CN101839908B - Quantitative detection device and detection method of biological fluid samples - Google Patents
Quantitative detection device and detection method of biological fluid samples Download PDFInfo
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- CN101839908B CN101839908B CN201010196737.7A CN201010196737A CN101839908B CN 101839908 B CN101839908 B CN 101839908B CN 201010196737 A CN201010196737 A CN 201010196737A CN 101839908 B CN101839908 B CN 101839908B
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Abstract
The invention discloses a quantitative detection device of biological fluid samples, which comprises a test strip arranged in a box body, a color intensity data memory and a color intensity data reader, wherein a substrate is arranged at the bottom layer of the test strip, and a porous test main film is arranged on the substrate; a second film is arranged at the upstream of the test main film, a sample application site and a tagged ligand site are arranged on the second film, and a biological fluid sample to be tested and compounds thereof and the tagged ligand can be promoted under the capillary action to flow; a third film is arranged at the downstream of the test main film, and the third film is used for adsorbing the biological fluid sample to be tested passing through the test main film at the downstream end of the test main film; and two sample injecting holes and a viewing window are arranged on the box cover of the box body. The detection device of the invention can be used for drawing a standard sample curve through the light reflection intensity of the standard sample with known concentration, thereby quickly, effectively and accurately carrying out quantitative analysis on the biological body fluid sample.
Description
Technical field
The present invention relates to a kind of system for detection of biological liquid sample, relate in particular to the concentration of thing to be checked in a kind of detection 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, after its principle is reagent-impregnated, by capillary action to the film that contains immobilized reagent, test strip detection zone be combined with visable indicia thing, as the determinand reaction of latex particle, metal composite.It is worth mentioning that and from metal-sol, obtain label, this label dealing from metal-sol can with ligand binding, be then further combined with determinand, part or antibody.Test-strips is by a large number for quantitatively detecting body fluid, as the determinand in urine and blood.This may be the most general the earliest method as the method for assignor's pregnancy to detect human chorionic gonadotrophin.Above-mentioned detection is all carried out in test-strips, and its result shows only needs a step to complete, and for example, liquid sample passes through absorbing membrane, inner determinand and corresponding ligand binding, and result can clearly be presented on the detection zone separated with sample loading zone.Carry out above-mentioned detection, conventional proving installation generally contains cutting and capsule at present, and user just can detect without extra utensil.Known pick-up unit adopts sandwich detection and competition to suppress to detect two kinds of detection methods substantially.
Sandwich detection method, is that the determinand in liquid sample reacts with mark or indicator ligands, generates complicated determinand or label.This reaction occurs to have determinand to enter detector bar, otherwise tagged ligand will be deposited on test-strips absorbing membrane completely.In test-strips, the determinand that is combined with label moves on to sample capture district by capillary action; There, by solidifying capture ligands, can obtain determinand complex.The in the situation that of golden mark, there is colour band in pattern detection district, shows to have determinand to exist.This pick-up unit also contains second and solidifies part band, generally not only as quality control band, is combined with tagged ligand, even, in the situation that there is no determinand, can show the performance of test-strips.
Competition suppresses detection method, is that determinand can react with the fixed ligand in sample capture district with tagged ligand, and determinand and tagged ligand are competed the fixed ligand in sample capture district jointly like this.The existence of any determinand all will replace the binding site of tagged ligand.Tagged ligand is caught and is occurred signal, represents that result is negative.
In prior art, based on test-strips, determine the device that determinand has or not, how the accurate amount of determinand in liquid sample can not be provided.Even if be furnished with the standard items of quantitative test in pick-up unit, also can be 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 to biological fluid samples carry 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 being 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, is provided with the main film of test with porous on described substrate; The main film of described test has a high standard band HC, a substandard band LC and a calibration tape T, in described high standard band HC and substandard band LC, has calibrating reagent, on described calibration tape T, has fixed ligands; The provided upstream of the main film of described test is equipped with the second film (54), described the second film (54) comprises at least two-layer non-activity of white lower and upper setting and has film and a filtering membrane of porous, on described the second film, be provided with a sample application site and a tagged ligand site, by capillary action, impel biological fluid samples to be measured and compound thereof, marker ligand physical efficiency to flow; 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 for contacting with biological fluid samples to be measured, by the capillary action of described tertiary membrane, impel biological fluid samples to be measured to flow through the main film of test, described tertiary membrane has passed through the biological fluid samples to be measured of the main film of test for the end absorption in test main film downstream; Described box body comprises lid, is provided with two wells and a form on described lid, the position of described two wells respectively with box on the second film sample application site and tagged ligand site align; Described form is consistent with the region that high standard band HC, substandard band LC on the main film of box body build-in test and calibration tape T limit; The color intensity that described storer produces for storing high standard band HC, substandard band LC and calibration tape T biological fluid samples to be measured, in described storer by measuring the luminous value of color intensity and the correlation that biological fluid samples concentration to be measured forms; Described storer is electric magnetic card or rfid card; Described reader is optical card reader, and described optical card reader is for providing respectively the concentration value of all analytes of biological fluid samples.
In the present invention, utilize the detecting step of above-mentioned biological fluid sample quantitative test device as follows:
Step 1, with transfer pipet to the biological fluid samples to be measured that drips 10 to 100 microlitres in the well on pick-up unit lid;
Step 2, described biological fluid samples to be measured are positioned at the second film absorption of test main film upstream, and from the second film, flow to the main film of test with capillary action; In this process, biological fluid samples to be measured is flowed through and is had the second diaphragm area of tagged ligand deposition, and described biological fluid samples to be measured is combined with tagged ligand, is formed with height and the low calibration part of known quantity on described the second film;
Step 3, with test main film contact, biological fluid samples to be measured just acts on calibration tape T and high standard band HC and substandard band LC with tagged ligand;
The result that the colored intensity comparison of step 4, the colored intensity based on biological fluid samples to be measured and index zone alignment reagent and obtaining quantitatively detects; That is: in biological fluid samples to be measured, determinand content is higher, the analyte of being combined with tagged ligand is just more, fixed ligands is combined in the main film calibration tape T of test tagged ligand or determinand compound amount are just higher, thereby the mark colored intensity on index zone increases; Along with analyte content in biological fluid samples to be measured increases, the colored intensity of mark just increases thereupon;
Step 5, the mark colored intensity that biological fluid samples to be measured in index zone and calibration tape is produced together store in an electric magnetic card or rfid card; When this electric magnetic card or rfid card are inserted in optical card reader, this card reader provides respectively the concentration value of all analytes in biological fluid samples to be measured;
Step 6, according to the light reflection strength drawing standard product curve of biological fluid samples concentration standard product band to be measured.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention's application solid phase chromatography detects, and as sandwich detection, one or more biological fluid samples to be measured are combined with mark bond, also can be combined with labelled reagent by the curing calibrating reagent above test-strips.On standard items band, described labelled reagent is hunted down by calibrating reagent, for measuring to detect, is with upper biological fluid samples concentration to be measured to create a platform.The curing calibrating reagent of standard items band is combined as liquid sample with corresponding labelled reagent and passes through detector bar.Determinand in liquid sample is just combined on sample capture band.Utilize these relative intensities that are marked with complex just can obtain the accurate concentration of biological fluid samples to be measured.Therefore, the quantity that the intensity of the label on film in various sample capture bands (as: gold size bond), the degree of depth have just reflected biological fluid samples to be measured, in like manner, standard items band also can reflect intensity and the concentration of calibrating reagent.So the commercial visualization device that utilizes is converted into measurement concentration by the light reflection strength of sample.Finally, use 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 biological fluid samples to be measured (hereinafter to be referred 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 of mentioning in sandwich assay, the same with determinand, label, tracer part in competition detection method.Bond can be chosen from the large molecule that can be combined with determinand or compound, as antigen-antibody complex, Antibody-antigen complex.
3, test section (or band), refers to that bond or determinand can adhere to the position on it, for example: a part for test-strips in pick-up unit.
4, test-strips (or detector bar), refers to and can pass through capillary action, makes the liquid sample that contains determinand need to determine the mobile perforated membrane of antigen of testing concentration with other.General perforated membrane has glass fibre, porous nitrocellulose, tygon.The test-strips of invention is all comprised of supporting film and filtrator.
5, tracer, refers to and is marked with determinand or the bond part that can survey, be preferably marked with special raadable mark thing (as: collaurum, latex, the liposome that comprises dyestuff, carbon black and analog).
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 any liquid that contains determinand.The sample detecting can be body fluid, comprise: the urine obtaining from fetus, neonate, youth, adult, blood, sweat, lymph liquid, abdomen intraluminal fluid, crude extracts or homogenate, the liquid of inanimate object activity, as the water obtaining from river, lake, test water etc.
8, label, refers to directly or indirectly large molecule or the compound of conditioning signal (as change color) pattern.This signal is used to indicate the concentration range that has that it's too late that detects target determinand in sample.Label may contain enzyme, fluorescer, liposome, red blood cell, polymer microcapsules, colour developing polymer particles (latex), also may be the colloid that contains metal composite.Various patents and be applied as and produce optical signal lot of documents is provided.In the present invention, can find the application of several label type patents.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 labeling thing; United States Patent (USP): 4,104,099 finds chemiluminescent labels; United States Patent (USP): 4,160,645 find without enzyme catalyst label; Patent attractive in appearance: 3,966,879 find that electrophoretic techniques can be used for antibody district; United States Patent (USP): 4,120,945 find that radioimmunoassay detects first, and in this method, the determinand of mark can be combined with solid support by antibody; United States Patent (USP): 4,233,402 find paired enzyme labeling thing; 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 can be also metal-sol; Metal or metal composite, mix or be coated in the metal composite at polymkeric substance center mutually as: metal oxide, oxyhydroxide, slaine, metal composite or with polymkeric substance.Any metal-sol that these metal marker things are mentioned above may being or the dewatering state of metal composite colloidal sol may be more the collaurums of dewatering state.
9, labeled reactant, refers to the signal intensity generating when solidified reagents links on label and calibration tape or index zone.The reflected light device generating with color is the good method of measurement markers reaction.
10, compound, refers to that (based on context) is any by determinand and one or more part, or the polymolecular compound being formed by tagged ligand and curing part.In sandwich immunoassay method, such as following compound can produce: first determinand/tagged ligand double combination produces (the first compound) in detection, determinand/tagged ligand/solidify part three in conjunction with appearing at (the second compound) in detection.
11, liquid exchanges, and refers to a kind of contacting with each other but not addition, allows the fluid by a passage, to be flowed to the structure of another passage.
12, chemical examination, refers to that use test bar can detect several dissimilar detecting patterns of determinand.For example, in sandwich immunoassay method, 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 a kind of can combination with target determinand, the molecule that also can be combined with label, and label can be metal marker thing, preferably collaurum.
13, capture zone (or catching site), refers to region or band in the stratographic analysis test-strips that at least contains a determinand binding reagents.Determinand binding reagents is conventionally fixed on district or is with, after detectable reagent reacting, district or with on generate the result that can survey, can reflect the situation of determinand content in sample or its existence." capture zone " may be comprised of the above trapping region that can catch a plurality of samples, and in the case, more than one determinand bonding agent can be used.For example, within the scope of the present invention, the combination of two detections is considered to a test set, can detect hepatitis C virus (HCV) and human immunodeficiency virus (HIV) simultaneously, or detect the antibody of hepatitis B surface antibody (HBsAg) and microspironema pallidum (TP) simultaneously.It is feasible also having other combination, and within the scope of the present invention.
14, bond and detection reagent, this can with combine detect reagent as: the antigen of developer, fluorescer, enzyme or chemiluminescence agent or antibody exchange.In practice of the present invention, " bond " and " detection reagent " is specifically in conjunction with determinand that will be definite or be fixed on the catches on capture zone.On capture zone, " bond " and " detection agent " produces the value of the current determinand content of reflection that can quantitative measurment.As will be explained hereinafter, the density that on capture zone, direct quantitative is measured differs and reflects surely the quantity that is combined in current determinand on capture zone, but the measured band strength of luminous value (RLU) but can reflect the content of current determinand in capture zone.
15, at this " index zone ", comprise the calibrating reagent that is fixed on test strip calibration district.Calibrating reagent is combined and forms calibration in conjunction with right with calibration binding reagents specifically.In the present invention, comprise two calibration tapes.Containing calibration is the effect that they can play internal calibration in conjunction with right advantage, and calibration agent may be by the cubage of current determinand in capture zone interior.Calibration agent may be 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 the LC product line that can be used for settling the standard.Standard items line can generate a regression equation by the RLU value of calibration agent, with this, describes two relations between variable, thereby is applied to each quantitative test experience.Although, conventional calibration agent is still being used, 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 be bought (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 the interior non-existent little molecule of human body but can be used as haptens, haptens refers to when itself and the coupling of macro-molecular protein carrier, 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 is solidified part, and to take bovine thyroglobulin antibody (BTG) be example; It is example that high calibration areas be take goat-anti rabbit protein antibodies.With the bond of curing ligand binding 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 being 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, is provided with the porous with sufficient porosity and tests main film 6 on described adhesive sticker substrate 1, can allow the check material that comprises determinand and compound thereof move 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 coated nitrocellulose filter, and these films are widely known by the people technically.
On the main film 6 of described test, there is 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, on described calibration tape T, there is fixed ligands, that is: detecting band T region, test and on main film, comprise the curing part that can be combined with determinand, and can better run through with the form of band the main film of test, just as the same through the main film formation of test index zone with the calibrating reagent on substandard band LC at high standard band HC.Curing part for calibration tape region and index zone region is different.On index zone fixedly the calibrating reagent of known quantity for the curve that settles the standard, thereby determine the quantity of determinand.Test after the ligand binding on main film, remaining avtive spot is closed, thereby allows determinand and compound, tagged ligand and compound thereof freely to pass through test-strips.
Test-strips porous in the present invention is tested the upstream of main film 6, also comprise second film, described the second film comprises at least two-layer non-activity arranging 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 ligand binding by film, in addition also comprise the curing part band for detection of fluid communication on film.This film 5 adopts the coated glass fibre membrane of collaurum, and this film more may be made by the glass fibre faling apart or polypropylene, has sufficient hole simultaneously, allows determinand and compound thereof, tagged ligand to depend on capillary action to flow.The top layer of described the second film is a filtering membrane 4, and this filtering layer 4 adopts glass fibre membrane, and this filtering membrane is applicable to from sample component, determinand be separated, and other components in sample may be disturbed the check to determinand.Therefore, when detecting blood, red blood cell or leucocyte can be separated from the serum that comprises determinand.Therefore the second film comprises a sample application site and a tagged ligand site, the upstream of the downstream of practical site and test membrane contact point, in sandwich reaction, react with determinand, or react with detection zone binding partner in competitive reaction, and reacting between complicated tagged ligand and correction part in index zone.Labelled reagent is attracted to the upstream of this film, when it contacts with liquid sample, can flow away from film, thereby form compound under the condition with analyte response, can continue to flow to test membrane by upstream film.
The downstream that is positioned at the main film 6 of test in test-strips also comprises tertiary membrane 2, this tertiary membrane 2 is an absorption layer, this tertiary membrane 2 contacts with liquid, for adsorbing by the liquid sample of test membrane in the downstream end of the main film 6 of test, also as driving force, by capillary action, impel liquid sample to flow through detection film.Such absorption layer is preferably made of efficient sorbing material, as: can adsorb sample simultaneously can be as the paper that adds any damping fluid in test-strips.
As shown in Figure 2, described box body comprises lid, is provided with two well S1, S2 and a form W on described lid, the position of described two well S1, S2 respectively with box on the second film sample application site and tagged ligand site align; Described form W is consistent with the region that high standard band HC, substandard band LC on the main film 6 of box body build-in test and calibration tape T limit.
As shown in Figure 1, the color intensity that storer in pick-up unit of the present invention produces for storing high standard band HC, substandard band LC and calibration tape T biological fluid samples to be measured, in described storer by measuring the luminous value of color intensity and the correlation that testing concentration forms; Described storer is electric magnetic card or rfid card; Described reader is optical card reader, and described optical card reader is for providing respectively the concentration value of all analytes of biological fluid samples.
As shown in Figure 4, utilize pick-up unit of the present invention to detect and consist essentially of following steps:
Step 1, with transfer pipet, in the well on pick-up unit lid, drip a certain amount of biological fluid samples that comprises suspicious determinand; Conventionally application of sample amount is 10 to 100 microlitres.
Step 2, in test-strips is installed in a box body time, on box body, will provide sample aperture for application of sample.Add the second film absorption that sample is positioned at test main film upstream, and from the second film, flow to the main film of test with capillary action; In this process, sample flow is through second diaphragm area that is 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 be removed red blood cell and leucocyte as filtration unit, only allows serum flow through.Add damping fluid can promote sample flowing in test-strips.The application of sample amount of damping fluid is generally between 2 to 5 times of sample size.Suitable damping fluid comprises the damping fluid that any pharmacy can received water, and it can and not control ligand reaction with test sample book.General phosphate buffer can be phosphoric acid one or disodium salt, and commercially available be basis, other apply also very extensive as citrate buffer solution and ringer's solution.
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 to detect the material that can produce color complexes in band and index zone at those.Although enzyme connection part or latex binding partner also can produce coloured product, part utilizes capillary flow principle for quantitatively detecting, and forming the best developer of part is golden bond, can be in conjunction with determinand and calibration agent.
Step 4, sizing technique of the present invention are the colored intensity comparisons of colored intensity based on determinand sample and the agent of index zone alignment and obtain result.Therefore in test sample book, determinand content is higher, and the analyte of being combined with tagged ligand is just more, and tagged ligand/determinand compound amount that fixed ligands is combined in test section is just higher.Along with analyte content in sample increases, colored intensity just increases thereupon.Yet for the actual concentrations of accurate Calculation determinand, other factors rather than testing concentration must be excluded within any result of calculation.
Tested entries of the present invention be to provide a kind of accurate method for quantitatively determining with the calibrating reagent in index zone.For higher degree of accuracy, the present invention uses two kinds of calibrating reagents on the standard regions separating before the test section of test membrane and afterwards.Because a certain amount of calibrating reagent is fixed on standard regions and excessive collimating marks bond is all deposited on the band of upstream, within the given time, will on index zone, generate the color of same intensity, be applicable to the different test-strips of doing by same method.Typical curve is used luminous value can obtain each high calibration and low calibration areas, for the quantitative measurement of analyte provides foundation.In general, the calibrating reagent of any routine can be used, first-selected calibrating reagent be in sample non-existent or not with sample in the reagent of compound generation immunological cross-reaction, as 2,4-dinitro benzene is for bovine serum albumin(BSA) (BSA-DNP), can buy from molecular probe (Eugene, OR, cat#A-23018) and use as calibrating reagent.2,2, 4-dinitrophenol (DNP) is the interior non-existent little molecule of human body but can be used as haptens, haptens refers to when itself and the coupling of macro-molecular protein carrier, 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 is solidified part, and to take bovine thyroglobulin antibody (BTG) be example; It is example that high calibration areas be take goat-anti rabbit protein antibodies.With the bond of curing ligand binding be BTG gold antigen and rabbit igg gold antigen.
In order to measure the content of determinand, be necessary to study the relation between testing concentration in the color intensity of test section and sample.This relation can be described with curve, and by the then constantly dilution of determinand solution of preparing a high concentration, and the variation of measuring color intensity can obtain.Obviously, this curve is also different in different time sections.Just measuring determinand content in sample, is to be mutually related between the typical curve that these curves and the calibrating reagent by concentration known obtain.Thereby the test of each unknown sample can obtain three different color intensities.The colored intensity of index zone and normal concentration curve are related, are applicable to the Measurement and Computation of determinand in sample and numerical value thereof.Relevant with the double exposure time in order to verify concentration standard value, to obtain desirable luminous value, in normal concentration value and catoptry, mark reacting phase closes, and testing concentration in sample is verified.
A kind of simple version that the present invention quantitatively detects is that on index zone, fixedly the determinand of concentration known can be accomplished as calibrating reagent.In this case, only need a kind of excessive single labelled part to go to be deposited on movably in the upstream film of accepting sample.Sample that contains determinand dispersibles mobile mark binding partner 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 reacts with curing antibody and is hunted down and occurrence flag reaction herein, is often the reaction of color form.Excessive tagged ligand reacts with the curing determinand of known quantity in standard regions and provides the reaction of Standard Colors intensity.On to the basis of the predefined color response of testing concentration and the agent of index zone alignment, in sample, the concentration of analyte can be determined.
Although necessity that step 5 index zone and concentration are calculated is proofreaied and correct can be by manually completing, business machine of the prior art can be measured the color intensity of calibration tape and index zone.The color intensity that in index zone and calibration tape, determinand produces can together store 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 respectively the concentration value of all any analytes in sample.
Step 6, according to the light reflection strength drawing standard product curve of biological fluid samples concentration standard product band to be measured.
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 mark bond could be combined with determinand like this.For example with gradient distribution test, determine hepatitis B surface antibody in blood sample (HBsAg), in capture zone, contain and be fixed on the HbsAg-antibody detecting on tape test film.
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, can be Mycosporum mushroom or aspergillus mushroom, and be also not limited only to these fungies.
Determinand is as being hormone, can from following typical monoid, screen: 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, however hormone on other physiology or on pathology also all can be used as determinand.
If determinand tumour or cancer markers can be screened from following typical monoid: prostate specific antigen (PSA), carcinomebryonic antigen (CEA), alpha-fetoprotein, other cancer or tumor markers also can be used as determinand.
If determinand Applications of Cardiac Markers, can from following typical monoid, screen: Troponin I, TnT, creatine kinase isozyme (CK-MB), myoglobins, c reactive protein (CRP), fatty acid binding protein (FABP), glycogen phosphorylation isodynamic enzyme (GPBB), Type B urine peptide (BNP), front BNP etc., other Applications of Cardiac Markers also can be used as analyte.
The present invention has a lot of examples, and lower example is only done finite specification.
A porous nitrocellulose membrane 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 PSA monoclonal specific antibody, respective concentration with golden mark are respectively 1 and 10ng/mL while mixing, will produce corresponding color intensity.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 that is equivalent to 1ng/mLPSA antigen, is and then high standard band HC, and the PSA antigen that can be 10ng/mL with equivalent is combined.The antigen depositing on index zone and antibody allow react and be permanently affixed on the index zone and calibration tape of test membrane with the antigenic determinant on nitrocellulose membrane.Then remaining antigenic determinant is used standard technique to seal.For specific test-strips, determinand and the color intensity correlativity producing between the reaction time are stored on storage card.
Nitrocellulose membrane (testing main film) is connected by fluid with nonwoven glass fibre membrane (being positioned at the second film of test main film upstream), is depositing antibody be used as golden mark in conjunction with PSA antigen on the main film of test with known technology.Analyzed sample is added in the upstream of glass fibre membrane, and downstream is depositing mark bond.Test-strips chemical examination also comprises that the absorption of sample pad of a test membrane end is connected by fluid-phase with downstream.
The blood sample that the suspection of about 30 microlitres is contained to PSA by the well on lid drops in the application of sample site of glass fibre membrane upstream, follows hard on the phosphate buffer that adds about 40 microlitres.It is enough thick in to filter red blood cell and leucocyte that glass fibre membrane is wanted, but serum can pass through.Serum and damping fluid arrive mark bond with capillary action by upstream film, golden labeling antibody bond redistribute and with sample in analyte 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, comprises enough calibrating reagents and catch equivalent in 1 or the determinand of 10ng/mL on index zone.If there is PSA in sample, can obtain the band of three different colours intensity.After by calibration tape T and index zone C, remaining buffering sample arrives absorption pad and is stored in there by after nitrocellulose membrane.
The etui that will contain color bar (test-strips) inserts in the card reader with storage card, in storage card by measuring the luminous value of color intensity and the correlation that testing concentration forms.The CHR100 card reader of using 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 in conjunction with figure, invention has been described above; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; rather than restrictive; those of ordinary skill in the art is under enlightenment of the present invention; in the situation that not departing from aim of the present invention, can also make a lot of distortion, within these all belong to protection of the present invention.
Claims (12)
1. a biological fluid sample quantitative test device, comprises the test-strips being 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), is provided with the main film of test (6) with porous on described substrate (1); The main film of described test (6) has 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, on described calibration tape T, there is fixed ligands, wherein said calibrating reagent is 2,4-dinitro benzene is for bovine serum albumin(BSA) (BSA-DNP) or 2,4-DNP (DNP);
The provided upstream of the main film of described test (6) is equipped with the second film, described the second film comprises at least two-layer non-activity arranging from bottom to top and has film (5) and a filtering membrane (4) of porous, on described the second film, be provided with a sample application site and a tagged ligand site, by capillary action, impel biological fluid samples to be measured and compound thereof, tagged ligand active;
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 biological fluid samples to be measured, by the capillary action of described tertiary membrane (2), impel biological fluid samples to be measured to flow through test main film (6), described tertiary membrane (2) has passed through the biological fluid samples to be measured of the main film of test (6) for the end absorption in test main film (6) downstream;
Described box body comprises lid, is provided with two wells (S1, S2) and a form on described lid, the position of described two wells respectively with box on the second film sample application site and tagged ligand site align; 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;
The color intensity that described storer produces for storing high standard band HC, substandard band LC and calibration tape T biological fluid samples to be measured, in described storer by measuring the luminous value of color intensity and the correlation that testing concentration forms; Described storer is electric magnetic card or rfid card; Described reader is optical card reader, and described optical card reader is for providing respectively the concentration value of all analytes of biological fluid samples.
2. biological fluid sample quantitative test device according to claim 1, it is characterized in that: the main film of described test (6) is coated nitrocellulose filter, non-activity in described the second film and the film (5) with porous are the coated glass fibre membrane of collaurum, and the filtering membrane (4) in described the second film is made by glass fibre or the polypropylene of non-woven shape; Described absorption layer is made by efficient sorbing material.
3. biological fluid sample quantitative test device according to claim 1, is characterized in that: between the main film of described test (6) and the second film, adopt an adhesive tape (3) fixing.
4. biological fluid sample quantitative test device according to claim 1, it is characterized in that: if biological fluid samples to be measured is whole blood,: the second film that is arranged in test main film upstream can remove by filter red blood cell and the leucocyte of biological fluid samples to be measured, only allows serum flow through.
5. biological fluid sample quantitative test device according to claim 1, is characterized in that: biological fluid samples to be measured 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.
6. biological fluid sample quantitative test device according to claim 5, is characterized in that: described biological fluid samples to be measured is antigen, and 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 the population of bacillus anthracis, Escherichia coli, helicobacter pylori, diplococcus, Salmonella or Shigella;
When infectious agent is fungi, be Mycosporum mushroom or aspergillus mushroom.
7. biological fluid sample quantitative test device according to claim 5, is characterized in that: described biological fluid samples to be measured is that 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.
8. biological fluid sample quantitative test device according to claim 5, is characterized in that: described biological fluid samples to be measured is that tumour or cancer markers are selected from: prostate specific antigen (PSA), carcinomebryonic antigen (CEA) or alpha-fetoprotein.
9. biological fluid sample quantitative test device according to claim 5, it is characterized in that: described biological fluid samples to be measured is Applications of Cardiac Markers, from following typical monoid, screen: Troponin I, TnT, creatine kinase isozyme (CK-MB), myoglobins, c reactive protein (CRP), fatty acid binding protein (FABP), glycogen phosphorylation isodynamic enzyme (GPBB), Type B urine peptide (BNP), front BNP.
10. a biological fluid samples quantitative detecting method, it is characterized in that, utilize pick-up unit as claimed in claim 1 to detect, wherein, the main film of described test (6) is coated nitrocellulose filter, non-activity in described the second film and the film (5) with porous are the coated glass fibre membrane of collaurum, and the filtering membrane (4) in described the second film is made by glass fibre or the polypropylene of non-woven shape; Described absorption layer is made by efficient sorbing material; The developer of described tagged ligand is golden bond, and this tagged ligand is used in conjunction with biological fluid samples to be measured and calibrating reagent;
Its detecting step is as follows:
Step 1, with transfer pipet to the biological fluid samples to be measured that drips 10 to 100 microlitres in the well on pick-up unit lid;
Step 2, described biological fluid samples to be measured are positioned at the second film absorption of test main film (6) upstream, and from the second film, flow to test main film (6) with capillary action; In this process, biological fluid samples to be measured is flowed through and is had the second diaphragm area of tagged ligand deposition, and described biological fluid samples to be measured is combined with tagged ligand, is formed with height and the low calibration part of known quantity on described the second film;
Step 3, with test main film (6) contact, biological fluid samples to be measured just acts on calibration tape T and high standard band HC and substandard band LC with tagged ligand;
The result that the colored intensity comparison of step 4, the colored intensity based on biological fluid samples to be measured and index zone alignment reagent and obtaining quantitatively detects; That is: in biological fluid samples to be measured, determinand content is higher, the analyte of being combined with tagged ligand is just more, fixed ligands is combined in the main film calibration tape T of test tagged ligand or determinand compound amount are just higher, thereby the mark colored intensity on index zone increases; Along with analyte content in biological fluid samples to be measured increases, the colored intensity of mark just increases thereupon;
Step 5, the mark colored intensity that biological fluid samples to be measured in index zone and calibration tape is produced together store in an electric magnetic card or rfid card; When this electric magnetic card or rfid card are inserted in optical card reader, this card reader provides respectively the concentration value of all analytes in biological fluid samples to be measured;
Step 6, according to the light reflection strength drawing standard product curve of biological fluid samples concentration standard product band to be measured; Calibrating reagent on wherein said high standard band HC and substandard band LC is that 2,4-dinitro benzene is for bovine serum albumin(BSA) (BSA-DNP) or 2,4-DNP (DNP).
11. biological fluid samples quantitative detecting methods according to claim 10, is characterized in that: on the second film, form after the height and low calibration part of known quantity, add damping fluid to promote described biological fluid samples to be measured flowing on the main film of test; Between 2 to 5 times that the application of sample amount of described damping fluid is sample size; Described damping fluid comprises the damping fluid that any pharmacy can received water, and described damping fluid does not react with sample and tagged ligand.
12. according to biological fluid samples quantitative detecting method described in claim 11, 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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| CN201010196737.7A CN101839908B (en) | 2010-05-28 | 2010-05-28 | Quantitative detection device and detection method of biological fluid samples |
| CN201410348806.XA CN104215757B (en) | 2010-05-28 | 2010-05-28 | Biological fluid sample quantitative test device and its detection method |
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| CN102680671A (en) * | 2012-05-15 | 2012-09-19 | 中国检验检疫科学研究院 | Test strip housing with data storage function |
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