CN1690711B - Immune chromatographic test paper bar based on up conversion luminescence technology - Google Patents
Immune chromatographic test paper bar based on up conversion luminescence technology Download PDFInfo
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- CN1690711B CN1690711B CN 200410034104 CN200410034104A CN1690711B CN 1690711 B CN1690711 B CN 1690711B CN 200410034104 CN200410034104 CN 200410034104 CN 200410034104 A CN200410034104 A CN 200410034104A CN 1690711 B CN1690711 B CN 1690711B
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Abstract
The invention discloses an immune chromatography test paper based on luminous technique with upside conversion. Use UCP as biological mark, show the result with format of visible light signal on the exposure of infrared light, interpret the result by device, then realize the quantitative detection to object. The structure main comprises sample pad, combination pad or conjugate pad, analysis membrane, sopping pad and plastic backboard. The invention also discloses the preparation and application of the test paper in biological sample quantitative detection. According to the difference of immunological reaction ways of material, classify the test paper to sandwich mode, competition mode and indirect mode; all modes can take qualitative and quantitative detection to different material quickly and acutely.
Description
Technical field
The present invention relates to a kind of immuno-chromatographic test paper strip, particularly relating to the employing up-conversion luminescent material is the immuno-chromatographic test paper strip of UCP as biomarker.The result of this kind test strips shows with the form of visible light light signal under the irradiation of infrared light, and can use and change optical biosensor and carry out interpretation, thereby realizes the qualitative and quantitative detection quick, sensitive to the target checking matter.
Background technology
(Up-Converting Phosphor is a kind ofly can go up the rare earth metal complex that changes to energy UCP) to up-conversion luminescent material, and promptly UCP can absorb low-energy (long wavelength) infrared light, but launches high-octane (short wavelength) visible light.UCP is doped in the lattice of some crystal by several thuliums to constitute.Three kinds of major ingredients are arranged: main matrix (host matrix), absorption (absorber) and emission (emitter) in this material.Crystalline material as main matrix has: oxysulfide is (as Y
2O
2S, GdO
2S, La
2O
2S etc.), fluoride is (as YF
3, GdF
3, LaF
3Deng), gallate is (as YGaO
3, Y
3Ga
5O
12Deng) and silicate (as YSi
2O
5, YSi
3O
7Deng) etc.; Being commonly used for the rare earth ion that absorbs son has: ytterbium ion (Yb
3+), erbium ion (Er
3+), samarium ion (Sm
3+) etc.; The rare earth ion that is commonly used for emission has: erbium ion (Er
3+), holmium ion (Ho
3+), thulium ion (Tm
3+), terbium ion (Tb
3+) etc.Spatial orientation and distance that absorption and this ion pair of emission suit in the main matrix lattice are the bases that produces up-conversion luminescence.
Different absorption, emission, main matrix are in conjunction with making UCP have different optical properties.Under the identical situation of: (1) main matrix, a series of UCP adopt identical absorption, different emission, and what then it can be by identical wavelength is infrared ray excited, produces emission light (as: the infrared ray excited absorption-Yb of 980nm of different wave length
3+-emission-Er
3+, absorb son-Yb
3+-emission-Tm
3+, launch the green visible light of 550nm, 475nm blue visible light respectively); Adopt different absorption, identical emission is though then via different exciting lights, can produce identical emission light.(2) under the different situation of main matrix, the UCP spectral signature also will change and (as: absorb son-Yb
3+-emission-Er
3+, at main matrix YF
3, GdF
3In, emission redness respectively and green visible light).The diversity of forming has determined the diversity of UCP spectrum (excitation spectrum, emission spectrum), and this becomes the basis of dirigibility in its use.Employed UCP provides by Shanghai Keyan Opto-electrical Technology Co., Ltd in the present technique.
UCP is as a kind of inorganic material a series of finishing of process and activation of complete inertia, comprising: UCP surface siliconization, UCP surface amination, surface-functionalized, the surperficial connection antibody of UCP of UCP, just can combine with immunochromatography technique, in biological field, fully play the various characteristics that himself has.
At present, in the immunochromatography technique employed label be generally enzyme, collaurum and coloring agent pearl label, these several labels are applied to have in the immunochromatography technique two common ground: the physisorption cross-linking method with by the color judged result.Wherein the fragility of physisorphtion (promptly preparing label by hydrophobicity and Electrostatic Absorption) makes based on the immunochromatography of this kind label very harsh for the conditional request of reaction, thereby some are non-special removal reagent effectively, as: polysorbas20 (Tween 20), blow logical 100 (Triton 100), sodium dodecylsulphonate (SDS) etc., owing to hydrophobicity and electrostatic influence are changed and can only use at low concentration, just caused thus based on the higher inevitable outcome of immune chromatography test paper false positive rate, the false negative rate of this kind label; Must be subjected to that observer's subjectivity influences greatly, sensitivity is low by the color judged result in easy and simple to handle using in addition, and can only rest on qualitative level, and definitely can't realize accurate quantification.These shortcomings have limited the range of application of immunochromatography technique in biological detection greatly.
The principle schematic that the UCP particle is used in field of biological detection has been described in the accompanying drawing 1.By the covalent bond preparation label that is connected, this label is fixed in the surface of solid phase carrier by immune response in the process of chromatography, and launch visible light under the exciting of infrared light with up-conversion luminescence particle and bioactive molecule.Realizing thus up-conversion luminescent material combines with immunochromatography technique, the target checking matter in the biological sample is detected.
Up-conversion luminescent material UCP is combined with immunochromatography technique, for traditional immunochromatography technique has brought breakthrough change:
The characteristics of A.UCP luminous marker, feasible immuno-chromatographic test paper strip with its thing that serves as a mark can combine with instrument, the target checking matter is carried out the high accurate quantification of sensitivity detect;
The various features spectrum that B.UCP had (excitation spectrum and emission spectrum) makes that the immune chromatography test paper based on up-converting phosphor technology can carry out the high multiple analysis of sensitivity, promptly disposable plurality of target checking matter in the biological sample is detected;
The up-conversion luminescence phenomenon of C.UCP uniqueness makes and has got rid of because biological sample autofluorescence to be detected causes the possibility of interference with the testing process of its thing that serves as a mark, and improves signal to noise ratio (S/N ratio), thereby has improved the sensitivity and stability that detects;
D. by the crosslinked bioactive molecule of covalent manner, under the prerequisite that guarantees detection sensitivity, improved the reliability and stability of system.
Summary of the invention
For biological sample is carried out fast, sensitive, detect quantitatively, the invention provides a kind of immuno-chromatographic test paper strip, it adopts up-conversion luminescent material is that UCP is as biomarker, the result shows with the form of visible light light signal under the irradiation of infrared light, and can use and change optical biosensor and carry out interpretation.Its structure comprises:
A. sample pad;
B. pad or bond discharge pad, are fixed with the bioactive molecule of UCP mark on it;
C. analyzing film has the band of detection and quality control band on it;
D. adsorptive pads provides flow of liquid to cross the power of whole test strips by syphonic effect;
E. plastic back plate wherein simultaneously scribbles viscose glue;
Above-mentioned sample pad, pad, film, adsorptive pads are fixed on the plastic back plate by viscose glue.
Be overlapped between the each part mentioned above, guaranteed the continuity that liquid flows on test strips.When detecting, sample drop is added on the sample pad, sample enters pad by infiltration and syphonic effect, make UCP bond wherein dissolve free again, and under the syphonic effect of adsorptive pads, leave pad and enter film, portion flows to the direction of adsorptive pads within it.Between UCP bond, target checking matter, detection band, the quality control band certain immune response will take place specifically in this process, and generation has tell-tale signal.Difference according to the immune response mode takes place on test strips can be divided into sandwich mode, competitive mode and indirect pattern.
A. sandwich mode:
Pathogen, microorganism and the big molecular antigen that can be used for existing in the test sample based on the test strips of sandwich mode, antibody etc.Wherein, the pattern that pathogen, microorganism and big molecular antigen are detected is a double-antibody sandwich, and antagonist detects and is double antigens sandwich.Below mainly be that example describes with the double-antibody sandwich.
In the preparation process of test strips, at first UCP and checking matter specific antibody A (that is, the antibody that can combine with the checking matter specificity, but it is incorporated into the A site on the checking matter only) are combined, and be fixed in the pad; Then, with checking matter specific antibody B (that is antibody that can combine, but it is incorporated into the B site on the checking matter only, with the checking matter specificity; Annotate: antibody as used herein also can be the checking matter specific polyclonal antibody.) be fixed on the film as detecting band, two anti-(that is, the antibody that can combine with checking matter specific antibody A specificity) are fixed on the film as quality control band, the accurate pH test paper of color change interval 5.5-9.0 is affixed on the adsorptive pads as the terminal point index strip.
Accompanying drawing 3 is not contain determinand in the biological sample, that is the synoptic diagram of negative sample when detecting.Owing to do not contain checking matter in the sample, thereby dissolving is free again, and has only the UCP-antibody A with what the fluid matrix of sample entered film.Thus, UCP-antibody A and two anti-immune responses only take place on quality control band, and the UCP-antibody A is fixed on it.Test strips in negative like this detection is under final UPT biology sensor interpretation, and having only has visible phosphorescence to produce on the quality control band.
Antibody A in the sandwich mode system and antibody B are changed to antigen A and antigen B, both can the antibody that certain pathogenic infection produced be detected double antigens sandwich.Reaction principle, as a result indication and above double-antibody sandwich to detect antigen identical.
B. competitive mode:
The micromolecule antigen that can be used for existing in the test sample based on the test strips of competitive mode.
In the preparation process of test strips, at first UCP is combined with checking matter specific antibody A, and be fixed in the pad; Then, antigen that will be identical with the checking matter A site of the combination of antibody A specificity (but all contain) is fixed on the film as detecting band, resist (promptly two, the antibody that can combine) be fixed on the film as quality control band, the accurate pH test paper of color change interval 5.5-9.0 is affixed on the adsorptive pads as the terminal point index strip with checking matter specific antibody A specificity.
When positive is detected, can be divided into two kinds of situations: checking matter concentration height, checking matter concentration are lower.
Accompanying drawing 4 is the competition immunoreactive synoptic diagram of checking matter concentration when high.When checking matter concentration is high in the sample, checking matter behind the application of sample in the sample and the whole combinations of UCP-antibody A in the pad, promptly the UCP-antibody A is incorporated into the A point on the checking matter.Thereby under the drive of adsorptive pads, what the fluid matrix in sample entered film has only UCP-antibody A-checking matter immune complex.When it flows through when detecting band because the antibody A of UCP mark combines with checking matter in the immune complex, so can not with detection with on contain the A site antigen combine.UCP-antibody A-checking matter immune complex continues to flow, and antibody A and two anti-immune responses in the immune complex take place when flowing through quality control band, and UCP-antibody A-checking matter immune complex is fixed on it.Remaining fluid matrix continues to flow to adsorptive pads, and with its on indicator in the pH test paper react, make it become green by yellow.Test strips after the variable color is carried out the sensor interpretation, and having only under the irradiation of infrared light has visible phosphorescence to produce on the quality control band.
The immunoreactive synoptic diagram of competition when accompanying drawing 5 hangs down for checking matter concentration.When checking matter concentration in the sample is low, the checking matter behind the application of sample in the sample at first with pad in the UCP-antibody A combine.Then, immune complex of the two formation (UCP-antibody A-checking matter) and free UCP-antibody A bond, the fluid matrix in sample enters film under the drive of adsorptive pads.When it flows through when detecting band because the antibody A of UCP mark combines with checking matter in the immune complex, thereby have only free UCP-antibody A bond can with detection with on contain the A site antigen combine, and be fixed.Remaining UCP-antibody A-checking matter immune complex continues to flow, and antibody A and two anti-immune responses in the immune complex take place when flowing through quality control band, and UCP-antibody A-checking matter immune complex is fixed on it.Test strips is under final infrared light irradiation like this, and detecting all has visible phosphorescence to produce on band and the quality control band, and its pairing signal intensity is respectively T and C.Detection is with the ratio of going up phosphorescence light intensity C on phosphorescence light intensity T and the quality control band, and promptly T/C is inversely proportional to the checking matter concentration that contains in the sample.
The accompanying drawing 6 immunoreactive synoptic diagram of competition when negative sample is detected.Owing to do not contain checking matter in the sample, thereby dissolving is free again, and has only the UCP-antibody A with what the fluid matrix of sample entered film.Its with detect with on contain the antigen in A site, two on the quality control band anti-all can in conjunction with.Test strips is under final infrared light irradiation like this, and detecting all has visible phosphorescence to produce on band and the quality control band.But the T/C value of this moment is compared during with the positive detection low concentration and is reached the strongest.
C. indirect pattern:
Can be used for detecting multiple animal (comprising the people) based on the test strips of indirect pattern and be subjected to the corresponding antibodies in the serum behind certain pathogenic infection.
In the preparation process of test strips, at first UCP is combined with staphylococcal protein A (SPA), and be fixed in the pad; Then, the surface antigen of certain pathogen is fixed on the film as detecting band, sheep IgG is fixed on the film as quality control band, the accurate pH test paper of color change interval 5.5-9.0 is affixed on the adsorptive pads as the terminal point index strip.
The accompanying drawing 7 indirect immune response synoptic diagram when the positive serum sample is detected.Fluid matrix behind the application of sample in the sample make the UCP-SPA bond dissolve free again and with blood serum sample in the various IgG (immunoglobulin G is comprising a part of pathogen specific IgG antibody) that exist combine.Under the drive of adsorptive pads, the UCP-SPA-IgG bond flows into film with free UCP-SPA.When flowing through when detecting band, the UCP-SPA-pathogen specific IgG antibody in the UCP-SPA-IgG bond by the pathogen specific IgG antibody with detect with on pathogen antigen special immune association reaction takes place, and in conjunction with thereon.Free UCP-SPA and other some UCp-SPA-IgG bonds (wherein not containing UCP-SPA-pathogen specific IgG antibody) will continue mobile, and the sheep IgG of UCP-SPA on it combines by quality control band the time.Remaining fluid matrix continues to flow to adsorptive pads, and with its on indicator in the pH test paper react, make it become green by yellow.Test strips after the variable color is carried out the sensor interpretation, and quality control band and detection are with visible phosphorescence are all arranged under the infrared light irradiation, and its pairing signal intensity is respectively T and C.Detection is with the ratio of going up phosphorescence light intensity C on phosphorescence light intensity T and the quality control band, and promptly T/C is directly proportional with the concentration of pathogen specific IgG antibody in the blood serum sample.(annotate: staphylococcal protein A, i.e. SPA, characteristic be to combine with multiple animal IgG.)
The accompanying drawing 8 indirect immune response synoptic diagram when the negative serum sample is detected.Owing to do not contain the pathogen specific IgG antibody in the sample, thereby dissolving is free again, and has only UCP-SPA and other some UCP-SPA-IgG bonds (wherein not containing UCP-SPA-pathogen specific IgG antibody) with what the fluid matrix of sample entered film.Wherein UCP-SPA combines with sheep IgG on the quality control band, and detect with on pathogen antigen do not participate in any reaction.Test strips is under final infrared light irradiation like this, and having only has visible phosphorescence to produce on the quality control band.
Comprise a shell in this test strips structure, comprise well on the described shell, as a result interpretation window and terminal point indication window.By well, biological sample is added on the sample pad; The interpretation window is corresponding to detection band and quality control band on the analyzing film as a result; The terminal point indication window is corresponding to the terminal point index strip on the adsorptive pads.
The bioactive molecule of UCP institute mark comprises that antigen, antibody, staphylococcal protein A, external source coagulate hormone, polynucleotide, receptors ligand, medicine, cell etc.
The diameter of UCP particle is 200-300nm.And, can modify the surface of UCP particle.
When being used for the double-antibody sandwich immunologic detection method, wherein, be fixed with first specific antibody of UCP mark on the pad, this specific antibody can combine with a site of determinand; The detection of analyzing film with on be fixed with second specific antibody that combines with another site of determinand, be fixed with the antibody that can combine on the quality control band with the first specific antibody generation specificity.
When being used for the double antigens sandwich immunologic detection method, wherein, be fixed with the antigen A of UCP mark on the pad; The detection of analyzing film is with and is fixed with antigen B, and antigen A and antigen B can be incorporated into the different loci of the tested antibody of target; Be fixed with the antibody that can combine on the quality control band with antigen A generation specificity.
When being used to compete immunologic detection method, wherein, be fixed with the specific antibody of UCP mark on the pad, this antibody can combine with a site of determinand; The detection of analyzing film with on be fixed with the antigen that has identical above-mentioned site with determinand, be fixed with the antibody that can combine on the quality control band with above-mentioned specific antibody generation specificity.
When being used for indirect immunologic detection method, wherein, be fixed with the staphylococcal protein A of UCP mark on the pad; The detection of analyzing film with on be fixed with the surface antigen of certain pathogen, be fixed with IgG on the quality control band.
The preparation method of described immune chromatography test paper based on up-converting phosphor technology is: adopting up-conversion luminescent material is that UCP carries out detection by quantitative as biomarker to the target checking matter; Be fixed with the staphylococcal protein A of UCP mark on the pad; The detection of analyzing film with on be fixed with the surface antigen of determinand, be fixed with IgG on the quality control band; It is pH=7.2 0.03mol/L phosphate buffer that described UCP preserves liquid, contains 0.05%-0.3%BSA, 0.01%-0.1%Tween20,0.01%-0.1%NaN
3The dilution of described UCP bioactive molecule is the 0.03mol/L phosphate buffer of pH=7.2, contains 0.5%-2% sucrose, 0.5%-3%BSA; Described sample pad confining liquid is a pH=7.2 0.03mol/L phosphate buffer, contains 1%-10%BSA, and 0.05%-0.2%Tween 20.
Another aspect of the present invention also relates to the preparation method of described test strips, and it may further comprise the steps:
1) preparation of UCP-bioactive molecule
The UCP particle is carried out finishing, be connected, preserve in the liquid at UCP and preserve with bioactive molecule;
Get the UCP-bioactive molecule bond in a certain amount of UCP of being stored in preservation liquid, centrifugal, and abandoning supernatant;
In the UCP of sedimentation bioactive molecule bond, add dilution, and abundant mixing, be prepared into suspension;
2) preparation of sample pad
Select for use cellulose membrane as the sample pad solid phase material, cut into band with certain specification;
Sample pad is put into the sample pad confining liquid soaks;
Then sample pad is taken out from confining liquid, and oven dry, make sample pad fully dry;
3) preparation of pad
Select for use the plain film of glass fibre as the pad solid phase material, cut into band with certain specification;
On this band, add the suspension of UCP-bioactive molecule bond;
Dry this band, make pad fully dry;
4) preparation of analyzing film
Select for use nitrocellulose filter as the analyzing film solid phase material, cut into band with certain specification;
Diverse location specking bioactive molecule on band is made and is detected band and quality control band;
Dry this band, make analyzing film fully dry;
5) assemble this test strips
The analyzing film of handling is pasted on the plastic back plate, and quality control band upwards detects band downwards;
The pad of handling is pasted on the plastic back plate, and the upper end is pressed on the lower end of analyzing film, and overlapped;
The sample pad of handling is pasted on the sub-plastic back plate, and the lower end is concordant with plastic back plate, and the upper end is pressed on the lower end of pad, and overlapped;
Adsorptive pads is pasted on the plastic back plate, and the upper end is concordant with plastic back plate, and the lower end is pressed on the upper end of analyzing film, and overlapped with analyzing film;
The band of assembling shaping is cut into the test strips of certain specification.
Be prepared into of the present inventionly based on the up-converting phosphor technology immuno-chromatographic test paper strip thus, this test strips can be packed in the shell, and is standby under drying condition.This shell can be plastics.
Wherein, it is pH=7.2 0.03mol/L phosphate buffer (PB) that described UCP preserves liquid, contains 0.1% BSA, 0.05%Tween20,0.02%NaN
3
Wherein, the dilution of described UCP bioactive molecule is a pH=7.2 0.03mol/L phosphate buffer (PB), contains 1% sucrose, 1%BSA.
Wherein, described sample pad confining liquid is a pH=7.2 0.03mol/L phosphate buffer (PB), contains 5%BSA, and 0.1%Tween 20.
Wherein, the accurate pH test paper of color change interval 5.5-9.0 can be affixed on adsorptive pads as the terminal point index strip.
Another aspect of the present invention also relates to the application of described test strips in the detection of biological sample.
With through the up-conversion luminescent material UCP of finishing and activation as biomarker, the development of new immune chromatography test paper makes stability, reappearance and the sensitivity that it can be high be applied to the quick bio detection.Final realize to plurality of target checking matters such as antibody that pathogen, pathogenic infection produced, illegal drug, major disease (tumour, cancer and diabetes etc.) mark qualitative, quantitatively reach multiple detection, with the auxiliary diagnosis that satisfies bio-terrorism agent detection, infectious disease and major disease and the demand of civilian health care.
Description of drawings
Accompanying drawing 1:UCP particle is used for the principle schematic of field of biological detection
Accompanying drawing 2: the reaction synoptic diagram that the sandwich immunoassay reaction is positive
Accompanying drawing 3: the reaction synoptic diagram that the sandwich immunoassay reaction is negative
Accompanying drawing 4: when the competition immune response was positive, determinand was the reaction synoptic diagram of high concentration
Accompanying drawing 5: when the competition immune response was positive, determinand was the reaction synoptic diagram of low concentration
Accompanying drawing 6: the negative reaction synoptic diagram of competition immune response
Accompanying drawing 7: the indirect positive reaction synoptic diagram of immune response
Accompanying drawing 8: the indirect negative reaction synoptic diagram of immune response
Accompanying drawing 9:UPT biology sensor is interpretation figure (left side is the negative detection of hepatitis B surface antigen, right positive detection) as a result
Accompanying drawing 10: hepatitis B virus surface antigen examination criteria working curve
Accompanying drawing 11:UPT biology sensor is interpretation figure (left side is the negative detection of SARS virus, right positive detection) as a result
Accompanying drawing 12:SARS virus examination criteria working curve
Accompanying drawing 13:UPT biology sensor is interpretation figure (left side is that plague FI-antigen negative detects right positive detection) as a result
Accompanying drawing 14: plague FI-Ag examination criteria working curve
Accompanying drawing 15:UPT biology sensor is interpretation figure (left side is the negative detection of amphetamine, right positive detection) as a result
Accompanying drawing 16: amphetamine examination criteria working curve diagram
Accompanying drawing 17:UPT biology sensor is interpretation figure (right positive detection is detected for the plague infects negative antibody in a left side) as a result
Accompanying drawing 18: plague antibodies examination criteria working curve
Accompanying drawing 19:UPT biology sensor is interpretation figure (right positive detection is detected for SARS virus infects negative antibody in a left side) as a result
Accompanying drawing 20:SARS antiviral antibody examination criteria working curve
Accompanying drawing 21: the structural representation of immuno-chromatographic test paper strip
Accompanying drawing 22: during the assembling test strips, the stickup synoptic diagram of each several part
Accompanying drawing 23: the structure of test strips and size
Accompanying drawing 24: the vertical view of test strips shell
Accompanying drawing 25: the inner structure of test strips shell is divided into slice and following sheet
Accompanying drawing 26: the perspective view of up-conversion luminescence biology sensor
Accompanying drawing 27: the up-conversion luminescence biology sensor comprises the planar structure synoptic diagram of excitation light path optical axis, test strips normal and phosphor pattern receiving light path optical axis
Accompanying drawing 28: the spectral radiation curves of up-conversion luminescent material
Accompanying drawing 29: the transmittance curve of optical filter
Experimental example 1: double-antibody sandwich mode detection hepatitis B surface antigen HBs-Ag:
One, as a result interpretation of UPT biology sensor:
Accompanying drawing 9:UPT biology sensor is interpretation figure (left side is the negative detection of hepatitis B surface antigen, right positive detection) as a result
Two, the drafting of standard working curve:
1. the hepatitis B surface antigen HBs-Ag standard items of purifying are disposed the series concentration standard items with the normal human serum (with the dilution of pH=7.20.03mol/L PB buffer solution) of dilution in 1: 10 as dilution, concentration is: 20 duplicate samples of 0pg/ml, 50pg/ml, 100pg/ml, 150pg/ml, 200pg/ml, 250pg/ml, 300pg/ml, 400pg/ml, 500pg/ml, 600pg/ml, 700pg/ml, 800pg/ml, 900pg/ml, 1000pg/ml, 1100pg/ml, 1200pg/ml, 1300pg/ml, 1400pg/ml, 1500pg/ml, 1600pg/ml;
2. each sample is used respectively 10 UPT ELISA test strips 10 times, and the sensor interpretation obtains in 10 detections T value and C value are averaged respectively, and finally the ratio according to the two draws the T/C result corresponding with each concentration, is listed in the table below 1:
| Concentration (pg/ml) | 0 | 50 | 100 | 150 | 200 | 250 | 300 |
| T mean value | 0.22259 | 0.24568 | 0.21078 | 0.36785 | 0.4199 | 0.4713 | 0.47517 |
| C mean value | 1.28843 | 1.32237 | 0.94927 | 1.0361 | 0.80599 | 0.84555 | 0.80156 |
| T/C | 0.17276 | 0.18579 | 0.22204 | 0.35503 | 0.52097 | 0.55739 | 0.59281 |
| Concentration (pg/ml) | 400 | 500 | 600 | 700 | 800 | 900 | 1000 |
| T mean value | 0.9892 | 1.16628 | 1.54263 | 1.50086 | 1.59532 | 2.03564 | 1.93305 |
| C mean value | 1.17636 | 1.11031 | 0.85193 | 0.68688 | 0.6764 | 0.81724 | 0.63325 |
| T/C | 0.8409 | 1.05041 | 1.81075 | 2.18504 | 2.35855 | 2.49087 | 3.05259 |
| Concentration (pg/ml) | 1100 | 1200 | 1300 | 1400 | 1500 | 1600 | |
| T mean value | 2.15422 | 4.80941 | 5.16321 | 3.57106 | 3.96671 | 2.62067 | |
| C mean value | 0.62623 | 1.28657 | 1.14657 | 0.66165 | 0.73204 | 0.45822 | |
| T/C | 3.43998 | 3.73816 | 4.50318 | 5.3972 | 5.41871 | 5.71925 |
Table 1: HBsAg examination criteria working curve
With the T/C value as X, as Y drawing standard working curve, the expression formula that fits by statistics standard working curve is with hepatitis B surface antigen HBs-Ag concentration: Y=268.73X+103.06, fit coefficient square be: R2=0.975; The results are shown in accompanying drawing 10: HBsAg examination criteria working curve.
4. the computing formula of hepatitis B surface antigen concentration is:
The hepatitis B surface antigen HBs-Ag concentration (pg/ml) that contains in the blood serum sample=10Y=10 * (268.73X+103.06)=2687.3X+1030.6
Three, actual testing result:
1. detection accuracy:
Identify that available from biological products the B-type hepatitis human blood liquidation (wherein containing 13 parts of positives, 37 parts of feminine genders) of institute uses colloidal gold immune chromatography test and native system (UPT test strips and sensor) to carry out the double blinding detection simultaneously with 50 parts:
Colloidal gold immune chromatography test method---11 parts of positives, 39 parts of feminine genders (that is, two parts of positives are undetected);
UPT test strips and sensor method---13 parts of positives, 37 parts of feminine genders fit like a glove with actual result;
Simultaneously relative with the qualitative detection of colloidal gold immune chromatography test, UPT test strips and sensor method have provided the final accurately concentration of every duplicate samples.
2. detect stability:
A B-type hepatitis human serum with 10 times of dilutions of pH=7.20.03mol/L PB buffer solution, is used UPT test strips and sensor duplicate detection 10 times, the results are shown in following table 2:
| The duplicate |
1 | 2 | 3 | 4 | 5 |
| T/C | 0.40447 | 0.39211 | 0.37042 | 0.4091 | 0.42247 |
| HBs-Ag concentration (pg/ml) | 2117.532 | 2084.317 | 2026.03 | 2129.974 | 2165.904 |
| The duplicate |
6 | 7 | 8 | 9 | 10 |
| T/C | 0.42068 | 0.41614 | 0.42471 | 0.39527 | 0.36826 |
| HBs-Ag concentration (pg/ml) | 2161.093 | 2148.893 | 2171.923 | 2092.809 | 2020.225 |
The coefficient of variation (CV)=2.621% with a serum duplicate measurements
Table 2: HBsAg detects repeatability
Conclusion: in hepatitis B surface antigen HBs-Ag detected, the UPT test strips is compared with the colloidal gold immune chromatography test method with sensor method had higher sensitivity, and was realizing having good stability when accurate quantification detects.
Experimental example 2: double-antibody sandwich mode detection coronavirus (SARS virus):
One, as a result interpretation of UPT biology sensor:
Accompanying drawing 11:UPT biology sensor is interpretation figure (left side is the negative detection of SARS virus, right positive detection) as a result
Two, the drafting of standard working curve:
1. will dispose the series concentration standard items with pH=7.2 0.03mol/L PB buffer solution as dilution through the SARS virus of the also deactivation of cultivating, count, concentration is: 21 duplicate samples of 0org/ml, 100org/ml, 200org/ml, 300org/ml, 400org/ml, 500org/ml, 600org/ml, 700org/ml, 800org/ml, 900org/ml, 1000org/ml, 1100org/ml, 1200org/ml, 1300org/ml, 1400org/ml, 1500org/ml, 1600org/ml, 1700org/ml, 1800org/ml, 1900org/ml, 2000org/ml;
2. each sample is used respectively 10 UPT ELISA test strips 10 times, and the sensor interpretation obtains in 10 detections T value and C value are averaged respectively, and finally the ratio according to the two draws the T/C result corresponding with each concentration, is listed in the table below 3:
| Concentration (org/ml) | 0 | 100 | 200 | 300 | 400 | 500 | 600 |
| T mean value | 0.35469 | 0.35455 | 0.46158 | 0.43979 | 0.61668 | 0.54227 | 0.63369 |
| C mean value | 1.78049 | 1.40951 | 1.30636 | 0.96737 | 0.99995 | 0.8215 | 0.86849 |
| T/C | 0.19921 | 0.25154 | 0.35333 | 0.45462 | 0.61671 | 0.6601 | 0.72965 |
| Concentration (org/ml) | 700 | 800 | 900 | 1000 | 1100 | 1200 | 1300 |
| T mean value | 0.77334 | 1.40432 | 1.63278 | 1.78659 | 1.78619 | 1.77043 | 2.09792 |
| C mean value | 0.78131 | 1.11981 | 1.0004 | 0.84633 | 0.73392 | 0.64788 | 0.68762 |
| Concentration (org/ml) | 0 | 100 | 200 | 300 | 400 | 500 | 600 |
| T/C | 0.9898 | 1.25407 | 1.63213 | 2.11098 | 2.43376 | 2.73265 | 3.05099 |
| Concentration (org/ml) | 1400 | 1500 | 1600 | 1700 | 1800 | 1900 | 2000 |
| T mean value | 2.17253 | 2.09461 | 4.87004 | 4.84787 | 2.82163 | 3.90254 | 2.80012 |
| C mean value | 0.63772 | 0.55493 | 1.24835 | 1.08773 | 0.58017 | 0.7367 | 0.49579 |
| T/C | 3.40671 | 3.77455 | 3.90118 | 4.45687 | 4.86345 | 5.29733 | 5.64779 |
Table 3:SARS virus examination criteria working curve
With the T/C value as X, as Y drawing standard working curve, the expression formula that fits by statistics standard working curve is with SARS virus concentration: Y=337.2X+216.12, fit coefficient square be: R2=0.9631; The results are shown in accompanying drawing 12:SARS virus examination criteria working curve.
4.SARS the computing formula of virus concentration is:
The SARS virus concentration (org/ml) that contains in the sample=10Y=10 * (337.2X+216.12)=3372X+2161.2
Three, actual testing result:
1. detection accuracy:
30 parts of samples to be checked (wherein containing 6 parts of positives, 24 parts of feminine genders) are carried out double blinding with native system (UPT test strips and sensor) to be detected:
UPT test strips and sensor method---6 parts of positives, 24 parts of feminine genders fit like a glove with actual result;
UPT test strips and sensor method have provided the final accurately concentration of every duplicate samples simultaneously.
2. detect stability:
Portion sample to be checked with 10 times of dilutions of pH=7.2 0.03mol/L PB buffer solution, is detected 10 times with UPT test strips and sensor, the results are shown in following table 4:
| The duplicate |
1 | 2 | 3 | 4 | 5 |
| T/C | 0.59887 | 0.57197 | 0.60519 | 0.5936 | 0.6024 |
| SARS virus concentration (org/ml) | 4180.59 | 4089.883 | 4201.901 | 4162.819 | 4192.493 |
| The duplicate |
6 | 7 | 8 | 9 | 10 |
| T/C | 0.59913 | 0.5926 | 0.62355 | 0.60257 | 0.59749 |
| SARS virus concentration (org/ml) | 4181.466 | 4159.447 | 4263.811 | 4193.066 | 4175.936 |
The coefficient of variation (CV)=1.03% with a sample duplicate measurements to be checked
Table 4:SARS virus detects repeatability
Conclusion: in SARS virus detected, UPT test strips and sensor method had good sensitivity and stability, and have realized accurate quantification.
Experimental example 3: competitive mode detects plague FI-antigen (plague FI-Ag):
One, as a result interpretation of UPT biology sensor:
Accompanying drawing 13:UPT biology sensor is interpretation figure (left side is that plague FI-antigen negative detects right positive detection) as a result
Two, the drafting of standard working curve:
1. the plague FI-Ag standard items of purifying are disposed the series concentration standard items with pH=7.2 0.03mol/L PB buffer solution as dilution, concentration is: 21 duplicate samples of 0pg/ml, 100pg/ml, 200pg/ml, 300pg/ml, 400pg/ml, 500pg/ml, 600pg/ml, 700ng/ml, 800pg/ml, 900pg/ml, 1000pg/ml, 1100pg/ml, 1200pg/ml, 1300pg/ml, 1400pg/ml, 1500pg/ml, 1600pg/ml, 1700pg/ml, 1800pg/ml, 1900pg/ml, 2000pg/ml;
2. each sample is used respectively 10 UPT ELISA test strips 10 times, and the sensor interpretation obtains in 10 detections T value and C value are averaged respectively, and finally the ratio according to the two draws the T/C result corresponding with each concentration, is listed in the table below 5:
| Concentration (pg/ml) | 0 | 100 | 200 | 300 | 400 | 500 | 600 |
| T mean value | 3.4065 | 3.97134 | 3.25825 | 6.11733 | 11.04111 | 5.0306 | 5.29534 |
| C mean value | 0.54259 | 0.6675 | 0.56455 | 1.1223 | 2.12737 | 1.02515 | 1.19673 |
| T/C | 6.27823 | 5.94958 | 5.77141 | 5.45071 | 5.19003 | 4.90718 | 4.42484 |
| Concentration (pg/ml) | 700 | 800 | 900 | 1000 | 1100 | 1200 | 1300 |
| T mean value | 5.18186 | 4.18439 | 4.57649 | 4.05474 | 4.30334 | 3.95632 | 3.51903 |
| C mean value | 1.24416 | 1.08102 | 1.28164 | 1.25251 | 1.43797 | 1.5151 | 1.50269 |
| T/C | 4.16495 | 3.87078 | 3.57081 | 3.23729 | 2.99265 | 2.61126 | 2.34182 |
| Concentration (pg/ml) | 1400 | 1500 | 1600 | 1700 | 1800 | 1900 | 2000 |
| T mean value | 3.2772 | 2.88279 | 2.07385 | 1.67005 | 1.63219 | 0.95272 | 0.54594 |
| C mean value | 1.59014 | 1.69625 | 1.51203 | 1.60722 | 2.07268 | 1.90214 | 2.16068 |
| T/C | 2.06095 | 1.69951 | 1.37157 | 1.03909 | 0.78748 | 0.50087 | 0.25267 |
Table 5: plague FI-Ag examination criteria working curve
With the T/C value as X, as Y drawing standard working curve, the expression formula that fits by statistics standard working curve is with plague FI-Ag concentration: Y=-324.63X+2058.5, fit coefficient square be: R2=0.9993; The results are shown in accompanying drawing 14: plague FI-Ag examination criteria working curve.
4. the computing formula of plague FI-Ag concentration is:
The plague FI-Ag concentration (pg/ml) that contains in the sample=10Y=10 * (324.63X+2058.5)=-3246.3X+20585
Three, actual testing result:
1. detection accuracy:
32 parts of samples to be checked (wherein containing 19 parts of positives, 13 parts of feminine genders) are carried out double blinding with native system (UPT test strips and sensor) to be detected:
UPT test strips and sensor method---19 parts of positives, 13 parts of feminine genders fit like a glove with actual result;
UPT test strips and sensor method have provided the final accurately concentration of every duplicate samples simultaneously.
2. detect stability:
Portion sample to be checked with 10 times of dilutions of pH=7.2 0.03mol/L PB buffer solution, is detected 10 times with UPT test strips and sensor, the results are shown in following table 6:
| The duplicate |
1 | 2 | 3 | 4 | 5 |
| T/C | 2.37458 | 2.3984 | 2.36769 | 2.38635 | 2.34121 |
| Plague FI-Ag concentration (pg/ml) | 12876.4 | 12799.07 | 12898.77 | 12838.19 | 12984.73 |
| The duplicate |
6 | 7 | 8 | 9 | 10 |
| T/C | 2.28906 | 2.38734 | 2.32011 | 2.28347 | 2.33309 |
| Plague FI-Ag concentration (pg/ml) | 13154.02 | 12834.98 | 13053.23 | 13172.17 | 13011.09 |
The coefficient of variation (CV)=1.034% with a sample duplicate measurements to be checked
Table 6: plague FI-Ag detects repeatability
Conclusion: in plague FI-Ag detected, UPT test strips and sensor method had good sensitivity and stability, and have realized the accurate quantification detection.
Experimental example 4: competitive mode detects illegal drug (is that example describes to detect amphetamine)
One, UPT biology sensor interpretation as a result:
Accompanying drawing 15:UPT biology sensor is interpretation figure (left side is the negative detection of amphetamine, right positive detection) as a result
Two, the drafting of standard working curve:
1. pure product amphetamine is disposed the series concentration standard items with pH=7.2 0.03mol/L PB damping fluid as dilution, concentration is: 20 duplicate samples of 0pg/ml, 10pg/ml, 20pg/ml, 30pg/ml, 40pg/ml, 50pg/ml, 70pg/ml, 90ng/ml, 110pg/ml, 130pg/ml, 150pg/ml, 170pg/ml, 190pg/ml, 210pg/ml, 230pg/ml, 250pg/ml, 270pg/ml, 290pg/ml, 310pg/ml, 330pg/ml;
2. each sample detects 10 times with 10 UPT test strips respectively, and the sensor interpretation obtains in 10 detections T value and C value are averaged respectively, and finally the ratio according to the two draws the T/C result corresponding with each concentration, is listed in the table below 7:
| Concentration (pg/ml) | 0 | 10 | 20 | 30 | 40 | 50 | 70 |
| T mean value | 3.25457 | 4.85874 | 3.85462 | 8.39048 | 5.46825 | 6.52519 | 6.30955 |
| C mean value | 0.54112 | 0.83405 | 0.68053 | 1.55738 | 1.07889 | 1.36111 | 1.5213 |
| T/C | 6.0145 | 5.82548 | 5.66415 | 5.38756 | 5.0684 | 4.79402 | 4.14747 |
| Concentration (pg/ml) | 90 | 110 | 130 | 150 | 170 | 190 | 210 |
| T mean value | 4.96756 | 4.44708 | 3.17447 | 4.08154 | 2.32299 | 1.88865 | 1.60525 |
| C mean value | 1.40311 | 1.43919 | 1.18685 | 1.81695 | 1.28834 | 1.191 | 1.21215 |
| T/C | 3.54039 | 3.08999 | 2.6747 | 2.24637 | 1.80309 | 1.58577 | 1.3243 |
| Concentration (pg/ml) | 230 | 250 | 270 | 290 | 310 | 330 | |
| T mean value | 2.05901 | 1.74944 | 1.1227 | 1.34347 | 0.86066 | 0.51467 | |
| C mean value | 1.84633 | 1.79797 | 1.51804 | 2.25774 | 1.89995 | 1.94995 | |
| T/C | 1.11519 | 0.97301 | 0.73957 | 0.59505 | 0.45299 | 0.26394 |
Table 7: amphetamine examination criteria working curve
With the T/C value as X, as Y drawing standard working curve, the expression formula that fits standard working curve by statistics is with amphetamine concentration: Y=-51.985X+296.45, what fit coefficient square is: R
2=0.9529; The results are shown in accompanying drawing 16: amphetamine examination criteria working curve.
4. the concentration computing formula of amphetamine is:
Amphetamine concentration (the pg/ml)=10Y=10 that contains in the sample * (51.985X+296.45)=-519.85X+2964.5
Three, actual detected result:
1. detection accuracy:
44 parts of samples to be checked (wherein containing 23 parts of positives, 21 parts of feminine genders) are carried out double blinding with native system (UPT test strips and sensor) to be detected:
UPT test strips and sensor method---23 parts of positives, 21 parts of feminine genders fit like a glove with actual result;
UPT test strips and sensor method have provided the final accurately concentration of every duplicate samples simultaneously.
2. detect stability:
Portion sample to be checked with 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid, is used UPT test strips and sensor 10 times, the results are shown in following table 8:
| The duplicate |
1 | 2 | 3 | 4 | 5 |
| T/C | 1.18037 | 1.18783 | 1.16444 | 1.18792 | 1.16883 |
| Amphetamine concentration (pg/ml) | 2350.885 | 2347.007 | 2359.166 | 2346.96 | 2356.884 |
| The duplicate |
6 | 7 | 8 | 9 | 10 |
| T/C | 1.2185 | 1.14839 | 1.18847 | 1.17538 | 1.19595 |
| Amphetamine concentration (pg/ml) | 2331.063 | 2367.509 | 2346.674 | 2353.479 | 2342.785 |
The coefficient of variation (CV)=0.423% with a sample duplicate measurements to be checked
Table 8: amphetamine detects repeatability
Conclusion: in amphetamine detected, UPT test strips and sensor method had good sensitivity and stability, and have realized the accurate quantification detection.
Experimental example 5: indirect pattern detects the plague and infects antibody (is that example describes to detect rabbit infection plague generation antibody)
One, UPT biology sensor interpretation as a result:
Accompanying drawing 17:UPT biology sensor is interpretation figure (right positive detection is detected for the plague infects negative antibody in a left side) as a result
Two, the drafting of standard working curve:
1. the anti-plague IgG standard items of rabbit of purifying are disposed the series concentration standard items with the normal rabbit serum (with the dilution of pH=7.2 0.03mol/L PB damping fluid) of dilution in 1: 10 as dilution, concentration is: 20 duplicate samples of 0ng/ml, 2ng/ml, 4ng/ml, 6ng/ml, 8ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 25ng/ml, 30ng/ml, 35ng/ml, 40ng/ml, 45ng/ml, 50ng/ml, 55ng/ml, 60ng/ml, 65ng/ml, 70ng/ml, 75ng/ml, 80ng/ml;
2. each sample detects 10 times with 10 UPT test strips respectively, and the sensor interpretation obtains in 10 detections T value and C value are averaged respectively, and finally the ratio according to the two draws the T/C result corresponding with each concentration, is listed in the table below 9:
| Concentration (ng/ml) | 0 | 2 | 4 | 6 | 8 | 10 | 15 |
| T mean value | 0.64465 | 0.78083 | 0.7329 | 0.75725 | 0.78867 | 0.86065 | 0.95173 |
| C mean value | 1.65228 | 1.96276 | 1.41627 | 1.4487 | 1.18135 | 1.05232 | 0.88773 |
| T/C | 0.39016 | 0.39782 | 0.51749 | 0.52271 | 0.6676 | 0.81786 | 1.07209 |
| Concentration (ng/ml) | 20 | 25 | 30 | 35 | 40 | 45 | 50 |
| T mean value | 1.14907 | 1.17353 | 1.72606 | 1.51789 | 1.70135 | 2.33072 | 2.13382 |
| Concentration (ng/ml) | 0 | 2 | 4 | 6 | 8 | 10 | 15 |
| C mean value | 0.83361 | 0.78799 | 0.79857 | 0.67269 | 0.65686 | 0.71419 | 0.62598 |
| T/C | 1.37843 | 1.48927 | 2.16144 | 2.25645 | 2.59013 | 3.26345 | 3.40877 |
| Concentration (ng/ml) | 55 | 60 | 65 | 70 | 75 | 80 | |
| T mean value | 2.04721 | 2.47774 | 2.88072 | 2.70869 | 3.18586 | 2.59155 | |
| C mean value | 0.56959 | 0.64625 | 0.63839 | 0.48019 | 0.64757 | 0.41861 | |
| T/C | 3.59418 | 3.83403 | 4.51248 | 5.64087 | 4.91972 | 6.19085 |
Table 9: plague antibodies examination criteria working curve
With the T/C value as X, as Y drawing standard working curve, the expression formula that fits standard working curve by statistics is with plague antibodies concentration: Y=14.129X-0.3076, what fit coefficient square is: R
2=0.9749; The results are shown in accompanying drawing 18: plague antibodies examination criteria working curve.
4. the computing formula of plague antibodies concentration is:
Plague antibodies concentration (the ng/ml)=10Y=10 that contains in the rabbit anteserum * (14.129X-0.3076)=141.29X-3.076
Three, actual detected result:
1. detection accuracy:
Using enzyme linked immunosorbent assay (ELISA) and native system (UPT test strips and sensor) to carry out double blinding simultaneously 100 parts of rabbit anteserums (wherein containing 31 parts of positives, 69 parts of feminine genders) detects:
ELISA method---23 parts of positives, 77 parts of feminine genders;
UPT test strips and sensor method---31 parts of positives (comprise 23 parts of feminine genders among the ELISA, and from 77 parts of negative samples that ELISA determines, detect 8 parts of positives again), 69 parts of feminine genders fit like a glove with actual result;
Simultaneously relative with the qualitative detection of ELISA, UPT test strips and sensor method have provided the final accurately concentration of every duplicate samples.
2. detect stability:
The serum that portion is infected plague rabbit is used UPT test strips and sensor 10 times with 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid, the results are shown in following table 10:
| The duplicate |
1 | 2 | 3 | 4 | 5 |
| T/C | 0.39897 | 0.39034 | 0.38739 | 0.38791 | 0.38983 |
| Plague antibodies concentration (ng/ml) | 53.29447 | 52.07514 | 51.65833 | 51.7318 | 52.00308 |
| The duplicate |
6 | 7 | 8 | 9 | 10 |
| The duplicate |
1 | 2 | 3 | 4 | 5 |
| T/C | 0.39754 | 0.40328 | 0.39182 | 0.39558 | 0.39116 |
| Plague antibodies concentration (ng/ml) | 53.09243 | 53.90343 | 52.28425 | 52.8155 | 52.191 |
The coefficient of variation (CV)=1.408% with a serum duplicate measurements
Table 10: plague antibodies examination criteria working curve
Conclusion: infect in the antibody test in the plague, the UPT test strips is compared with the ELISA method with sensor method has higher sensitivity, and has good stability when realizing accurate quantification.
Experimental example 6: indirect pattern detects SARS virus and infects antibody:
One, UPT biology sensor interpretation as a result:
Accompanying drawing 19:UPT biology sensor is interpretation figure (right positive detection is detected for SARS virus infects negative antibody in a left side) as a result
Two, the drafting of standard working curve:
1. the people's anti-SARS virus IgG standard items that will purify from the SARS patients serum dispose the series concentration standard items with the normal human serum (with the dilution of pH=7.2 0.03mol/L PB damping fluid) of dilution in 1: 10 as dilution, and concentration is: 21 duplicate samples of 0ng/ml, 1ng/ml, 2ng/ml, 3ng/ml, 4ng/ml, 5ng/ml, 6ng/ml, 8ng/ml, 10ng/ml, 12ng/ml, 14ng/ml, 16ng/ml, 18ng/ml, 20ng/ml, 22ng/ml, 24ng/ml, 26ng/ml, 28ng/ml, 30ng/ml, 32ng/ml, 34ng/ml;
2. each sample detects 10 times with 10 UPT test strips respectively, and the sensor interpretation obtains in 10 detections T value and C value are averaged respectively, and finally the ratio according to the two draws the T/C result corresponding with each concentration, is listed in the table below 11:
| Concentration (ng/ml) | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| T mean value | 0.69876 | 0.64241 | 0.57418 | 0.45505 | 0.61317 | 0.52559 | 0.58502 |
| C mean value | 1.88067 | 1.57133 | 1.25642 | 0.92109 | 1.05531 | 0.77997 | 0.80162 |
| T/C | 0.37155 | 0.40883 | 0.457 | 0.49403 | 0.58103 | 0.67386 | 0.7298 |
| Concentration (ng/ml) | 8 | 10 | 12 | 14 | 16 | 18 | 20 |
| T mean value | 0.77607 | 1.51038 | 2.08572 | 1.82193 | 1.77487 | 1.87806 | 2.28666 |
| C mean value | 0.80191 | 1.101 | 1.16543 | 0.79298 | 0.68393 | 0.63044 | 0.73583 |
| T/C | 0.96778 | 1.37183 | 1.78966 | 2.29757 | 2.59511 | 2.97897 | 3.10759 |
| Concentration (ng/ml) | 22 | 24 | 26 | 28 | 30 | 32 | 34 |
| Concentration (ng/ml) | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| T mean value | 2.25097 | 2.13682 | 6.16953 | 5.22465 | 3.57491 | 4.73442 | 2.72789 |
| C mean value | 0.65485 | 0.57506 | 1.47597 | 1.10033 | 0.6907 | 0.82293 | 0.44401 |
| T/C | 3.43738 | 3.71582 | 4.17998 | 4.74826 | 5.17578 | 5.75312 | 6.14375 |
Table 11:SARS antiviral antibody examination criteria working curve
With the T/C value as X, as Y drawing standard working curve, the expression formula that fits standard working curve by statistics is with the SARS virus antibody concentration: Y=5.7365X+0.8012, what fit coefficient square is: R
2=0.9841; The results are shown in accompanying drawing 20:SARS antiviral antibody examination criteria working curve.
4.SARS the computing formula of antiviral antibody concentration is:
SARS virus antibody concentration (the ng/ml)=10Y=10 that contains in the human serum * (5.7365X+0.8012)=57.365X+8.012
Three, actual detected result:
1. detection accuracy:
Use enzyme linked immunosorbent assay (ELISA) and native system (UP test strips and sensor) to carry out the double blinding detection simultaneously patients serum's (finally make a definite diagnosis and wherein contain 17 parts of positives, 28 parts of feminine genders) of 45 parts of PI SARS virus:
ELISA method---17 parts of positives, 28 parts of feminine genders fit like a glove with actual result, detect and last about 2 hours;
UPT test strips and sensor method---17 parts of positives, 28 parts of feminine genders fit like a glove with actual result, detect and last about half an hour;
Compare with the ELISA method, UPT test strips and sensor method detect more quick, and have provided the final accurately concentration of every duplicate samples on the basis of ELISA method qualitative detection quantitatively.
2. detect stability:
A SARS patients serum with 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid, is used UPT test strips and sensor 10 times, the results are shown in following table 12:
| The duplicate |
1 | 2 | 3 | 4 | 5 |
| T/C | 0.70227 | 0.69938 | 0.69776 | 0.70511 | 0.71272 |
| SARS virus antibody concentration (ng/ml) | 48.29772 | 48.13193 | 48.039 | 48.46064 | 48.89718 |
| The duplicate |
6 | 7 | 8 | 9 | 10 |
| T/C | 0.70485 | 0.70306 | 0.70199 | 0.72136 | 0.70459 |
| SARS virus antibody concentration (ng/ml) | 48.44572 | 48.34304 | 48.28166 | 49.39282 | 48.43081 |
The coefficient of variation (CV)=0.819% with a serum duplicate measurements
Table 12:SARS antiviral antibody detects repeatability
Conclusion: in SARS virus infected antibody test, that UPT test strips and sensor method are compared with the ELISA method was more quick, can realize that accurate quantification detects, and stable fine.
Embodiment
Below in conjunction with drawings and Examples the present invention is explained in detail.
The general structure that has shown test strips in the accompanying drawing 21 comprises: sample pad 10 (Sample Pad), pad 11 (Conjugate Pad or bond discharge pad Conjugate Release Pad), analyzing film 12 (AnalyticalMembrane), adsorptive pads 13 (Wicking Pad), plastic back plate 14 (wherein one side scribbles viscose glue) are (LaminatingCard).Sample pad 10, pad 11, analyzing film 12, adsorptive pads 13 are fixed on the plastic back plate 14 by viscose glue 15.
B. be fixed with UCP-bioactive molecule bonds such as UCP-antibody, UCP-antigen in the pad 11, after adding test sample, immune response can take place in it on test strips;
D. adsorptive pads 13 provides flow of liquid to cross the power of whole test strips by syphonic effect in whole testing process.
Overlapping region 16 between the various piece has guaranteed the continuity that liquid flows on test strips.When detecting, sample drop is added on the sample pad 10, sample enters pad 11 by infiltration and syphonic effect, make UCP bond wherein dissolve free again, and under the syphonic effect of adsorptive pads 13, leave pad 11 and enter analyzing film 12, portion flows to the direction of adsorptive pads 13 within it.UCP bond in this process, target checking matter, detect with 17, certain immune response will take place specifically between the quality control band 18, and produce and have tell-tale signal.
The assembling of test strips among the present invention may further comprise the steps:
A. plastic back plate 14 is cut into the band of 7.4 * 30cm specification;
B. the analyzing film 12 that will handle sticks at the position of 21mm-46mm on the plastic back plate 14, quality control band 18 upwards, detect be with 17 downward;
C. the pad 11 that will handle sticks at the position of 12mm on the plastic back plate 14, and the upper end is pressed on the lower end of analyzing film 12, overlapping 1mm;
D. the sample pad 10 that will handle sticks on the plastic back plate 14, and the lower end is concordant with plastic back plate 14, and the upper end is pressed on the lower end of pad 11, overlapping 3mm;
E. adsorptive pads 13 is sticked on the plastic back plate 14, the upper end is concordant with plastic back plate 14, and the lower end is pressed on the upper end of analyzing film 12, overlapping 2mm;
F. will assemble the band that is shaped and cut into the wide test strips of 4mm with cutting cutter;
G. the plastic casing of test strips being packed into is preserved standby in the exsiccator.
Overlapping relation when being stickup in the accompanying drawing 22 between the each several part.
Accompanying drawing 23 assembles the structure and the size of the back each several part that finishes for test strips, wherein the sample pad 10 clean length of exposure after overlapping is 15mm, clean length of exposure after pad 11 is overlapping is 7mm, and the clean length of exposure after analyzing film 12 is overlapping is 22mm, and the clean length of exposure after adsorptive pads 13 is overlapping is 30mm.Assembling finishes and just can pack in the shell of test strips through the test strips of shearing.
The shell of test strips is referring to accompanying drawing 24, and it comprises well 20, interpretation window 21 and terminal point indication window 22 as a result.Wherein, as a result in the interpretation window 21 corresponding detect be with 17, quality control band 18.Corresponding terminal point index strip 19 in the described terminal point indication window 22.After wherein being added to sample spot on the test strips by well 20, under the syphonic effect of adsorptive pads 13 fluid sample be with 17 through detecting successively, quality control band 18.After terminal point index strip 19 becomes green, with biology sensor on the shell as a result each band in the interpretation window 21 carry out interpretation, just can obtain the result.
The test strips shell is divided into slice and following sheet, referring to accompanying drawing 25.23,24 are the interlock finger, the two are compressed the sheet up and down of test strips shell is integrated; 25 is baffle plate, and 26 for falling nail, and six baffle plates 25 and three follow closely 26 and test strips can be stuck in the shell securely, prevent to move; 27 is pressing plate, and after sheet covered tightly up and down, the position of pressing plate 27 was in sample pad 10 and pad 11, pad 11 and analyzing film 12 overlappings on the test strips, increases the connection of test strips each several part with this, guarantees the continuity of liquid flow in the detection.
As shown in figure 25, test strips is put into following of shell, with last slice with following sheet lid tightly, just become the finished product that can be used to detect.
Embodiment 1:The structure of up-conversion luminescence biology sensor
Consult Figure 26 and Figure 27.As seen from the figure, up-conversion luminescence biology sensor of the present invention comprises excitation light path, phosphor pattern receiving light path, image processing system, the phosphor pattern that is respectively applied for illumination test strips 3, receives phosphor pattern that test strips 3 sends, test strips 3 is sent is analyzed and is handled, and the test strips structural representation is consulted Figure 24 in the up-conversion luminescence biology sensor.
On excitation light path, set gradually infrared excitation light source 1, one-dimensional focusing mirror 2 along optical axis, its optical axis is 010.On the phosphor pattern receiving light path, set gradually preceding mirror group 5, optical filter 6, back mirror group 7 and picture receiver 8 along optical axis, its optical axis is 002.The object plane of phosphor pattern receiving light path overlaps with the upper surface of test strips 3, and the image planes of phosphor pattern receiving light path overlap with the sensitive area of picture receiver 8.Test strips 3 is installed in the special shell 4, and its normal is 003.The focal line AA illumination test strips 3 that excitation light path forms, focal line AA is parallel with the long limit of test strips 3, and overlaps with the line of symmetry of test strips 3.The optical axis 002 of the optical axis 010 of excitation light path, the normal 003 of test strips 3 and phosphor pattern receiving light path is positioned at same plane, the optical axis 010 of excitation light path is B1 with the angle of the normal 003 of test strips 3, and the optical axis 002 of phosphor pattern receiving light path is B2 with the angle of the normal 003 of test strips 3.
Said excitation light path is made up of infrared excitation light source 1, one-dimensional focusing mirror 2, and its effect is that intensity of generation is 0.15 watt of/square centimeter (W/cm
2) elongated infrared light focal line AA, the illumination test strips 3 all functions band.Infrared excitation light source 1 normally through the semiconductor laser of collimation, sends parallel beam, and its wavelength is generally near the 980nm.One-dimensional focusing mirror 2 can be that cylindrical lens, prism or other can produce the optical element of focal line.
Contain two function bands on the said test strips 3, promptly detect and be with 17, quality control band 18.Wherein detecting with 17 and will specific immune response take place according to target checking matter in different detecting patterns and the detected sample and UCP label, is T in conjunction with the signal that UCP produced on it; Quality control band 18 is C by immune response in conjunction with the signal that UCP produced, the ratio of T and C, be that T/C is and variable concentrations target checking matter corresponding detection result, it will be certain linear dependence with the concentration of target checking matter in the detected sample, and C has supervisory function bit for the biologically performance of test strips simultaneously;
Said phosphor pattern receiving light path is made up of preceding mirror group 5, optical filter 6, back mirror group 7 and picture receiver 8; The object space aperture half-angle of phosphor pattern receiving light path is U2.Preceding mirror group 5 is collimated into directional light with the phosphorescence that each function band on the test strips 3 sends.The veiling glare that comprises in the optical filter 6 filtering phosphorescent signal is to improve signal to noise ratio (S/N ratio); It has high as far as possible transmitance (greater than 90%) to phosphorescent signal, and exciting light is had alap transmitance (less than 10
-5).The phosphorescent signal focal imaging of back mirror group 7 after with the filtering veiling glare is on the sensitive area of picture receiver 8.Picture receiver 8 can be a linear array CCD camera, also can be an one dimension photodiode array, and the orientation of its sensitive element is consistent with the direction of elongate of test strips 3.So picture receiver 8 can be measured the phosphorescence intensity that each function band sends on the test strips 3 accordingly.
The angle of said excitation light path optical axis 010 and test strips 3 normals 003 is that the angle of B1 and phosphor pattern receiving light path optical axis 002 and test strips 3 normals 003 is B2, and B1 ≠ B2, usually B1＞B2-U2, or B1＜B2-U2, U2 is the object space aperture half-angle of phosphor pattern receiving light path.The purpose of this design is to stop the reflected light of illuminating ray to enter the phosphor pattern receiving light path, to reduce veiling glare.
Compare with technology formerly, characteristics of the present invention are: excitation light path produces high strength infrared laser focal line AA, simultaneously with each the function band illumination on the test strips 3; The phosphor pattern receiving light path is to each the function band imaging simultaneously on the test strips 3; Included angle B 1 does not wait with included angle B 2, and satisfies B1＞B2-U2 or B1＜B2-U2.
These characteristics make the present invention have interpretation efficient height, interpretation highly sensitive, can carry out advantages such as multiple quantitative detection.
The course of work of up-conversion luminescence biology sensor of the present invention is: the test strips 3 that at first will be installed in the shell 4 is put into the interpretation position.The parallel beam that is sent by infrared excitation light source 1 forms focal line AA through one-dimensional focusing mirror 2, and focal line AA is positioned at the upper surface of test strips 3, and parallel with the direction of elongate of test strips 3, like this each the function band on the test strips 3 is all thrown light on.The phosphorescent signal of being sent by each function band on the test strips 3 becomes directional light behind preceding mirror group 5 collimations, quilt back mirror group 7 focal imagings are on the sensitive area of picture receiver 8 behind optical filter 6 filtering veiling glares.The test strips phosphor pattern of 9 pairs of picture receivers of image processing system, 8 outputs is analyzed and is handled, and provides the amplitude of each function band phosphorescent signal on the test strips 3, and then provides the attribute and the content of tested biomolecule.
Figure 26 and Figure 27 are most preferred embodiments of the present invention, and its concrete structure and statement parameter are as follows:
The sectional dimension of the parallel beam that the infrared excitation light source 1 in the excitation light path sends is 4mm * 1mm, and centre wavelength is 980nm, and power is 30mW; One-dimensional focusing mirror 2 is the plano-convex cylindrical lens, and its focal length is 20mm.Focal line AA is of a size of 20mm * 1mm, is slightly less than the window of interpretation as a result 21 sizes of test strips 3.Two bands on the test strips 3 are respectively in the interpretation window 21 as a result: from home indicating window one side is with 17 along 12.2mm place for detecting in the interpretation window 21 as a result, the 7.2mm place is quality control band 18.The up-converting phosphor material that adopts in the research is (YYbEr) F
3(yttrium fluoride ytterbium erbium), at the infrared ray excited phosphorescence spectrum that sends down as shown in figure 28, its main peak value wavelength is 541.5nm.The focal length of the preceding mirror group 5 in the phosphor pattern receiving light path is 40mm, object space aperture half-angle U2=20 °; The spectrum transmitting rate curve of optical filter 6 as shown in figure 29, in the transmitance at 541.5nm wavelength place greater than 90%, and in the transmitance at 980nm wavelength place less than 10
-5The focal length of back mirror group 7 is 40mm, so the enlargement ratio of phosphor pattern receiving light path is-1 times.Picture receiver 8 is linear array CCD cameras, and its sensitive area length is 22mm, contains 2200 pixels altogether, and Pixel Dimensions is 10 μ m * 10 μ m.So the space interpretation resolution of 8 pairs of test strips 3 of picture receiver is 10 μ m.
Most preferred embodiment is better than 1 μ g/L to the interpretation sensitivity of pure up-converting phosphor material, can detect the biological substance more than 4 kinds simultaneously.
Embodiment 2:Double-antibody sandwich detects hepatitis B surface antigen HBs-Ag:
(1) the immunochromatography liquid phase material is prepared:
The A.UCP-antibody conjugates:
A. utilize the finishing set up and activation method that the UCP particle of diameter 200-300nm is carried out finishing, and be connected with anti-hepatitis B surface antigen HBs-Ag monoclonal antibody, preserve liquid at UCP and (in the pH=7.2 0.03mol/L PB damping fluid, contain 0.1%BSA, 0.05%Tween20,0.02%NaN
3) in concentration 1mg/mL, 4 ℃ of preservations are standby;
B. will be stored in the 1mg/mL UCP-antibody conjugates 6mL in the UCP preservation liquid, 12000r/min, 4 ℃, centrifugal 30min abandons most supernatant as far as possible;
C. the UCP-antibody conjugates sedimentation in centrifuge tube adds 3mL bond dilution (in the pH=7.20.03mol/LPB damping fluid, containing 1% sucrose, 1%BSA) the abundant mixing of vortex (final concentration: 2mg/mL UCP-antibody conjugates)
D. suspension is poured in the reagent bottle, 4 ℃ of preservations are standby;
B. sample pad confining liquid:
A. with the accurate weighing BSA of balance (bovine serum albumin(BSA)) 1g, put into small beaker;
B. add pH=7.2 0.03mol/L PB damping fluid 20mL in the beaker, glass bar stirs abundant mixing;
Add Tween 20 (polysorbas20) 20 μ L in the c.BAS solution, glass bar stirs abundant mixing (final concentration: 5%BAS, 0.1% Tween 20);
D.4 ℃ preservation is standby;
C. detect band albumen:
A. with the anti-HBs-Ag polyclonal antibody of the rabbit 2mg of the accurate weighing purifying of balance, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
D. quality control band albumen;
A. with the sheep anti-mouse igg antibody 2mg of the accurate weighing purifying of balance, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
(2) the immunochromatography solid support material is prepared:
A. sample pad:
A. select for use cellulose membrane (Cellulose Membrane) as the sample pad solid phase material, it is cut into the band of 1.5 * 30.0cm specification;
B. sample pad is put into microscler plate, the sample pad confining liquid adds thereon, soak at room temperature 30min;
C. with sample pad by taking out in the confining liquid, be put in the clean plate;
D.37 ℃ oven dry is 3 hours, makes sample pad fully dry;
E. the sample pad after the sealing is preserved standby in the environment of drying;
B. pad:
A. select for use the plain film of glass fibre (Glass Fiber) as the pad solid phase material, it is cut into the band of 1.0 * 30.0cm specification;
B. preserve the standby ultrasonic 10s of 2mg/mL UCP-antibody conjugates (pH=7.2 0.03mol/L PB damping fluid contains 1% sucrose, 1%BSA) with 4 ℃;
C. pad is put into microscler plate, UCP-antibody conjugates suspension adds thereon;
D. pad is taken out and be put in the clean plate;
E.37 ℃ oven dry is 2.5 hours, makes pad fully dry;
F. the pad after handling is preserved standby in the environment of drying;
C. analyzing film:
A. with the aperture be the nitrocellulose filter (Nitrocellulose Membrane) of 12 μ m as solid phase material, it is cut into the band of 2.5 * 30cm specification;
B. use point sample instrument on the wide analyzing film of 2.5cm, the anti-HBs-Ag polyclonal antibody of the specking 2mg/mL of 1cm place rabbit from bottom to top, 2 μ L/cm are as detecting band;
C. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1.5cm place sheep anti-mouse igg antibody from bottom to top, 2 μ L/cm are as quality control band;
D.37 ℃ oven dry is 2 hours, makes analyzing film fully dry;
E. the analyzing film behind the point sample is preserved standby in the environment of drying;
D. adsorptive pads:
A. select for use cellulose membrane (Cellulose Membrane) as the adsorptive pads solid phase material, it is cut into the band of 3.0 * 30cm specification;
B. the accurate pH test paper with color change interval 5.5-9.0 is fixed in adsorptive pads 2.0cm place from bottom to top, as the terminal point index strip;
C. adsorptive pads is preserved standby in the environment of drying;
(3) immuno-chromatographic test paper strip detects
A. with blood serum sample to be detected 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid;
B.100 the sample after the μ L dilution is added in the well on the test strips shell;
C. after treating that terminal point index strip in the terminal point indication window becomes green, just can be with detection band and the quality control band in the interpretation window as a result on the sensor interpretation shell, to obtain a result.
Embodiment 3:Double-antibody sandwich detects coronavirus (SARS virus):
(1) the immunochromatography liquid phase material is prepared:
The A.UCP-antibody conjugates:
A. utilize the finishing set up and activation method that the UCP particle of diameter 200-300nm is carried out finishing, and be connected with the rabbit anti-SARS virus antibody of purifying, preserve liquid at UCP and (in the pH=7.2 0.03mol/L PB damping fluid, contain 0.1%BSA, 0.05%Tween20,0.02%NaN
3) in concentration 1mg/mL, 4 ℃ of preservations are standby;
B. will be stored in the 1mg/mL UCP-antibody conjugates 6mL in the UCP preservation liquid, 12000r/min, 4 ℃, centrifugal 30min abandons most supernatant as far as possible;
C. the UCP-antibody conjugates sedimentation in centrifuge tube adds 3mL bond dilution (in the pH=7.2 0.03mol/LPB damping fluid, containing 1% sucrose, 1%BSA) the abundant mixing of vortex (final concentration: 2mg/mL UCP-antibody conjugates)
D. suspension is poured in the reagent bottle, 4 ℃ of preservations are standby;
B. sample pad confining liquid:
A. with the accurate weighing BSA of balance (bovine serum albumin(BSA)) 1g, put into small beaker;
B. add pH=7.2 0.03mol/L PB damping fluid 20mL in the beaker, glass bar stirs abundant mixing;
Add Tween 20 (polysorbas20) 20 μ L in the c.BAS solution, glass bar stirs abundant mixing (final concentration: 5%BAS, 0.1%Tween 20);
D.4 ℃ preservation is standby;
C. detect band albumen:
A. with the goat-anti SARS virus antibody 2mg of the accurate weighing purifying of balance, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
D. quality control band albumen;
A. with the goat anti-rabbit igg antibody 2mg of the accurate weighing purifying of balance, place the 1.5mL micro-centrifuge tube;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
(2) the immunochromatography solid support material is prepared:
A. sample pad:
A. select for use cellulose membrane (Cellulose Membrane) as the sample pad solid phase material, it is cut into the band of 1.5 * 30.0cm specification;
B. sample pad is put into microscler plate, the sample pad confining liquid adds thereon, soak at room temperature 30min;
C. with sample pad by taking out in the confining liquid, be put in the clean plate;
D.37 ℃ oven dry is 3 hours, makes sample pad fully dry;
E. the sample pad after the sealing is preserved standby in the environment of drying;
B. pad:
A. select for use the plain film of glass fibre (Glass Fiber) as the pad solid phase material, it is cut into the band of 1.0 * 30.0cm specification;
B. preserve the standby ultrasonic 10s of 2mg/mL UCP-antibody conjugates (pH=7.2 0.03mol/L PB damping fluid contains 1% sucrose, 1%BSA) with 4 ℃;
C. pad is put into microscler plate, UCP-antibody conjugates suspension adds thereon;
D. pad is taken out and be put in the clean plate;
E.37 ℃ oven dry is 2.5 hours, makes pad fully dry;
F. the pad after handling is preserved standby in the environment of drying;
C. analyzing film:
A. with the aperture be the nitrocellulose filter (Nitrocellulose Membrane) of 12 μ m as solid phase material, it is cut into the band of 2.5 * 30cm specification;
B. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1cm place goat-anti SARS virus antibody from bottom to top, 2 μ L/cm are as detecting band;
C. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1.5cm place goat anti-rabbit igg antibody from bottom to top, 2 μ L/cm are as quality control band;
D.37 ℃ oven dry is 2 hours, makes analyzing film fully dry;
E. the analyzing film behind the point sample is preserved standby in the environment of drying;
D. adsorptive pads:
A. select for use cellulose membrane (Cellulose Membrane) as the adsorptive pads solid phase material, it is cut into the band of 3.0 * 30cm specification;
B. the accurate pH test paper with color change interval 5.5-9.0 is fixed in adsorptive pads 2.0cm place from bottom to top, as the terminal point index strip;
C. adsorptive pads is preserved standby in the environment of drying;
(3) immuno-chromatographic test paper strip detects:
A. with blood serum sample to be detected 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid;
B.100 the sample after the μ L dilution is added in the well on the test strips shell;
C. after treating that terminal point index strip in the terminal point indication window becomes green, just can be with detection band and the quality control band in the interpretation window as a result on the sensor interpretation shell, to obtain a result.
Embodiment 4:Plague FI-Ag (plague FI-antigen) detects:
(1) the immunochromatography liquid phase material is prepared:
The A.UCP-antibody conjugates:
A. utilize the finishing set up and activation method that the UCP particle of diameter 200-300nm is carried out finishing, and be connected with the anti-plague antibodies of the rabbit of purifying, preserve liquid at UCP and (in the pH=7.2 0.03mol/L PB damping fluid, contain 0.1%BSA, 0.05%Tween20,0.02%NaN
3) in concentration 1mg/mL, 4 ℃ of preservations are standby;
B. will be stored in the 1mg/mL UCP-antibody conjugates 6mL in the UCP preservation liquid, 12000r/min, 4 ℃, centrifugal 30min abandons most supernatant as far as possible;
C. the UCP-antibody conjugates sedimentation in centrifuge tube adds 3mL bond dilution (in the pH=7.2 0.03mol/LPB damping fluid, containing 1% sucrose, 1%BSA) the abundant mixing of vortex (final concentration: 2mg/mL UCP-antibody conjugates)
D. suspension is poured in the reagent bottle, 4 ℃ of preservations are standby;
B. sample pad confining liquid:
A. with the accurate weighing BSA of balance (bovine serum albumin(BSA)) 1g, put into small beaker;
B. add pH=7.2 0.03mol/L PB damping fluid 20mL in the beaker, glass bar stirs abundant mixing;
Add Tween 20 (polysorbas20) 20 μ L in the c.BAS solution, glass bar stirs abundant mixing (final concentration: 5%BAS, 0.1%Tween 20);
D.4 ℃ preservation is standby;
C. detect band albumen:
A. with the accurate weighing plague of balance FI-antigen 2mg, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
D. quality control band albumen:
A. with the goat anti-rabbit igg antibody 2mg of the accurate weighing purifying of balance, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
(2) the immunochromatography solid support material is prepared:
A. sample pad:
A. select for use cellulose membrane (Cellulose Membrane) as the sample pad solid phase material, it is cut into the band of 1.5 * 30.0cm specification;
B. sample pad is put into microscler plate, the sample pad confining liquid adds thereon, soak at room temperature 30min;
C. with sample pad by taking out in the confining liquid, be put in the clean plate;
D.37 ℃ oven dry is 3 hours, makes sample pad fully dry;
E. the sample pad after the sealing is preserved standby in the environment of drying;
B. pad:
A. select for use the plain film of glass fibre (Glass Fiber) as the pad solid phase material, it is cut into the band of 1.0 * 30.0cm specification;
B. preserve the standby ultrasonic 10s of 2mg/mL UCP-antibody conjugates (pH=7.2 0.03mol/L PB damping fluid contains 1% sucrose, 1%BSA) with 4 ℃;
C. pad is put into microscler plate, UCP-antibody conjugates suspension adds thereon;
D. pad is taken out and be put in the clean plate;
E.37 ℃ oven dry is 2.5 hours, makes pad fully dry;
F. the pad after handling is preserved standby in the environment of drying;
C. analyzing film:
A. with the aperture be the nitrocellulose filter (Nitrocellulose Membrane) of 12 μ m as solid phase material, it is cut into the band of 2.5 * 30cm specification;
B. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1cm place plague FI-antigen from bottom to top, 2 μ L/cm are as detecting band;
C. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1.5cm place goat anti-rabbit igg antibody from bottom to top, 2 μ L/cm are as quality control band;
D.37 ℃ oven dry is 2 hours, makes film fully dry;
E. the analyzing film behind the point sample is preserved standby in the environment of drying;
D. adsorptive pads:
A. select for use cellulose membrane (Cellulose Membrane) as the adsorptive pads solid phase material, it is cut into the band of 3.0 * 30cm specification;
B. the accurate pH test paper with color change interval 5.5-9.0 is fixed in adsorptive pads 2.0cm place from bottom to top, as the terminal point index strip;
D. adsorptive pads is preserved standby in the environment of drying;
(3) immuno-chromatographic test paper strip detects:
A. with detected sample 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid;
B.100 the sample after the μ L dilution is added in the well on the test strips shell;
C. after treating that terminal point index strip in the terminal point indication window becomes green, just can be with detection band and the quality control band in the interpretation window as a result on the sensor interpretation shell, to obtain a result.
Embodiment 5:Illegal drug detects
(1) the immunochromatography liquid phase material is prepared:
The A.UCP-antibody conjugates:
A. utilize the finishing set up and activation method that the UCP particle of diameter 200-300nm is carried out finishing, and with the anti-illegal drug antibody of the rabbit of purifying or in conjunction with aglucon (illegal drug comprises: drug molecules such as amphetamine, methyl amphetamine, Hog and laudanum) be connected, preserve liquid at UCP and (in the pH=7.2 0.03mol/L PB damping fluid, contain 0.1%BSA, 0.05%Tween20,0.02%NaN
3) in concentration 1mg/mL, 4 ℃ of preservations are standby;
B. will be stored in the 1mg/mL UCP-antibody conjugates 6mL in the UCP preservation liquid, 12000r/min, 4 ℃, centrifugal 30min abandons most supernatant as far as possible;
C. the UCP-antibody conjugates sedimentation in centrifuge tube adds 3mL bond dilution (in the pH=7.2 0.03mol/LPB damping fluid, containing 1% sucrose, 1%BSA) the abundant mixing of vortex (final concentration: 2mg/mL UCP-antibody conjugates)
D. suspension is poured in the reagent bottle, 4 ℃ of preservations are standby;
B. sample pad confining liquid:
A. with the accurate weighing BSA of balance (bovine serum albumin(BSA)) 1g, put into small beaker;
B. add pH=7.2 0.03mol/L PB damping fluid 20mL in the beaker, glass bar stirs abundant mixing;
Add Tween 20 (polysorbas20) 20 μ L in the c.BAS solution, glass bar stirs abundant mixing (final concentration: 5%BAS, 0.1%Tween 20);
D.4 ℃ preservation is standby;
C. detect band albumen:
A. with the accurate weighing BSA-of balance illegal drug molecular complex 2mg, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
D. quality control band albumen:
A. with the goat anti-rabbit igg antibody 2mg of the accurate weighing purifying of balance, place the 1.5mL micro-centrifuge tube;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing (final concentration: 2mg/mL) of vortex;
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
(2) the immunochromatography solid support material is prepared:
A. sample pad:
A. select for use cellulose membrane (Cellulose Membrane) as the sample pad solid phase material, it is cut into the band of 1.5 * 30.0cm specification;
B. sample pad is put into microscler plate, the sample pad confining liquid adds thereon, soak at room temperature 30min;
C. with sample pad by taking out in the confining liquid, be put in the clean plate;
D.37 ℃ oven dry is 3 hours, makes sample pad fully dry;
E. the sample pad after the sealing is preserved standby in the environment of drying;
B. pad:
A. select for use the plain film of glass fibre (Glass Fiber) as the pad solid phase material, it is cut into the band of 1.0 * 30.0cm specification;
B. preserve the standby ultrasonic 10s of 2mg/mL UCP-antibody conjugates (pH=7.2 0.03mol/L PB damping fluid contains 1% sucrose, 1%BSA) with 4 ℃;
C. pad is put into microscler plate, UCP-antibody conjugates suspension adds thereon;
D. pad is taken out and be put in the clean plate;
E.37 ℃ oven dry is 2.5 hours, makes pad fully dry;
F. the pad after handling is preserved standby in the environment of drying;
C. analyzing film:
A. with the aperture be the nitrocellulose filter (Nitrocellulose Membrane) of 12 μ m as solid phase material, it is cut into the band of 2.5 * 30cm specification;
B. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1cm place BSA-illegal drug molecular complex from bottom to top, 2 μ L/cm are as detecting band;
C. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1.5cm place goat anti-rabbit igg antibody from bottom to top, 2 μ L/cm are as quality control band;
D.37 ℃ oven dry is 2 hours, makes analyzing film fully dry;
E. the analyzing film behind the point sample is preserved standby in the environment of drying;
D. adsorptive pads:
A. select for use cellulose membrane (Cellulose Membrane) as the adsorptive pads solid phase material, it is cut into the band of 3.0 * 30cm specification;
B. the accurate pH test paper with color change interval 5.5-9.0 is fixed in adsorptive pads 2.0cm place from bottom to top, as the terminal point index strip;
C. adsorptive pads is preserved standby in the environment of drying;
(3) immuno-chromatographic test paper strip detects:
A. with detected sample 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid;
B.100 the sample after the μ L dilution is added in the well on the test strips shell;
C. after treating that terminal point index strip in the terminal point indication window becomes green, just can be with detection band and the quality control band in the interpretation window as a result on the sensor interpretation shell, to obtain a result.
Embodiment 6:The plague infects antibody test:
(1) the immunochromatography liquid phase material is prepared:
The A.UCP-SPA bond:
A. utilize the finishing set up and activation method that the UCP particle of diameter 200-300nm is carried out finishing, and be connected with SPA (staphylococcal protein A), preserve liquid at UCP and (in the pH=7.2 0.03mol/L PB damping fluid, contain 0.1%BSA, 0.05%Tween20,0.02%NaN
3) in concentration 1mg/mL, 4 ℃ of preservations are standby;
B. will be stored in the 1mg/mL UCP-SPA bond 6mL in the UCP preservation liquid, 12000r/min, 4 ℃, centrifugal 30min abandons most supernatant as far as possible;
C. the UCP-SPA bond sedimentation in centrifuge tube adds 3mL bond dilution (in the pH=7.2 0.03mol/LPB damping fluid, containing 1% sucrose, 1%BSA) the abundant mixing of vortex (final concentration: 2mg/mL UCP-SPA bond)
D. suspension is poured in the reagent bottle, 4 ℃ of preservations are standby;
B. sample pad confining liquid:
A. with the accurate weighing BSA of balance (bovine serum albumin(BSA)) 1g, put into small beaker;
B. add pH=7.2 0.03mol/L PB damping fluid 20mL in the beaker, glass bar stirs abundant mixing;
Add Tween 20 (polysorbas20) 20 μ L in the c.BAS solution, glass bar stirs abundant mixing (final concentration: 5%BAS, 0.1%Tween 20);
D.4 ℃ preservation is standby;
C. detect band albumen:
A. with the accurate weighing plague of balance FI-Ag (plague FI-Ag) 2mg, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing of vortex (final concentration: 2mg/mL plague FI-Ag);
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
D. quality control band albumen:
A. with the accurate weighing sheep of balance IgG 2mg, place the micro-centrifuge tube of 1mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing of vortex (final concentration: 2mg/mL sheep IgG);
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
(2) the immunochromatography solid support material is prepared:
A. sample pad:
A. select for use cellulose membrane (Cellulose Membrane) as the sample pad solid phase material, it is cut into the band of 1.5 * 30.0cm specification;
B. sample pad is put into microscler plate, the sample pad confining liquid adds thereon, soak at room temperature 30min;
C. with sample pad by taking out in the confining liquid, be put in the clean plate;
D.37 ℃ oven dry is 3 hours, makes sample pad fully dry;
E. the sample pad after the sealing is preserved standby in the environment of drying;
B. pad:
A. select for use the plain film of glass fibre (Glass Fiber) as the pad solid phase material, it is cut into the band of 1.0 * 30.0cm specification;
B. preserve the standby ultrasonic 10s of 2mg/mL UCP-SPA (pH=7.2 0.03mol/L PB damping fluid contains 1% sucrose, 1%BSA) with 4 ℃;
C. pad is put into microscler plate, UCP-SPA bond suspension adds thereon;
D. pad is taken out and be put in the clean plate;
E.37 ℃ oven dry is 2.5 hours, makes pad fully dry;
F. the pad after handling is preserved standby in the environment of drying;
C. analyzing film:
A. with the aperture be the nitrocellulose filter (Nitrocellulose Membrane) of 12 μ m as solid phase material, it is cut into the band of 2.5 * 30cm specification;
B. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1cm place pestis F 1-Ag from bottom to top, 2 μ L/cm are as detecting band;
C. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1.5cm place sheep IgG from bottom to top, 2 μ L/cm are as quality control band;
D.37 ℃ oven dry is 2 hours, makes analyzing film fully dry;
E. the analyzing film behind the point sample is preserved standby in the environment of drying;
D. adsorptive pads:
A. select for use cellulose membrane (Cellulose Membrane) as the adsorptive pads solid phase material, it is cut into the band of 3.0 * 30cm specification;
B. the accurate pH test paper with color change interval 5.5-9.0 is fixed in adsorptive pads 2.0cm place from bottom to top, as the terminal point index strip;
C. adsorptive pads is preserved standby in the environment of drying;
(3) immuno-chromatographic test paper strip detects:
A. blood serum sample to be detected 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid;
B.100 the sample after the μ L dilution is added in the well on the test strips shell;
C. after treating that terminal point index strip in the terminal point indication window becomes green, just can be with detection band and the quality control band in the interpretation window as a result on the sensor interpretation shell, to obtain a result.
Embodiment 7:SARS virus infects antibody test:
(1) the immunochromatography liquid phase material is prepared:
The A.UCP-SPA bond:
A. utilize the finishing set up and activation method that the UCP particle of diameter 200-300nm is carried out finishing, and be connected with SPA (staphylococcal protein A), preserve liquid at UCP and (in the pH=7.2 0.03mol/L PB damping fluid, contain 0.1%BSA, 0.05%Tween20,0.02%NaN
3) in concentration 1mg/mL, 4 ℃ of preservations are standby;
B. will be stored in the 1mg/mL UCP-SPA bond 6mL in the UCP preservation liquid, 12000r/min, 4 ℃, centrifugal 30min abandons most supernatant as far as possible;
C. the UCP-SPA bond sedimentation in centrifuge tube adds 3mL bond dilution (in the pH=7.2 0.03mol/LPB damping fluid, containing 1% sucrose, 1%BSA) the abundant mixing of vortex (final concentration: 2mg/mL UCP-SPA bond)
D. suspension is poured in the reagent bottle, 4 ℃ of preservations are standby;
B. sample pad confining liquid:
A. with the accurate weighing BSA of balance (bovine serum albumin(BSA)) 1g, put into small beaker;
B. add pH=7.2 0.03mol/L PB damping fluid 20mL in the beaker, glass bar stirs abundant mixing;
Add Tween 20 (polysorbas20) 20 μ L in the c.BAS solution, glass bar stirs abundant mixing (final concentration: 5%BAS, 0.1%Tween 20);
D.4 ℃ preservation is standby;
C. detect band albumen:
A. with the SARS virus surface N albumen 2mg of the accurate weighing purifying of balance, place the micro-centrifuge tube of 1.5mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing of vortex (final concentration: 2mg/mL SARS virus surface N antigen);
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
D. quality control band albumen:
A. with the sheep IgG 2mg of the accurate weighing purifying of balance, place the micro-centrifuge tube of 1mL;
B. add 1mL pH=7.2 0.03mol/L PB damping fluid in the micro-centrifuge tube, the abundant mixing of vortex (final concentration: 2mg/mL sheep IgG);
C. be packed as the every pipe of 50 μ L ,-20 ℃ frozen standby;
(2) the immunochromatography solid support material is prepared:
A. sample pad:
A. select for use cellulose membrane (Cellulose Membrane) as the sample pad solid phase material, it is cut into the band of 1.5 * 30.0cm specification;
B. sample pad is put into microscler plate, the sample pad confining liquid adds thereon, soak at room temperature 30min;
C. with sample pad by taking out in the confining liquid, be put in the clean plate;
D.37 ℃ oven dry is 3 hours, makes sample pad fully dry;
E. the sample pad after the sealing is preserved standby in the environment of drying;
B. pad:
A. select for use the plain film of glass fibre (Glass Fiber) as the pad solid phase material, it is cut into the band of 1.0 * 30.0cm specification;
B. preserve the standby ultrasonic 10s of 2mg/mL UCP-SPA bond (pH=7.2 0.03mol/L PB damping fluid contains 1% sucrose, 1%BSA) with 4 ℃;
C. pad is put into microscler plate, UCP-SPA bond suspension adds thereon;
D. pad is taken out and be put in the clean plate;
E.37 ℃ oven dry is 2.5 hours, makes pad fully dry;
F. the pad after handling is preserved standby in the environment of drying;
C. analyzing film:
A. with the aperture be the nitrocellulose filter (Nitrocellulose Membrane) of 12 μ m as solid phase material, it is cut into the band of 2.5 * 30cm specification;
B. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1cm place SARS virus surface N albumen from bottom to top, 2 μ L/cm are as detecting band;
C. use point sample instrument on the wide analyzing film of 2.5cm, the specking 2mg/mL of 1.5cm place sheep IgG from bottom to top, 2 μ L/cm are as quality control band;
D.37 ℃ oven dry is 2 hours, makes analyzing film fully dry;
E. the analyzing film behind the point sample is preserved standby in the environment of drying;
D. adsorptive pads:
A. select for use cellulose membrane (Cellulose Membrane) as the adsorptive pads solid phase material, it is cut into the band of 3.0 * 30cm specification;
B. the accurate pH test paper with color change interval 5.5-9.0 is fixed in adsorptive pads 2.0cm place from bottom to top, as the terminal point index strip;
C. adsorptive pads is preserved standby in the environment of drying;
(3) immuno-chromatographic test paper strip detects:
A. with blood serum sample to be detected 10 times of dilutions of pH=7.2 0.03mol/L PB damping fluid;
B.100 the sample after the μ L dilution is added in the well on the test strips shell;
C. after treating that terminal point index strip in the terminal point indication window becomes green, just can be with detection band and the quality control band in the interpretation window as a result on the sensor interpretation shell, to obtain a result.
The foregoing description helps to understand the present invention, but is not limitation of the present invention.Those of ordinary skill in the art can make suitable modification and change to the present invention according to the foregoing description, all belongs to protection scope of the present invention.
Claims (2)
1. preparation method based on the immune chromatography test paper of up-converting phosphor technology is characterized in that this method may further comprise the steps:
The preparation of UCP-bioactive molecule: the UCP particle is carried out finishing, be connected, preserve in the liquid at UCP and preserve with bioactive molecule; Get the UCP-bioactive molecule bond in a certain amount of UCP of being stored in preservation liquid, centrifugal, and abandoning supernatant; In the UCP of sedimentation bioactive molecule bond, add dilution, and abundant mixing, be prepared into suspension; The preparation of sample pad: select for use cellulose membrane as the sample pad solid phase material, cut into band with certain specification; Sample pad is put into the sample pad confining liquid soaks; Then sample pad is taken out from confining liquid, and oven dry, make sample pad fully dry; The preparation of pad: select for use the plain film of glass fibre as the pad solid phase material, cut into band with certain specification; On this band, add the suspension of UCP-bioactive molecule bond; Dry this band, make pad fully dry; The preparation of analyzing film: select for use nitrocellulose filter as the analyzing film solid phase material, cut into band with certain specification; Diverse location specking bioactive molecule on band is made and is detected band and quality control band; Dry this band, make analyzing film fully dry; Assemble this test strips: the analyzing film that will handle is pasted on the plastic back plate, and quality control band upwards detects band downwards; The pad of handling is pasted on the plastic back plate, and the upper end is pressed on the lower end of analyzing film, and overlapped; The sample pad of handling is pasted on the plastic back plate, and the lower end is concordant with plastic back plate, and the upper end is pressed on the lower end of pad, and overlapped; Adsorptive pads is pasted on the plastic back plate, and the upper end is concordant with plastic back plate, and the lower end is pressed on the upper end of analyzing film, and overlapped with analyzing film; The band of assembling shaping is cut into the test strips of certain specification; Wherein, it is the pH=7.20.03mol/L phosphate buffer that described UCP preserves liquid, contains 0.1%BSA, 0.05%Tween20,0.02%NaN
3The dilution of described UCP bioactive molecule is the pH=7.20.03mol/L phosphate buffer, contains 1% sucrose, 1%BSA; Described sample pad confining liquid is the pH=7.20.03mol/L phosphate buffer, contains 5%BSA, and 0.1%Tween 20.
2. the preparation method of immune chromatography test paper as claimed in claim 1 is characterized in that this method is further comprising the steps of: the accurate pH test paper of color change interval 5.5-9.0 is affixed on adsorptive pads as the terminal point index strip.
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| US6399397B1 (en) * | 1992-09-14 | 2002-06-04 | Sri International | Up-converting reporters for biological and other assays using laser excitation techniques |
| CN1425918A (en) * | 2003-01-14 | 2003-06-25 | 江苏省血吸虫病防治研究所 | Quick test paper strip diagnostic reagent kits for bilharziasis and its preparing method and use |
| CN1438486A (en) * | 2002-12-31 | 2003-08-27 | 江西中德生物工程有限公司 | Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and use |
-
2004
- 2004-04-23 CN CN 200410034104 patent/CN1690711B/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6399397B1 (en) * | 1992-09-14 | 2002-06-04 | Sri International | Up-converting reporters for biological and other assays using laser excitation techniques |
| CN2486973Y (en) * | 2001-06-20 | 2002-04-17 | 陈岩松 | Multiple combined fast detection reagent box |
| CN1438486A (en) * | 2002-12-31 | 2003-08-27 | 江西中德生物工程有限公司 | Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and use |
| CN1425918A (en) * | 2003-01-14 | 2003-06-25 | 江苏省血吸虫病防治研究所 | Quick test paper strip diagnostic reagent kits for bilharziasis and its preparing method and use |
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| CN1690711A (en) | 2005-11-02 |
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