CN104215757B - Biological fluid sample quantitative test device and its detection method - Google Patents
Biological fluid sample quantitative test device and its detection method Download PDFInfo
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- CN104215757B CN104215757B CN201410348806.XA CN201410348806A CN104215757B CN 104215757 B CN104215757 B CN 104215757B CN 201410348806 A CN201410348806 A CN 201410348806A CN 104215757 B CN104215757 B CN 104215757B
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of biological fluid sample quantitative test devices, including are arranged in the intracorporal test-strips of a box, the memory of color intensity data and the reader of color intensity data；The bottom of test-strips is a substrate, is provided on substrate with the porous main film of test；The upstream for testing main film is provided with the second film, and the same this application site and a tagged ligand site are provided on the second film, promotes tested organism liquid sample and its compound, tagged ligand that can flow through capillary action；The downstream for testing main film is provided with tertiary membrane, and tertiary membrane, which is used to adsorb in the end for testing main film downstream, has passed through the tested organism liquid sample for testing main film；There are two well and a forms for setting on the box cover of box body, standard items curve can be drawn with the light reflected intensity of known concentration standard items band using detection device of the present invention, to carry out quick, effective, accurate quantitative analysis to biological fluid samples.
Description
It is on May 28th, 2010 that the application, which is application No. is the 201010196737.7, applying date, entitled " biology
The divisional application of the application for a patent for invention of body fluid sample quantitative testing device and its detection method ".
Technical field
The system more particularly to a kind of detection and quantitative measurment sample that the present invention relates to a kind of for detecting biological liquid sample
The system of the concentration of object to be checked in this.
Background technique
The quantitative analysis of antigen, steroids antibody, endocrine albumen and other types albumen can usually provide most important
Clinical data.In biological field, after immunoassay is it is well known that its principle is reagent-impregnated, arrives contain through capillary action
Have on the film of immobilized reagent, in test strip detection zone and is combined with visable indicia object, such as: latex particle, metal composite
Determinand reaction.It is worth mentioning that marker is obtained from metal-sol, this mark dealt from metal-sol
Remember object can with ligand binding, then further in conjunction with determinand, ligand or antibody.Test-strips are largely used to detection body
Liquid, such as the determinand in urine and blood.The method that detection human chorionic gonadotrophin is pregnant as assignor this may be
Earliest most common method.Above-mentioned detection all carries out on the test strip, only needs a step to complete as the result is shown, for example, liquid
Body sample is by absorbing membrane, as a result internal determinand and corresponding ligand binding can be most clearly shown in and sample loading zone
On isolated detection zone.Above-mentioned detection is carried out, currently used test device typically contains cutting and capsule, and user is not necessarily to volume
Outer utensil can be carried out detecting.Known detection device substantially detects two kinds of detections using sandwich detection and Competitive assays
Method.
Sandwich-type detection procedures are that the determinand in liquid sample is reacted with label or indicator ligands, generate the to be measured of complexity
Object or marker.This reaction occurs to have determinand to enter detector bar, and otherwise, tagged ligand will be completely deposited on test-strips
On absorbing membrane.On the test strip, the determinand for being combined with marker moves on to sample capture area through capillary action；There, lead to
Crossing solidification capture ligands can be obtained determinand complex.In the case where gold label, there is colour band in pattern detection area, then shows have
Determinand exists.This detection device also contains Article 2 solidification ligand band, generally serves not only as quality control band and tagged ligand knot
It closes, or even in the presence of no determinand, can show the performance of test-strips.
Competitive assays detection method is that determinand can be reacted with tagged ligand with the fixed ligand in sample capture area,
Determinand and the tagged ligand fixed ligand that competes sample capture area jointly in this way.The presence of any determinand, which will all replace, marks
Remember the binding site of ligand.There is signal in tagged ligand capture, then it represents that result is feminine gender.
In the prior art, based on test-strips determine determinand whether there is or not device, determinand in liquid sample cannot be mostly provided
Correct amount.Even if being furnished with the standard items of quantitative test in detection device, as temperature, air humidity, time passage, survey
Strip difference, change in signal strength and inaccuracy.
Summary of the invention
For the above-mentioned prior art, the present invention provides a kind of biological fluid sample quantitative test device and method, the present invention
It can provide and quick, effective, accurate quantitative analysis is carried out to biological fluid samples.
In order to solve the above-mentioned technical problem, the technical solution that biological fluid sample quantitative test device of the present invention is achieved
It is: the detection device, including be arranged in the intracorporal test-strips of a box, it further include the memory and color intensity of color intensity data
The reader of data；The bottom of the test-strips is a substrate, is provided on the substrate with the porous main film of test；It is described
Main film is tested in high standard band HC, substandard band LC and calibration tape a T, the high standard band HC and substandard band LC
With calibrating reagent, there are fixed ligands on the calibration tape T；The upstream of the main film of the test is provided with the second film, and described the
Two films include that be arranged from bottom to top at least two layers is inactive and have porous film and a filter membrane, are arranged on second film
Have the same this application site and a tagged ligand site, promote through capillary action tested organism liquid sample and its compound,
Tagged ligand can flow；The downstream of the main film of test is provided with tertiary membrane, and the tertiary membrane is an absorption layer, the tertiary membrane
For with tested organism liquid sample contact, promote tested organism liquid sample to flow through survey by the capillarity of the tertiary membrane
Main film is tried, the tertiary membrane, which is used to adsorb in the end for testing main film downstream, has passed through the tested organism liquid sample for testing main film
This；The box body includes box cover, and there are two well and a form, the positions point of described two wells for setting on the box cover
Not with the second film loading this application site and tagged ligand site in box to just；High standard on the form and the main film of box internal test
Quasi- band HC, substandard band LC are consistent with region defined by calibration tape T；The memory is for storing high standard band HC, low mark
Color intensity caused by tested organism liquid sample in quasi- band LC and calibration tape T has measurement color intensity in the memory
Luminous value and tested organism liquid concentration of specimens composition correlation；The memory is electric magnetic card or RFID card；The reading
Taking device is optical card reader, and the optical card reader is used to provide the concentration value of all analytes in biological fluid samples respectively.
In the present invention, the detecting step using above-mentioned biological fluid sample quantitative test device is as follows:
Step 1: 10 to 100 microlitres of biology to be measured is added dropwise in the well on detection device box cover with pipette
Body fluid sample；
Step 2: the tested organism liquid sample is adsorbed by the second film for testing main film upstream is located at, and made with capillary
Main film is tested with from the second film flow direction；In the process, tested organism liquid sample flows through second with tagged ligand deposition
Diaphragm area, the tested organism liquid sample are formed with the high and low school of known quantity in conjunction with tagged ligand on second film
Quasi- ligand；
Step 3: contacting with main film is tested, tested organism liquid sample is just and tagged ligand is in calibration tape T and high standard band
It is acted on HC and substandard band LC；
Step 4: the colored intensity of colored intensity and index zone alignment reagent based on tested organism liquid sample compares
And obtain the result of quantitative detection；That is: determinand content is higher in tested organism liquid sample, the analysis in conjunction with tagged ligand
Object is more, and the tagged ligand or the compound object amount of determinand in conjunction with fixed ligands in the main film calibration tape T of test are higher, thus
Label colored intensity on index zone increases；As analyte content increases in tested organism liquid sample, the colour developing of label is strong
Degree just increases therewith；
Step 5: label colored intensity caused by tested organism liquid sample in index zone and calibration tape is stored together
Into an electric magnetic card or RFID card；It is inserted into optical card reader when by the electric magnetic card or RFID card, which provides respectively
The concentration value of all analytes in tested organism liquid sample；
Step 6: drawing standard items curve according to the light reflected intensity of tested organism liquid concentration of specimens standard items band.
Compared with prior art, the beneficial effects of the present invention are:
The detection of present invention application solid phase chromatography, such as sandwich detection, one or more tested organism liquid samples and label are tied
Object is closed to combine, it can also be in conjunction with solidification calibrating reagent or labelled reagent above test-strips.On index zone, the labelled reagent
It is captured by calibrating reagent, tested organism liquid concentration of specimens, which is taken, for measurement detection creates a platform.Index zone is solid
Change calibrating reagent is combined as liquid sample with corresponding labelled reagent and passes through detector bar.Determinand in liquid sample just combines
On sample capture band.It can be obtained by tested organism liquid sample using these relative intensities for being marked with complex
Actual concentrations.Therefore, it is reflected if the intensity, depth of marker on film in various sample capture bands (such as gold size conjugate)
The quantity of tested organism liquid sample, similarly, index zone can also reflect the intensity and concentration of calibrating reagent.So commercially sharp
Measurement concentration is converted by the light reflected intensity of sample with visualization device.Finally, it is reflected with the light of known concentration standard items band
Intensity draws standard items curve.
Detailed description of the invention
Fig. 1 is the composition block diagram of test device of the present invention；
Fig. 2 is the surface structure schematic diagram of test-strips box body in test device of the present invention；
Fig. 3 is test-strips structural decomposition diagram in test device of the present invention；
Fig. 4 is test device detection method flow chart of the present invention.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
Of the invention for ease of understanding, related word is defined as follows:
1, the antigen in tested organism liquid sample (hereinafter referred to as determinand), referring to can be with the substance in conjunction with antibody.It is anti-
Original can be compound, polypeptide, carbohydrate, nucleic acid, lipid and its analog, as virion, subunit, parasitic animal and plant,
Host protein etc..
2, conjugate refers to the determinand ligand mentioned in sandwich assay, with determinand, the label in competition detection method
Object, tracer ligand are the same.Conjugate can from can in conjunction with determinand macromolecular or compound in choose, as antigen-is anti-
Nanocrystal composition, Antibody-antigen complex.
3, test section (or band) refers to that conjugate or determinand can adhere to position thereon, such as: it is tested in detection device
A part of item.
4, test-strips (or detector bar) is to refer to through capillary action, make containing the liquid sample of determinand and other
It needs to be determined that the perforated membrane that the antigen of testing concentration flows.General perforated membrane has glass fibre, porous nitrocellulose, poly- second
Alkene.The test-strips of invention are all made of support film and filter.
5, tracer, refer to be marked with it is measurable, preferably be marked with special raadable mark object (such as: colloidal gold, latex,
Comprising dyestuff, carbon black, and the like liposome) determinand or conjugate ligand.
6, sample loading zone refers to determinand detection zone, that is, is moved to liquid sample in test-strips.
7, liquid sample to be tested refers to any liquid containing determinand.The sample of detection can be body fluid, packet
It includes: the urine that is obtained from fetus, newborn, youth, adult, blood, sweat, lymph, intraperitoneal liquid, crude extracts
Or homogenate, the liquid of inactive, such as the water obtained in river, the lake, test water etc..
8, marker refers to the macromolecular or compound of direct or indirect adjustment signal (such as color change) mode of energy.This
A signal is used to indicate the presence or absence of target determinand and concentration range in detection sample.Marker may contain enzyme, fluorescer, rouge
Plastid, polymer microcapsules, colour developing polymer particles (latex), is also likely to be the colloid containing metal composite at red blood cell.Respectively
Patent and the application for planting various kinds provide lot of documents for production optical signal.In the present invention, several markers can be found
The application of types patent.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 that enzyme marks；United States Patent (USP): 3,996,345 discovery fluorescent markers；United States Patent (USP): 3,996,
345 discovery radioactively labelled substances；United States Patent (USP): 4,067,959 discovery fluorescence or enzyme marker；United States Patent (USP): 4,104,099 hairs
Existing chemiluminescent labels；United States Patent (USP): 4,160,645 discoveries are without enzyme catalyst marker；United States Patent (USP): 3,966,879 hairs
Existing electrophoretic techniques can be used for antibody district；United States Patent (USP): 4,120,945 discovery radioimmunoassay detections for the first time, in this approach,
The determinand of label can be in conjunction with solid support by antibody；United States Patent (USP): the 4,233,402 pairs of enzyme markers of discovery；Beauty
State's patent: 4,720,450 discovery chemical induction fluorescent markers；United States Patent (USP): 4,287,300 discovery enzyme negative electrical charge markers.
In addition, marker is also possible to metal-sol；Metal or metal composite, such as: metal oxide, hydroxide, metal salt, gold
Belong to compound or the metal composite of polymer core is mixed or be coated in polymer phase.On these metal marker objects may be
The dewatering state of any metal-sol or metal composite colloidal sol that face is previously mentioned, it is more likely that the colloid of dewatering state
Gold.
9, label reaction refers to that signal generated is strong when marker links with solidified reagents on calibration tape or index zone
Degree.It is the good method of measurement markers reaction with the reflection electro-optical device that color generates.
10, compound refers to that (based on context) is any by determinand and one or more ligands, or by tagged ligand with
Solidify the multi-molecular complex of ligand formation.In sandwich immunoassays, for example following compound can generate determinand/label
Ligand double combination generates (the first compound) in the detection first, and determinand/tagged ligand/solidification ligand three combines and appears in inspection
In survey (the second compound).
11, fluid communication, refer to it is a kind of be in contact with each other but not addition, allow fluid to flow to another channel by a channel
Structure.
12, it chemically examines, refers to the several different types of detection patterns that can detecte determinand using test-strips.For example,
In sandwich immunoassays, in the presence of target determinand in sample, it just in conjunction with the tracer of label and is moved to test
Item (containing absorbing membrane) indicates in area, forms the first compound.Indicant be it is a kind of can be in conjunction with target determinand, also can be with mark
Remember that the molecule that object combines, marker can be metal marker object, preferably colloidal gold.
13, capture zone (or capture site), refers to the chromatography test-strips at least containing a determinand binding reagents
On region or band.Determinand binding reagents are normally held in area or take, after reacting with detectable reagent, in area or band
It is upper generate it is measurable as a result, it is possible to reflect in sample determinand content or its there are the case where." capture zone " may be by one
The trapping region composition of multiple samples can be captured above, 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 as a detection group, Hepatitis C Virus can be detected simultaneously
(HCV) and human immunodeficiency virus (HIV) hepatitis B surface antibody (HBsAg) and microspironema pallidum are detected, or simultaneously
(TP) antibody.It is feasible there are also others combination, and within the scope of the present invention.
14, conjugate and detection reagent, herein can with combine detection reagent such as: color developing agent, fluorescer, enzyme or chemistry send out
The antigen or antibody of photo etching exchange.In the practice of the invention, " conjugate " and " detection reagent " specifically combination to be determined
Determinand or the catches being fixed in capture zone.In capture zone, " conjugate " and " detection agent " generates can quantitative measurment
Reflect the value of current determinand content.It is as will be explained hereinafter, the different surely reflection knot of the density that direct quantitative measures in capture zone
Close in capture zone current determinand quantity, but band strength measured by luminous value (RLU) can but reflect in capture zone when
The content of preceding determinand.
15, " index zone " includes the calibrating reagent for being fixed on test strip calibration area herein.Calibrating reagent specifically with calibration
Binding reagents in conjunction with and formed calibration combine pair.It include two calibration tapes in the present invention.Advantage containing calibration combination pair is it
Can play the role of internal calibration, i.e. calibration agent may count the content of determinand current in capture zone.Calibration agent
It is possibly used for the variability of test strip calibration.One of them can be designated as high calibration (HC), another can be designated as low school
Quasi- (LC).In addition to this, the density of HC and LC can be used for determining standard items line.Standard items line can be with by the RLU value of calibration agent
A regression equation is generated, describes the relationship between two variables with this, is tested to be applied to each quantitative detection.Though
So, conventional calibration agent is still using, but generally people are still ready using being not present in sample or without immune friendship
The calibration compound of reaction is pitched, such as 2,4_ dinitrobenzene can be bought for bovine serum albumin (BSA-DNP) from molecular probe
(Eugene, OR, cat#A-23018) and as calibrating reagent come using.2,4- dinitrophenol (DNP) is not present in human body
Small molecule but can be used as haptens, haptens refers to when itself and macro-molecular protein carrier conjugation, or is injected into and can generate
When in the mammal such as rat of antibody, mouse, rabbit, ox, horse, sheep or goat body, the substance of immunogenicity is generated.It is inefficient
Calibration areas solidifies ligand by taking bovine thyroglobulin antibody (BTG) as an example；High calibration areas is by taking goat-anti rabbit protein antibodies as an example.With it is solid
The conjugate for changing ligand binding is BTG gold antigen and rabbit igg gold antigen.
As shown in Figure 1, biological fluid sample quantitative test device of the present invention, including be arranged in the intracorporal test-strips of a box,
It further include the memory of color intensity data and the reader of color intensity data.
As shown in figure 3, the bottom of the test-strips is an adhesive sticker substrate 1, being provided on the adhesive sticker substrate 1 has
The main film 6 of the porous test of sufficient porosity can allow the inspection substance comprising determinand and its compound to move on to this through capillary action
On film.The common main film 6 of test has porous cellulose nitrate, porous polypropylene or paper film, and the main film 6 of test is preferably adopted
With nitrocellulose filter is coated with, these films are technically well-known.
There is high standard band HC, a substandard band LC and calibration tape T, the high standard band on the main film 6 of test
There is calibrating reagent in HC and substandard band LC, there are fixed ligands on the calibration tape T, it may be assumed that in the detection band region T, test
It, and can be preferably in strip form through the main film of test, just as in height comprising can be with solidification ligand determinand ins conjunction on main film
Index zone HC is passed through with the calibrating reagent on LC with substandard and is tested as main film formation index zone.For testing region and mark
The solidification ligand of quasi- region is different.The calibrating reagent of known quantity is fixed on index zone for determining standard curve, thus
Determine the quantity of determinand.After testing the ligand binding on main film, remaining active site is closed, thus allow determinand and
Its compound, tagged ligand and its compound are freely through test-strips.
The upstream of the porous main film 6 of test of test-strips in the present invention, also includes second film, second film includes certainly
At least two layers of lower and upper setting is inactive and has porous film 5 and a filter membrane 4, described inactive and have porous film
5 without can with the position by the ligand binding of film, in addition to this also comprising the solidification ligand for detecting the exchange of film upper fluid
Band.The film 5 is coated with glass fibre membrane using colloidal gold, which is more likely to be made of scattered glass fibre or polypropylene, have simultaneously
There is sufficient hole, allows determinand and its compound, tagged ligand that capillarity is depend on to flow.The top layer of second film is
One filter membrane 4, the filter layer 4 use glass fibre membrane, the filter membrane suitable for determinand is separated from sample components,
Other components in sample may interfere with the inspection to determinand.Therefore, when detecting blood, red blood cell or leucocyte can be with
It is separated from the serum comprising determinand.Therefore second film includes a sample practical site and a tagged ligand position
Point, the downstream of practical site and the upstream of test film contact point, react in sandwich reaction with determinand, or in competitive reaction
Reacted with detection zone binding partner and index zone in reacting between complicated tagged ligand and correction ligand.Labelled reagent quilt
It is adsorbed on the upstream of the film, can be flowed away from film when it is contacted with liquid sample, is formed under conditions of with analyte response
Compound is so as to continuing to flow to test film by upstream film.
The downstream for being located at the main film 6 of test in test-strips also includes tertiary membrane 2, which is an absorption layer, the tertiary membrane
2 contact with liquid, for that can adsorb the liquid sample by test film in the downstream end for testing main film 6, also as drive
Power promotes liquid sample to flow through detection film through capillary action.Such absorption layer preferably with efficient adsorbent material come
It does, such as: sample can be adsorbed while can be as adding the paper of any buffer in test-strips.
As shown in Fig. 2, the box body includes box cover, it is arranged on the box cover there are two well S1, S2 and a form W,
The position of described two well S1, S2 are respectively with the second film loading this application site and tagged ligand site in box to just；Institute
It is consistent with region defined by high standard band HC, substandard band LC and calibration tape T on the main film 6 of box internal test to state form W.
As shown in Figure 1, the memory in detection device of the present invention is for storing high standard band HC, substandard band LC and test
Color intensity caused by tested organism liquid sample in band T, have in the memory luminous value of measurement color intensity with to
Survey the correlation of object concentration composition；The memory is electric magnetic card or RFID card；The reader is optical card reader, the light
Learn the concentration value that card reader is used to provide all analytes in biological fluid samples respectively.
As shown in figure 4, carrying out detection using detection device of the present invention consists essentially of following steps:
Step 1: being added dropwise in the well on detection device box cover with pipette a certain amount of comprising suspicious determinand
Biological fluid samples；Usual sample-adding amount is 10 to 100 microlitres.
Step 2: will provide sample aperture when test-strips are installed in a box body on box body and be used to be loaded.Added sample
It is located at the second film absorption for testing main film upstream, and main film is tested from the second film flow direction with capillarity；In the process, sample
Originally the second diaphragm area positioned at upstream of label binding partner deposition is flowed through.In addition to can other than the tagged ligand in conjunction with determinand,
The high and low calibration ligand of known quantity is also had on the second film of the upstream.
If sample is whole blood, the second film of the upstream can also remove red blood cell and leucocyte as filter device, only allow
Serum flows through.Buffer, which is added, can promote the flowing of sample on the test strip.The sample-adding amount of buffer is usually the 2 of sample size
To between 5 times.Suitable buffer include any pharmacy can received water buffer, it will not be with test sample and control
Ligand reaction.General phosphate buffer can be phosphoric acid one or disodium salt, other such as lemon based on commercially available
Acid buffer and ringer's solution application are also very extensive.
Step 3: contacting with main film is tested, sample is just acted on calibration tape and index zone with tagged-ligand.The present invention
Most suitable marker be that can generate the substances of color complexes in those detection bands and index zone.Although enzyme-linked ligand or
Latex binding partner also can produce color products, and ligand utilizes capillary flow principle for quantitative detection, but constitutes ligand most
Good color developing agent is golden conjugate, can combine determinand and calibration agent.
Step 4: sizing technique of the invention is the colour developing of the colored intensity based on determinand sample and index zone alignment agent
Intensity, which compares, obtains result.Therefore determinand content is higher in test sample, and the analyte in conjunction with tagged ligand is got over
More, the compound object amount of tagged ligand/determinand in conjunction with fixed ligands in test section is higher.With analyte content in sample
Increase, colored intensity just increases therewith.However in order to accurately calculate the actual concentrations of determinand, other factors rather than determinand
Concentration must be excluded except any calculated result.
Tested entries of the invention are that a kind of accurate quantitative determination side is provided using the calibrating reagent in index zone
Method.For higher accuracy, the present invention uses two kinds on the separated standard regions before and after test section of test film
Calibrating reagent.Since a certain amount of calibrating reagent is fixed on standard regions and excessive calibration mark conjugate is all deposited on
In upstream belt, the color of same intensity will be generated on index zone within the given time, suitable for making of same process
Different test-strips.Standard curve can obtain each height calibration and low calibration areas using luminous value, for determining for analyte
Fixed offer foundation is provided.It is, in general, that any conventional calibrating reagent can use, preferred calibrating reagent is not deposited in sample
Or in sample compound occur immunological cross-reaction reagent, such as 2,4- dinitrobenzene for bovine serum albumin(BSA)
(BSA-DNP), can be bought from molecular probe (Eugene, 0R, cat#A-23018) and as calibrating reagent come using.2,4- bis-
Nitrophenol (DNP) is the small molecule being not present in human body but can be used as haptens, and haptens refers to when itself and macromolecular egg
White matter carrier conjugation, or it is injected into mammal such as rat, mouse, rabbit, ox, horse, sheep or the goat body that can generate antibody
When interior, the substance of immunogenicity is generated.Inefficient calibration areas solidification ligand is by taking bovine thyroglobulin antibody (BTG) as an example；Colleges and universities
Quasi- area is by taking goat-anti rabbit protein antibodies as an example.It is BTG gold antigen and rabbit igg gold antigen with the conjugate for solidifying ligand binding.
In order to measure the content of determinand, it is necessary to study in the color intensity and sample of test section between testing concentration
Relationship.This relationship can be described with curve, by preparing the determinand solution then constantly dilution of a high concentration, and survey
The variation for determining color intensity can obtain.Obviously, this curve is also different in different time period.With regard to be measured in measurement sample
It is to be mutually related between these curves and the standard curve obtained by the calibrating reagent of known concentration for object content.Cause
And the test of each unknown sample can get three different color intensities.The colored intensity and standard concentration curve of index zone
It is related, the test and calculating of determinand and its numerical value suitable for sample.In order to verify concentration standard value and expose twice
Related between light time, to obtain ideal luminous value, the standard density marks reaction related in catoptry, in sample to
Object concentration is surveyed to be verified.
A kind of simple version of quantitative detection of the present invention is that the determinand of known concentration is fixed on index zone as calibration examination
Agent can be accomplished.In this case, it is only necessary to which the excessive single labelled ligand of one kind, which goes movably to be deposited on, receives sample
Upstream film on.One sample containing determinand can be dispersed mobile label binding partner and allow determinand and ligand reaction
And form compound.Compound and excess ligand are brought in test film through capillary action, and determinand binding marker is matched herein
Nanocrystal composition is reacted with curing antibody and is captured and occurrence flag reaction, is often the reaction of colored forms.Excessive label is matched
Body reacts with the solidification determinand of known quantity in standard regions and provides a standard color intensity reaction.To testing concentration and
On the basis of the preset color response of index zone alignment agent, the concentration of analyte can be determined in sample.
Step 5: although the necessary correction of index zone and concentration calculation can be by completing, in the prior art manually
Business machine can measure the color intensity of calibration tape and index zone.Color caused by determinand in index zone and calibration tape
Intensity can be stored together in a memory (such as electric magnetic card or RFID card).When electric magnetic card or RFID card are inserted into for business
On optical card reader, such as the card reader that Kaiwood Technology company produces, card reader will provide in sample respectively
The concentration value of all any analytes.
Step 6: drawing standard items curve according to the light reflected intensity of tested organism liquid concentration of specimens standard items band.
With the development of determinand qualitative detection technology, if determinand antigen, corresponding compound is exactly antibody；If to
Surveying object is antibody, and corresponding compound is exactly antigen, and label conjugate in this way could be in conjunction with determinand.Such as it is chemically examined with gradient
Hepatitis B surface antibody (HBsAg) in blood sample is measured, is resisted in capture zone containing the HbsAg- being fixed on detection tape test film
Body.
Suitable determinand is not limited only to antigen, antibody, hormone, addictive drug, cell protein, DNA, cardiac muscle mark
Object, tumour or cancer markers, autoimmune disease marker or any macromolecular substances that can generate antibody.Determinand is such as
For antigen, antigen can be the antigen for the infectious agent that associated.Infectious agent can be virus, bacterium, fungi or prion.When
When infectious agent is virus, virus can be HIV, hepatitis virus A, B, C, D, single herpesviral, cytomegalovirus, mamillary
Tumor virus, Ebola virus, SARS virus, rhinovirus and vaccinia virus etc. are viral, and are not limited only to these viruses.When infect because
When son is bacterium, it can be Gram-positive or Gram-negative bacteria.These bacteriums can be bacillus anthracis, Escherichia coli,
Helicobacter pylori, diplococcus, Salmonella and Shigella population, and be not limited only to these bacteriums.Work as infection
When the factor is fungi, then it can be Mycosporum mushroom or aspergillus mushroom, and be also not limited to these fungies.
Determinand for example hormone can then screen: human chorionic gonadotrophin, thyroid gland from following typical monoid
Element, glucagon, insulin, relaxain, interstitialcellstimulating hormone (ICSH), melanotropin, growth hormone, promotees ovarian follicle at thyroid-stimulating hormone
Generation element, gastrin, bradykinin, antidiuretic hormone and other releasing factor, however other hormones physiologically or pathologically
Also it all can be used as determinand.
Determinand is if tumour or cancer markers, then can screen from following typical monoid: prostate specific is anti-
Original (PSA), carcinomebryonic antigen (CEA), alpha-fetoprotein, other cancers or tumor markers can also be used as determinand.
Determinand can then be screened if Applications of Cardiac Markers from following typical monoid: Troponin I, troponin T,
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), preceding BNP etc., other Applications of Cardiac Markers also can be used as analyte.
The present invention has many examples, and lower example only makees finite specification.
A porous nitrocellulose film in test-strips is as the main film of test, enough human prostate specific antigen
(PSA) it is fixed on two index zones (high standard band HC and substandard band LC), when anti-with the PSA monoclonal specificity of gold label
When body, respective concentration are respectively 1 and l0ng/mL mixing, corresponding color intensity will be generated.In addition, PSA monoclonal antibody
It can be deposited on the calibration tape T of nitrocellulose membrane.Index zone is designed as substandard band of the sample first with energy display color intensity
LC contact, this substandard are accompanied by the gold mark conjugate for being equivalent to 1ng/mLPSA antigen with LC, are and then high standard band
HC, the PSA antigen binding that can be l0ng/mL with equivalent.In antigen and the antibody permission deposited on index zone and nitrocellulose membrane
Antigenic determinant react and be permanently affixed on the index zone and calibration tape of test film.Then remaining antigenic determinant makes
It is closed with standard technique.For specific test-strips, the color intensity correlation that is generated between determinand and reaction time
It is stored on storage card.
Nitrocellulose membrane (testing main film) and nonwoven glass fibre membrane (being located at the second film for testing main film upstream)
By fluidly connecting, antibody is deposited to combine PSA antigen as gold label with known technology on testing main film.It is analyzed
Sample be added in the upstream of glass fibre membrane, downstream then deposits label conjugate.Test-strips chemical examination further includes a test film
The sample absorption pad of end is connected with downstream by fluid.
Blood sample of the suspection containing PSA of one about 30 microlitres of drop is dripped on glass fibre membrane by the well on box cover
The sample-adding site of trip follows hard on the phosphate buffer for adding about 40 microlitres of a drop.Glass fibre membrane wants sufficiently thick red thin to filter
Born of the same parents and leucocyte, but serum can pass through.Serum and buffer, which are reached with capillarity by upstream film, marks conjugate, Jin Biao
Antibody conjugates are redistributed and form compound with analyte in sample.The diluted serum of buffer is arrived by glass fibre membrane
Up to porous nitrocellulose membrane, after passing through entire nitrocellulose membrane with capillarity.
Gold containing determinand marks the PSA antibody capture that compound is deposited on calibration tape T and shows blush, and color is strong
Spend corresponding testing concentration in counter sample.Excessive gold conjugate can also be captured by high and low index zone LC, index zone
Upper includes enough calibrating reagents to capture equivalent in the determinand of 1 or l0ng/mL.PSA if it exists in sample, then can obtain
The band of three different colours intensity.By after calibration tape T and index zone C, remaining Buffer samples by nitrocellulose membrane it
Absorption pad is reached afterwards and is stored in there.
By the etui insertion containing color bar (test-strips) in the card reader of storage card, there is measurement face in storage card
The correlation of luminous value and the testing concentration composition of intensity of colour.The CHRl00 card reader used is by Kaiwood Industries
Manufacture, it can convert concentration value for luminous intensity using storage card.The measurement of luminous intensity carries out after 10 and 15 minutes after sample-adding
Measurement, as follows:
10 minutes test results:
| Determinand | PSA concentration | RLU |
| Height calibration | 10ng/ml | 10 |
| Low calibration | 1ng/ml | 1 |
| Sample | X | 4.04 |
According to two o'clock standard curve, card reader shows that PSA concentration is 4ng/mL in sample.
15 minutes test results:
| Determinand | PSA concentration | RLU |
| Height calibration | 10ng/ml | 23.9 |
| Low calibration | 1ng/ml | 10.3 |
| Sample | X | 19.49 |
According to two o'clock standard curve, card reader shows that PSA concentration is 4ng/mL in sample.
Although above in conjunction with figure, invention has been described, and the invention is not limited to above-mentioned specific embodiment parties
Formula, the above mentioned embodiment is only schematical, rather than restrictive, and those skilled in the art are in this hair
Under bright enlightenment, without deviating from the spirit of the invention, many variations can also be made, these belong to guarantor of the invention
Within shield.
Claims (15)
1. a kind of biological fluid sample quantitative test device comprising test-strips, the test-strips include the survey being arranged on
Main film is tried, the second film of the main film upstream of test is set in a manner of fluid communication, and is set in a manner of fluid communication
Set the tertiary membrane in the main film downstream of test, wherein
It is provided with the sample practical site for applying the biological fluid samples on second film and is answered positioned at the sample
With the downstream in site, it is deposited with the tagged ligand site of tagged ligand thereon, the tagged ligand includes can be with determinand knot
The tagged ligand of conjunction and high calibration ligand and low calibration ligand；
There is high standard band HC, substandard band LC and calibration tape T, high standard band HC and the substandard band on the main film of test
The calibrating reagent of known concentration is respectively and fixedly provided on LC, the calibrating reagent is not present in the sample or does not occur immunological cross
Reaction, and the high standard can generate color intensity with the high calibration ligand binding with the calibrating reagent on HC, it is described low
Calibrating reagent on index zone LC can generate color intensity, and be used for the high standard band with the low calibration ligand binding
HC and the substandard are different substance with the calibrating reagent of LC, have fixed ligands, the fixed ligands on the calibration tape T
Color intensity can be generated in conjunction with the determinand for being combined with tagged ligand；
The tertiary membrane absorbs the biological fluid samples for flowing through the main film of test through capillary action；
Described device further includes memory and reader, and the memory is for storing high standard band HC, substandard band LC and survey
Generated color intensity in test strip T, and high standard band HC, substandard band LC and calibration tape T are stored in the memory
On color intensity and testing concentration between correlation, the reader provides biological fluid samples according to the correlation
The concentration value of middle determinand.
2. biological fluid sample quantitative test device according to claim 1, wherein for high standard band HC and low
Immunological cross-reaction does not occur for the calibrating reagent of index zone LC.
3. biological fluid sample quantitative test device according to claim 1, wherein the tagged ligand is golden label
's.
4. biological fluid sample quantitative test device according to claim 1, wherein the high standard is with the calibration on HC
Reagent is goat-anti rabbit protein antibodies.
5. biological fluid sample quantitative test device according to claim 1, wherein the substandard is with the calibration on LC
Reagent is bovine thyroglobulin antibody.
6. biological fluid sample quantitative test device according to claim 1, wherein the main film of test is that coating nitric acid is fine
Tie up plain film.
7. biological fluid sample quantitative test device according to claim 1, wherein second film includes from bottom to top
At least two layers be arranged is inactive and has porous film and filter membrane.
8. biological fluid sample quantitative test device according to claim 7, wherein inactive in second film and
Be that colloidal gold is coated with glass fibre membrane with porous film, the filter membrane in second film by non-woven shape glass fibre or
Polypropylene production.
9. biological fluid sample quantitative test device according to claim 1, wherein the device further includes box body, described
Test-strips are arranged in the box body, and the box body includes box cover, and setting is there are two well and form on the box cover, and described two
The position of a well is respectively with the second film loading this application site and tagged ligand site in box to just；The form and box body
High standard band HC, substandard band LC are consistent with region defined by calibration tape T on the interior main film of test.
10. biological fluid sample quantitative test device according to claim 7, wherein the tested organism liquid sample is
Whole blood, the filter membrane can be filtered to remove red blood cell and leucocyte in tested organism liquid sample, only serum be allowed to flow through.
11. biological fluid sample quantitative test device according to claim 1, wherein tested organism liquid sample is selected from: anti-
Original, antibody, hormone, addictive drug, cell protein, DNA, Applications of Cardiac Markers, tumour or cancer markers or autoimmunity
Disease marker, in which:
The antigen is the antigen of infectious agent of having associated；Wherein, the infectious agent is virus, bacterium or fungi；
When infectious agent is virus, virus is HIV, hepatitis virus A, B, C, D, single herpesviral, cytomegalovirus, nipple
Shape tumor virus, Ebola virus, SARS virus, rhinovirus, vaccinia virus or prion；
When infectious agent is bacterium, bacterium is gram-positive bacteria or Gram-negative bacteria；
It is then Mycosporum mushroom or aspergillus mushroom when infectious agent is fungi；
The hormone is selected from: human chorionic gonadotrophin, thyroxine, thyroid-stimulating hormone, glucagon, insulin, pine
Relax element, interstitialcellstimulating hormone (ICSH), melanotropin, growth hormone, follicle-stimulating hormone, gastrin, bradykinin, antidiuretic hormone；
The tumour or cancer markers are selected from: prostate-specific antigen (PSA), carcinomebryonic antigen (CEA) or alpha-fetoprotein；
The Applications of Cardiac Markers is selected from: Troponin I, troponin T, creatine kinase isozyme (CK-MB), myoglobins, C are anti-
Answer albumen (CRP), fatty acid binding protein (FABP), glycogen phos isodynamic enzyme (GPBB), Type B urine peptide (BNP), preceding BNP.
12. biological fluid sample quantitative test device according to claim 11, wherein the gram-positive bacteria is anthrax
Bacillus, the Gram-negative bacteria are Escherichia coli, helicobacter pylori, diplococcus, Salmonella or Shigella
Population.
13. a kind of raw using biological fluid sample quantitative test device quantitative detection described in any one of any one of claims 1 to 10 10
The method of object body fluid sample, described method includes following steps:
Step 1: biological fluid samples are applied to the sample practical site, flow through the biological fluid samples and sink thereon
Product has the tagged ligand site of tagged ligand, thus make determinand in the biological fluid samples and it is described can be with determinand
In conjunction with tagged ligand combine；
Step 2: contacting the biological fluid samples for flowing through the tagged ligand site with main film is tested, to make the high standard
The quasi- calibrating reagent on HC and the high calibration ligand binding generate color intensity, make the substandard with the calibration examination on LC
Agent and the low calibration ligand binding generate color intensity, and make fixed ligands on the calibration tape T and be combined with label
The determinand of ligand, which combines, generates color intensity；
Step 3: using high standard band HC, substandard in the memory storing step two with generated in LC and calibration tape T
Color intensity；
Step 4: using the reader according to the high standard stored in memory with HC, substandard on LC and calibration tape T
Correlation between color intensity and testing concentration obtains the concentration value of determinand in biological fluid samples.
14. the 3 biological fluid samples quantitative detecting method according to claim 1, wherein the method also includes will be biological
Body fluid sample is applied to after the sample practical site, buffer is added further to promote in the sample practical site
Tested organism liquid sample is stated in the flowing tested on main film；The sample-adding amount of the buffer is between 2 to 5 times of sample size；
The buffer include any pharmacy can received water buffer, the buffer do not react with sample and tagged ligand.
15. the 4 biological fluid samples quantitative detecting method according to claim 1, wherein the buffer is phosphoric acid buffer
Liquid, the phosphate buffer are monosodic alkaliine or disodic alkaliine；Or the buffer is citrate buffer solution or woods grignard
Liquid.
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| CN201410348806.XA CN104215757B (en) | 2010-05-28 | 2010-05-28 | Biological fluid sample quantitative test device and its detection method |
| HK15103636.7A HK1203231A1 (en) | 2010-05-28 | 2015-04-14 | Quantitative detection device and detection method of biological fluid samples |
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| CN201010196737.7A CN101839908B (en) | 2010-05-28 | 2010-05-28 | Quantitative detection device and detection method of biological fluid samples |
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| CN105403709A (en) * | 2015-10-27 | 2016-03-16 | 上海芯超生物科技有限公司 | Whole-blood one-step alpha fetoprotein collaurum detection kit and detection method thereof |
| 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 |
| 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 |
| 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 |
| US10605741B2 (en) | 2018-06-28 | 2020-03-31 | International Business Machines Corporation | Accurate colorimetric based test strip reader system |
| CN115060779B (en) * | 2022-06-27 | 2023-03-24 | 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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| 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 |
| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
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| DE602005023183D1 (en) * | 2004-07-29 | 2010-10-07 | Relia Diagnostic Systems Llc | CROSS-CIRCUIT SYSTEM AND ASSAY |
| EP2261673B1 (en) * | 2008-03-31 | 2018-01-24 | Terumo Kabushiki Kaisha | Method for preparing calibrating reagent, calibrating reagent, and method for calibrating blood component measuring device using said calibrating reagent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
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