 have found radioactively labelled substance; United States Patent (USP): 3,654,090,3,791,932 and 3,817,838 have found enzyme labeling; United States Patent (USP): 3,996,345 find fluorescent marker; United States Patent (USP): 3,996,345 find radioactively labelled substance; United States Patent (USP): 4,067,959 find fluorescence or enzyme marker; United States Patent (USP): 4,104,099 finds chemiluminescent labels; United States Patent (USP): 4,160,645 find without enzyme catalyst label; United States Patent (USP): 3,966,879 find that electrophoretic techniques can be used for antibody district; United States Patent (USP): 4,120,945 Late Cambrian radioimmunoassays detect, and in this approach, the determinand of mark can be combined with solid support by antibody; United States Patent (USP): 4,233,402 find paired enzyme marker; United States Patent (USP): 4,720,450 find chemical induction fluorescent marker; United States Patent (USP): 4,287,300 find enzyme negative charge label.In addition, label also can be metal-sol; Metal or metal composite, as: metal oxide, oxyhydroxide, slaine, metal composite or mix mutually with polymkeric substance or be coated in the metal composite of polymer core.The dewatering state of any one metal-sol that these metal marker things are mentioned above may being or metal composite colloidal sol may be more the collaurum of dewatering state.
9, labeled reactant, refers to the signal intensity generated when solidified reagents links on label and calibration tape or index zone.The reflected light device generated with color is the good method that measurement markers is reacted.
10, compound, refers to that (based on context) is any by determinand and one or more part, or the multi-molecular complex formed with solidification part by tagged ligand.In sandwich immunoassays, such as following compound can produce determinand/tagged ligand double combination and produce (the first compound) first in the detection, and determinand/tagged ligand/solidification part three combines and occurs in the detection (the second compound).
11, fluid communication, refers to a kind of contacting with each other but not addition, allows fluid to be flowed to the structure of another passage by a passage.
12, chemically examine, refer to several the dissimilar detecting patterns using test-strips can detect determinand.Such as, in sandwich immunoassays, when in sample, target determinand exists, it is just combined with the tracer of mark and moves on test-strips (containing absorbing membrane) indicator, forms the first compound.Indicant is that one can be combined with target determinand, and the molecule that also can be combined with label, label can be metal marker thing, preferably collaurum.
13, catch band (or catching site), refer at least containing the region in the stratographic analysis test-strips of a determinand binding reagents or band.Determinand binding reagents is usually fixed on district or brings, and after detectable reagent reacting, in district or bring and generate the result that can survey, can reflect determinand content or its situation about existing in sample.The trapping region of " catching band " and may can catch multiple sample by more than one forms, and in the case, more than one determinand bonding agent can be used.Such as, within the scope of the present invention, two combinations detected are considered to a test set, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) can be detected simultaneously, or detect the antibody of hepatitis B surface antibody (HBsAg) and microspironema pallidum (TP) simultaneously.The combination also having other is feasible, and within the scope of the present invention.
14, bond and detection reagent, can detect reagent such as the antigen of: developer, fluorescer, enzyme or chemiluminescence agent or antibody and exchanges with combining at this.In the practice of the invention, " bond " and " detection reagent " combines the determinand that will determine specifically or is fixed on the catches of catching and bringing.Bring catching, " bond " and " detection agent " produces can the value of the current determinand content of reflection of quantitative measurment.As will be explained hereinafter, catch the density bringing direct quantitative to measure differ surely reflection be combined in the quantity of catching and bringing current determinand, but the band strength measured by luminous value (RLU) but can reflect the content of catching current determinand in band.
15, comprise at this " index zone " calibrating reagent being fixed on test strip calibration district.Calibrating reagent is combined with calibration binding reagents specifically and forms calibration in conjunction with right.Two calibration tapes are comprised in the present invention.Be that they can play the effect of internal calibration containing calibration in conjunction with right advantage, namely calibration agent may will catch the cubage of current determinand in band interior.Calibration agent may be used for the variability of test strip calibration.One of them can be designated as high calibration (HC), and another can be designated as low calibration (LC).In addition, the density of HC and LC can be used for confirmed standard product line.Standard items line can generate a regression equation by the RLU value of calibration agent, describes the relation between Two Variables with this, thus is applied to each quantitative test experience.Although, conventional calibration agent is still in use, but people are still ready to use non-existent in sample or without the calibration compound of immunological cross-reaction generally, as 2,4_ dinitro benzene can buy (Eugene from molecular probe for bovine serum albumin (BSA-DNP), OR, cat#A-23018) and use as calibrating reagent.2,2, 4-dinitrophenol (DNP) is non-existent Small molecular but can as haptens in human body, haptens refers to when itself and macro-molecular protein carrier conjugation, or be expelled to the mammal that can produce antibody as in rat, mouse, rabbit, ox, horse, sheep or goat body time, produce immunogenic material.Poor efficiency calibration areas solidification part is for bovine thyroglobulin antibody (BTG); High calibration areas is for goat-anti rabbit protein antibodies.With solidification ligand binding bond be BTG gold antigen and rabbit igg gold antigen.
As shown in Figure 1, biological fluid sample quantitative test device of the present invention, comprises the test-strips be arranged in a box body, also comprises the storer of color intensity data and the reader of color intensity data.
As shown in Figure 3, the bottom of described test-strips is an adhesive sticker substrate 1, described adhesive sticker substrate 1 is provided with the porous with sufficient porosity and tests main film 6, and the inspection material comprising determinand and compound thereof can be allowed to be moved on on this film by capillary action.The conventional main film 6 of described test has porous cellulose nitrate, porous polypropylene or paper film, and the main film 6 of described test preferably adopts bag by nitrocellulose filter, and these films are widely known by the people technically.
The main film 6 of described test have a high standard band HC, a substandard band LC and a calibration tape T, in described high standard band HC and substandard band LC, there is calibrating reagent, described calibration tape T has fixed ligands, that is: in detection zone T region, test the solidification part main film comprising and can be combined with determinand, and the main film of test can better be run through in strip form, just as the same through test main film formation index zone with the calibrating reagent on substandard band LC at high standard band HC.Solidification part for calibration tape region and index zone region is different.Index zone is fixed the calibrating reagent of known quantity for the curve that settles the standard, thus determine the quantity of determinand.After testing the ligand binding on main film, remaining avtive spot is closed, thus allows determinand and compound, tagged ligand and compound thereof freely to pass through test-strips.
Test-strips porous in the present invention tests the upstream of main film 6, also comprise second film, described second film comprises at least two-layer non-activity that arranges from bottom to top and has film 5 and a filtering membrane 4 of porous, described non-activity and the film 5 with porous containing can with the position of the ligand binding by film, in addition also comprise the solidification part band for detecting fluid communication on film.This film 5 adopts collaurum bag by glass fibre membrane, and this film more may be made up of the glass fibre faling apart or polypropylene, has sufficient hole simultaneously, allows determinand and compound thereof, tagged ligand depends on capillary action to flow.The top layer of described second film is a filtering membrane 4, and this filtering layer 4 adopts glass fibre membrane, and this filtering membrane is applicable to be separated by determinand from sample components, and other components in sample may disturb the inspection to determinand.Therefore, when detecting blood, red blood cell or leucocyte can be separated from the serum comprising determinand.Therefore second film comprises a sample application site and a tagged ligand site, the downstream of practical site and the upstream of test membrane contact point, react with determinand in sandwich reaction, or react with detection zone binding partner in competitive reaction, and the reaction in index zone between complicated tagged ligand and correction part.Labelled reagent is attracted to the upstream of this film, can flow away when it contacts with liquid sample from film, with the condition of analyte response under form compound thus can be continued to flow to test membrane by upstream film.
Be positioned at the downstream testing main film 6 in test-strips and also comprise tertiary membrane 2, this tertiary membrane 2 is an absorption layer, this tertiary membrane 2 and liquid comes into contact, for adsorbing by the liquid sample of test membrane in the downstream end of the main film 6 of test, also as driving force, liquid sample is impelled to flow through detection film by capillary action.Such absorption layer preferably makes of efficient sorbing material, as: can adsorb sample simultaneously can as paper test-strips being added any damping fluid.
As shown in Figure 2, described box body comprises lid, described lid is provided with two wells S1, S2 and a form W, and the position of described two wells S1, S2 aligns with sample application site on the second film in box and tagged ligand site respectively; Described form W is consistent with the region that high standard band HC, substandard band LC and calibration tape T on the main film of box body build-in test 6 limit.
As shown in Figure 1, storer in pick-up unit of the present invention for storing the color intensity that in high standard band HC, substandard band LC and calibration tape T, tested organism fluid samples produces, by the correlation measured the luminous value of color intensity and testing concentration and form in described storer; Described storer is electric magnetic card or rfid card; Described reader is optical card reader, and described optical card reader is used for the concentration value providing all analysis things in biological fluid samples respectively.
As shown in Figure 4, utilize pick-up unit of the present invention to carry out detection and consist essentially of following steps:
Step one, in the well on pick-up unit lid, drip a certain amount of biological fluid samples comprising suspicious determinand with transfer pipet; Usual application of sample amount is 10 to 100 microlitres.
Step 2, when test-strips is installed in a box body, box body will provide sample aperture be used for application of sample.Added sample is positioned at the second film testing main film upstream and is adsorbed, and flows to the main film of test with capillary action from the second film; In the process, sample flows through second diaphragm area being positioned at upstream of mark binding partner deposition.Except the tagged ligand that can be combined with determinand, also have the height of known quantity and low calibration part on the second film of this upstream.
If sample is whole blood, the second film of this upstream can also remove red blood cell and leucocyte as filtration unit, only allows serum flow through.Add damping fluid and can promote sample flowing on the test strip.Between 2 to 5 times that the application of sample amount of damping fluid is generally sample size.Suitable damping fluid comprises any pharmacy can the damping fluid of received water, and it with test sample book and can not control ligand reaction.General phosphate buffer can be phosphoric acid one or disodium salt, based on commercially available, other as citrate buffer solution and ringer's solution application also very extensive.
Step 3, with test main film contact, sample just acts on calibration tape and index zone with tagged-ligand.The suitableeest label of the present invention is the material that can produce color complexes in those detection zone and index zone.Although enzyme connection part or latex binding partner also can produce color products, part utilizes capillary flow principle for quantitatively detecting, and the developer forming part best is golden bond, can in conjunction with determinand and calibration agent.
Step 4, sizing technique of the present invention compare based on the colored intensity of determinand sample and the colored intensity of index zone alignment agent and obtain result.Therefore in test sample book, determinand content is higher, and the analysis thing be combined with tagged ligand is more, and the tagged ligand that fixed ligands is combined in test section/determinand compound amount is higher.Increase along with analyzing thing content in sample, colored intensity just increases thereupon.But in order to the actual concentrations of accurate Calculation determinand, other factors instead of testing concentration must be excluded outside any result of calculation.
Tested entries of the present invention be use the calibrating reagent in index zone to provide a kind of accurate method for quantitatively determining.In order to higher degree of accuracy, the standard regions that separate of the present invention before and after the test section of test membrane uses two kinds of calibrating reagents.All be deposited in upstream belt because a certain amount of calibrating reagent is fixed on collimating marks bond on standard regions and excessive, the color of same intensity will be generated within the given time, be applicable to the different test-strips done by same method on index zone.Typical curve uses luminous value can obtain each high calibration and low calibration areas, for the quantitative measurement analyzing thing provides foundation.In general, the calibrating reagent of any routine can use, first-selected calibrating reagent be in sample non-existent or not with the reagent of compound generation immunological cross-reaction in sample, as 2,4-dinitro benzene is for bovine serum albumin(BSA) (BSA-DNP), can (Eugene, 0R, cat#A-23018) be bought from molecular probe and use as calibrating reagent.2,2, 4-dinitrophenol (DNP) is non-existent Small molecular but can as haptens in human body, haptens refers to when itself and macro-molecular protein carrier conjugation, or be expelled to the mammal that can produce antibody as in rat, mouse, rabbit, ox, horse, sheep or goat body time, produce immunogenic material.Poor efficiency calibration areas solidification part is for bovine thyroglobulin antibody (BTG); High calibration areas is for goat-anti rabbit protein antibodies.With solidification ligand binding bond be BTG gold antigen and rabbit igg gold antigen.
In order to measure the content of determinand, be necessary to study the relation in the color intensity of test section and sample between testing concentration.This relation can be described with curve, is then constantly diluted by the determinand solution preparing a high concentration, and the change measuring color intensity can obtain.Obviously, this curve is also different in different time sections.With regard to measuring determinand content in sample, be mutually related between the typical curve that these curves and the calibrating reagent by concentration known obtain.Thus the test of each unknown sample can obtain three different color intensities.The colored intensity of index zone and standard concentration curve are related, are applicable to the Measurement and Computation of determinand and numerical value thereof in sample.In order to verify that concentration standard value is relevant with the double exposure time, obtaining desirable luminous value, marking reacting phase pass in normal concentration value and catoptry, testing concentration in sample is verified.
A kind of simple version that the present invention quantitatively detects is, determinand index zone being fixed concentration known can be accomplished as calibrating reagent.In this case, a kind of excessive single labelled part is only needed to go to be deposited on movably to accept in the upstream film of sample.Sample containing determinand dispersibles the mark binding partner of movement and allows determinand and ligand reaction and form compound.Compound and excess ligand are brought on test membrane by capillary action, and determinand incorporation of markings ligand complex and curing antibody react and to be captured and occurrence flag is reacted herein, are often the reaction of colored forms.Excessive tagged ligand reacts at the solidification determinand of standard regions and known quantity and provides a Standard Colors intensity and reacts.On the basis of the color response preset to testing concentration and the agent of index zone alignment, the concentration analyzing thing in sample can be determined.
Although necessity that step 5 index zone and concentration calculate corrects manually to have come, business machine of the prior art can measure the color intensity of calibration tape and index zone.The color intensity that in index zone and calibration tape, determinand produces can together be stored in a storer (as electric magnetic card or rfid card).When electric magnetic card or rfid card are inserted into for coml optical card reader, as the card reader that Kaiwood Technology company produces, card reader will provide the concentration value of all any analysis things in sample respectively.
Step 6, light reflection strength drawing standard product curve according to tested organism fluid samples concentration standards band.
Along with the development of determinand qualitative detection technology, if determinand antigen, corresponding compound is exactly antibody; If determinand is antibody, corresponding compound is exactly antigen, and marking bond like this could be combined with determinand.Such as determine hepatitis B surface antibody in blood sample (HBsAg) with gradient distribution test, catch in band containing the HbsAg-antibody be fixed on detection zone test membrane.
Suitable determinand is not limited only to antigen, antibody, hormone, addictive drug, cell protein, DNA, Applications of Cardiac Markers, tumour or cancer markers, autoimmune disease mark or any macromolecular substances that can produce antibody.Determinand is as being antigen, and antigen can be the antigen of infectious agent of having associated.Infectious agent can be virus, bacterium, fungi or prion.When infectious agent is virus, virus can be the viruses such as HIV, hepatitis virus A, B, C, D, single herpesviral, cytomegalovirus, papillomavirus, Ebola virus, SARS virus, rhinovirus and vaccinia virus, and is not limited only to these virus.When infectious agent is bacterium, it can be Gram-positive or Gram-negative bacteria.These bacteriums can be the populations of bacillus anthracis, Escherichia coli, helicobacter pylori, diplococcus, Salmonella and Shigella, and are not limited only to these bacteriums.When infectious agent is fungi, then can be Mycosporum mushroom or aspergillus mushroom, and be also not limited only to these fungies.
Determinand is as being hormone, then can screen from following typical monoid: human chorionic gonadotrophin, thyroxine, thyroid-stimulating hormone, hyperglycemic factor, insulin, relaxain, interstitialcellstimulating hormone (ICSH), melanotropin, growth hormone, FSH, gastrin, bradykinin, antidiuretic hormone and other releasing factor, but the hormone on other physiology or on pathology also all can be used as determinand.
If determinand tumour or cancer markers, then can screen from following typical monoid: prostate specific antigen (PSA), carcinomebryonic antigen (CEA), alpha-fetoprotein, other cancer or tumor markers also can as determinands.
If determinand Applications of Cardiac Markers, then can screen from following typical monoid: Troponin I, TnT, creatine kinase isozyme (CK-MB), myoglobins, c reactive protein (CRP), fatty acid binding protein (FABP), glycogen phos isodynamic enzyme (GPBB), Type B urine peptide (BNP), front BNP etc., other Applications of Cardiac Markers also can be used as analysis thing.
The present invention has a lot of example, and lower example only does finite specification.
A porous nitrocellulose film in test-strips is as the main film of test, enough PSAs (PSA) are fixed on two index zones (high standard band HC and substandard band LC), when the PSA monoclonal specific antibody marked with gold, respective concentration are respectively 1 and l0ng/mL mixes, corresponding color intensity will be produced.In addition, PSA monoclonal antibody also can be deposited on the calibration tape T of nitrocellulose membrane.Index zone is designed to sample and first contacts with the substandard band LC of energy Show Color intensity, and this substandard band LC is attended by the gold mark bond being equivalent to 1ng/mLPSA antigen, is and then high standard band HC, and the PSA antigen that can be l0ng/mL with equivalent is combined.The antigen that index zone deposits and antibody allow to react with the antigenic determinant on nitrocellulose membrane and on the index zone being permanently affixed at test membrane and calibration tape.Then remaining antigenic determinant uses standard technique to close.For specific test-strips, the color intensity correlativity produced between determinand and reaction time is stored on storage card.
Nitrocellulose membrane (testing main film) is connected by fluid with nonwoven glass fibre membrane (being namely positioned at the second film testing main film upstream), the main film of test deposits antibody with known technology and is used as gold mark in conjunction with PSA antigen.Analyzed sample is added in the upstream of glass fibre membrane, and downstream then deposits mark bond.The absorption of sample pad that test-strips chemical examination also comprises a test membrane end is connected by fluid-phase with downstream.
By the well on lid, the blood sample that the suspection of about 30 microlitres contains PSA is dropped in the application of sample site of glass fibre membrane upstream, follow hard on the phosphate buffer adding about 40 microlitres.Glass fibre membrane is enough thick in filtering red blood cell and leucocyte, but serum can pass through.Serum and damping fluid to be arrived by upstream film with capillary action and mark bond, golden labeling antibody bond redistribute and with sample in analyze thing and form compound.The serum of damping fluid dilution arrives the nitrocellulose membrane of porous by glass fibre membrane, continue with capillary action by whole nitrocellulose membrane.
Gold mark compound containing determinand is deposited over the PSA antibody capture on calibration tape T and shows blush, corresponding testing concentration in color intensity counter sample.Excessive golden bond also can be caught by high and substandard band LC, index zone comprises enough calibrating reagents to catch equivalent in 1 or the determinand of l0ng/mL.If there is PSA in sample, then can obtain the band of three different colours intensity.After passing through calibration tape T and index zone C, remaining Buffer samples is by arriving absorption pad after nitrocellulose membrane and being stored in there.
Etui containing color bar (test-strips) is inserted with in the card reader of storage card, the correlation be made up of the luminous value and testing concentration of measuring color intensity in storage card.The CHRl00 card reader used is manufactured by Kaiwood Industries, and it uses storage card light intensity can be converted into concentration value.The measurement of light intensity is measured after 10 and 15 minutes after application of sample, as follows:
10 minutes test results:
| Determinand | PSA concentration | RLU |
| High calibration | 10ng/ml | 10 |
| Low calibration | 1ng/ml | 1 |
| Sample | X | 4.04 |
According to 2 typical curves, in card reader show sample, PSA concentration is 4ng/mL.
15 minutes test results:
| Determinand | PSA concentration | RLU |
| High calibration | 10ng/ml | 23.9 |
| Low calibration | 1ng/ml | 10.3 |
| Sample | X | 19.49 |
According to 2 typical curves, in card reader show sample, PSA concentration is 4ng/mL.
Although invention has been described for composition graphs above; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; when not departing from present inventive concept, can also make a lot of distortion, these all belong within protection of the present invention.
Claims (7)
1. a biological fluid sample quantitative test device, comprises the test-strips be arranged in a box body, it is characterized in that: also comprise the storer of color intensity data and the reader of color intensity data;
The bottom of described test-strips is a substrate (1), described substrate (1) is provided with the main film of the test with porous (6); The main film of described test (6) has a high standard band HC, a substandard band LC and a calibration tape T, all there is calibrating reagent in described high standard band HC and substandard band LC, described calibrating reagent corresponds respectively to the determinand of concentration known, described calibration tape T has the fixed ligands that can be combined with determinand;
The upstream of the main film of described test (6) is provided with the second film, described second film comprises at least two-layer non-activity that arranges from bottom to top and has film (5) and a filtering membrane (4) of porous, described second film is provided with a sample application site and a tagged ligand site, impels tested organism fluid samples and compound thereof, the flowing of marker ligand physical efficiency by capillary action;
The downstream of the main film of described test (6) is provided with tertiary membrane (2), described tertiary membrane (2) is an absorption layer, described tertiary membrane (2) is for contacting with tested organism fluid samples, impel tested organism fluid samples to flow through the main film of test (6) by the capillary action of described tertiary membrane (2), described tertiary membrane (2) is for having passed through the tested organism fluid samples of the main film of test (6) in the end absorption in test main film (6) downstream;
Described box body comprises lid, described lid is provided with two wells (S1, S2) and a form, and the position of described two wells aligns with sample application site on the second film in box and tagged ligand site respectively; Described form is consistent with the region that the main film of box body build-in test (6) upper high standard band HC, substandard band LC and calibration tape T limit;
Described storer is for storing the color intensity that in high standard band HC, substandard band LC and calibration tape T, tested organism fluid samples produces, and described reader is used for the concentration value providing all analysis things in biological fluid samples respectively.
2. biological fluid sample quantitative test device according to claim 1, it is characterized in that: the main film of described test (6) is for bag is by nitrocellulose filter, non-activity in described second film and the film (5) with porous are for collaurum bag is by glass fibre membrane, and the filtering membrane (4) in described second film is made by the glass fibre of non-woven shape or polypropylene; Described absorption layer is made by efficient sorbing material.
3. biological fluid sample quantitative test device according to claim 1, it is characterized in that: if tested organism fluid samples is whole blood, then: be arranged in the second film testing main film upstream and can cross the red blood cell and leucocyte that filter tested organism fluid samples, only allow serum flow through.
4. biological fluid sample quantitative test device according to claim 1, it is characterized in that: tested organism fluid samples is selected from: antigen, antibody, hormone, addictive drug, cell protein, DNA, Applications of Cardiac Markers, tumour or cancer markers, autoimmune disease mark or any macromolecular substances that can produce antibody, wherein:
Described antigen is the antigen of infectious agent of having associated; Wherein, described infectious agent is virus, bacterium, fungi or prion;
When infectious agent is virus, virus is HIV, hepatitis virus A, B, C, D, single herpesviral, cytomegalovirus, papillomavirus, Ebola virus, SARS virus, rhinovirus or vaccinia virus;
When infectious agent is bacterium, bacterium is Gram-positive or Gram-negative bacteria; Or bacillus anthracis, Escherichia coli, helicobacter pylori, diplococcus, Salmonella or Shigella population;
When infectious agent is fungi, be then Mycosporum mushroom or aspergillus mushroom;
Described hormone is selected from: human chorionic gonadotrophin, thyroxine, thyroid-stimulating hormone, hyperglycemic factor, insulin, relaxain, interstitialcellstimulating hormone (ICSH), melanotropin, growth hormone, FSH, gastrin, bradykinin, antidiuretic hormone;
Described tumour or cancer markers are selected from: prostate specific antigen (PSA), carcinomebryonic antigen (CEA) or alpha-fetoprotein;
Described Applications of Cardiac Markers is selected from: Troponin I, TnT, creatine kinase isozyme (CK-MB), myoglobins, c reactive protein (CRP), fatty acid binding protein (FABP), glycogen phos isodynamic enzyme (GPBB), Type B urine peptide (BNP), front BNP.
5. a biological fluid samples quantitative detecting method, is characterized in that, utilizes the pick-up unit according to any one of Claims 1-4 to detect;
Its detecting step is as follows:
Step one, in the well on pick-up unit lid, drip tested organism fluid samples;
Step 2, described tested organism fluid samples are positioned at the second film absorption of test main film (6) upstream, and flow to the main film of test (6) with capillary action from the second film; In the process, tested organism fluid samples flows through second diaphragm area with tagged ligand deposition, and described tested organism fluid samples is combined with tagged ligand, described second film is formed with the height of known quantity and low calibration part;
Step 3, with test main film (6) contact, tested organism fluid samples just acts on calibration tape T and high standard band HC and substandard band LC with tagged ligand;
Step 4, compare based on the colored intensity of tested organism fluid samples and the colored intensity of index zone alignment reagent and obtain the result that quantitatively detects;
Step 5, the mark colored intensity produced by tested organism fluid samples in index zone and calibration tape are together stored in storer; And the concentration value of all analysis things in tested organism fluid samples is provided by reader;
Step 6, the mark colored intensity drawing standard curve produced according to described index zone and described calibration tape.
6. biological fluid samples quantitative detecting method according to claim 5, is characterized in that: after the height forming known quantity on the second film and low calibration part, add damping fluid to promote the flowing of described tested organism fluid samples on the main film of test; Between the application of sample amount of described damping fluid is 2 to 5 times of sample size; Described damping fluid comprises any pharmacy can the damping fluid of received water, and described damping fluid does not react with sample and tagged ligand.
7. biological fluid samples quantitative detecting method according to claim 6, it is characterized in that: described damping fluid does not react with sample and tagged ligand, and described damping fluid is phosphate buffer, is monosodic alkaliine or disodic alkaliine; Or described damping fluid is citrate buffer solution or ringer's solution.
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| HK15103636.7A HK1203231A1 (en) | 2010-05-28 | 2015-04-14 | Quantitative detection device and detection method of biological fluid samples |
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| CN201410348806.XA CN104215757B (en) | 2010-05-28 | 2010-05-28 | Biological fluid sample quantitative test device and its detection method |
| CN201010196737.7A CN101839908B (en) | 2010-05-28 | 2010-05-28 | Quantitative detection device and detection method of biological fluid samples |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017071092A1 (en) * | 2015-10-27 | 2017-05-04 | 上海芯超生物科技有限公司 | Whole blood one-step alpha-fetoprotein colloidal gold detection kit and detection method thereof |
| CN107367526A (en) * | 2017-07-31 | 2017-11-21 | 重庆微奥云生物技术有限公司 | A kind of RNA detection method and rna probe method for coating |
| CN107447002A (en) * | 2017-07-31 | 2017-12-08 | 重庆微奥云生物技术有限公司 | A kind of DNA detection method and DNA probe method for coating |
| CN107449807A (en) * | 2017-07-31 | 2017-12-08 | 重庆微奥云生物技术有限公司 | A kind of detection method of enzyme |
| WO2020003021A1 (en) * | 2018-06-28 | 2020-01-02 | International Business Machines Corporation | Accurate colorimetric based test strip reader system |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1215163A (en) * | 1997-10-20 | 1999-04-28 | 李金波 | Method and apparatus for single step analysis of whole blood |
| CN1608207A (en) * | 2001-12-24 | 2005-04-20 | 金伯利-克拉克环球有限公司 | Internal calibration system for flow-through assays |
| CN101031798A (en) * | 2004-07-29 | 2007-09-05 | 瑞莱诊断体系有限公司 | Quantitative lateral flow system and assay |
| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
| WO2009123069A1 (en) * | 2008-03-31 | 2009-10-08 | テルモ株式会社 | Method for preparing calibrating reagent, calibrating reagent, and method for calibrating blood component measuring device using said calibrating reagent |
-
2010
- 2010-05-28 CN CN201410348806.XA patent/CN104215757B/en active Active
-
2015
- 2015-04-14 HK HK15103636.7A patent/HK1203231A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1215163A (en) * | 1997-10-20 | 1999-04-28 | 李金波 | Method and apparatus for single step analysis of whole blood |
| CN1608207A (en) * | 2001-12-24 | 2005-04-20 | 金伯利-克拉克环球有限公司 | Internal calibration system for flow-through assays |
| CN101031798A (en) * | 2004-07-29 | 2007-09-05 | 瑞莱诊断体系有限公司 | Quantitative lateral flow system and assay |
| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
| WO2009123069A1 (en) * | 2008-03-31 | 2009-10-08 | テルモ株式会社 | Method for preparing calibrating reagent, calibrating reagent, and method for calibrating blood component measuring device using said calibrating reagent |
Cited By (10)
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|---|---|---|---|---|
| WO2017071092A1 (en) * | 2015-10-27 | 2017-05-04 | 上海芯超生物科技有限公司 | Whole blood one-step alpha-fetoprotein colloidal gold detection kit and detection method thereof |
| CN107367526A (en) * | 2017-07-31 | 2017-11-21 | 重庆微奥云生物技术有限公司 | A kind of RNA detection method and rna probe method for coating |
| CN107447002A (en) * | 2017-07-31 | 2017-12-08 | 重庆微奥云生物技术有限公司 | A kind of DNA detection method and DNA probe method for coating |
| CN107449807A (en) * | 2017-07-31 | 2017-12-08 | 重庆微奥云生物技术有限公司 | A kind of detection method of enzyme |
| WO2020003021A1 (en) * | 2018-06-28 | 2020-01-02 | International Business Machines Corporation | Accurate colorimetric based test strip reader system |
| US10605741B2 (en) | 2018-06-28 | 2020-03-31 | International Business Machines Corporation | Accurate colorimetric based test strip reader system |
| GB2586939A (en) * | 2018-06-28 | 2021-03-10 | Ibm | Accurate colorimetric based test strip reader system |
| GB2586939B (en) * | 2018-06-28 | 2021-06-30 | Ibm | Accurate colorimetric based test strip reader system |
| US11307147B2 (en) | 2018-06-28 | 2022-04-19 | International Business Machines Corporation | Accurate colorimetric based test strip reader system |
| CN115060779A (en) * | 2022-06-27 | 2022-09-16 | 上海德琪医药科技有限公司 | Blood anti-tumor drug metabolite concentration detection device and monitoring method thereof |
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| Publication number | Publication date |
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| HK1203231A1 (en) | 2015-10-23 |
| CN104215757B (en) | 2018-12-04 |
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