CN102680692B - Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker - Google Patents
Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker Download PDFInfo
- Publication number
- CN102680692B CN102680692B CN201210152948.XA CN201210152948A CN102680692B CN 102680692 B CN102680692 B CN 102680692B CN 201210152948 A CN201210152948 A CN 201210152948A CN 102680692 B CN102680692 B CN 102680692B
- Authority
- CN
- China
- Prior art keywords
- antibody
- antigen
- infrared fluorescent
- test paper
- pad
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention relates to an immunochromatographic quantitative test reagent based on a near-infrared fluorescent marker. A near-infrared fluorescent molecule is adopted as a marker, and the immunochromatographic technology is adopted to prepare a near-infrared fluorescent immunochromatographic test strip. In the test process, a conventional near-infrared test instrument is used for respectively scanning a quality control line and a sample line through near-infrared rays, and after being corrected by utilizing the fluorescent strength of the quality control line, the fluorescent strength of a detection line is substituted in a standard curved line in a fluorescent analysis instrument, so that the concentration of an object to be tested in a test specimen can be analyzed. The reagent can be applied to the detection of microorganism, the food safety detection, the drug detection and the quick detection of dangerous chemicals. The immunochromatographic quantitative test reagent has characteristics of high sensitivity, accuracy in quantization and convenience in operation.
Description
Technical field
The present invention has set up a kind of immunochromatography quantitative detecting reagent based on near-infrared fluorescent label.Comprise the preparation method of near-infrared fluorescent test strip and corresponding reagent.The present invention adopts near-infrared fluorescent molecule to serve as a mark, adopt immunochromatography technique, prepare near-infrared fluorescent immuno-chromatographic test paper strip, then form the test card that comprises sample pad/glass fibre membrane/nitrocellulose filter and thieving paper, wherein on nitrocellulose filter, be fixed with detection line and nature controlling line.In testing process, adopt near infrared light to scan respectively nature controlling line and sample wire, fluorescence excitation mulecular luminescence, the fluorescence of launching is through optical filter, convert digital signal to through photomultiplier, with the typical curve in substitution fluorescence analyser after nature controlling line fluorescence intensity correct detection line fluorescence intensity, get final product the concentration of the determinand in analyzing and testing sample.This system can be applied in microorganism detection, food safety detection, illicit drugs inspection and hazardous chemical fast detecting.Highly sensitive, quantitatively accurate easy to operate feature that the present invention has.
Background technology
Immunochromatographic method is widely used in fields such as disease quick diagnosis, little Molecular Detection.Its principle is a certain district band that special antigen or antibody is first fixed on to nitrocellulose membrane, when this dry nitrocellulose filter one end is immersed after sample, due to capillarity, sample will move forward along this film, in the time moving to the region that is fixed with antibody, in sample corresponding antigen with this antibody generation specific binding, if can make this region show certain color with immune colloid gold, thereby realize specific immunodiagnosis.Conventional label is colloid gold particle, enzyme and painted microballoon.Colloid gold particle and painted microballoon be by Electrostatic Absorption by antigen or antibody labeling at particle surface, by observing colloid gold grain or microballoon, the gathering colour developing on detection line judges testing result.Enzyme labeling develops the color by catalytic substrate, shows testing result.Above labelling technique exists sensitivity low, shortcoming that can not accurate quantification.Limit immunochromatography range of application.
The features such as fluorescence labeling technology has highly sensitive, simple to operate, are widely used in field of biological detection.As determined dna sequence, protein expression analysis, clinical diagnosis etc.Conventional fluorescent marker spectral range is in visible region (400-750nm) mostly.Because albumen, the nucleic acid etc. of biosome can produce fluorescence under the exciting of ultraviolet light, therefore, use the fluorescent marker of visible region can produce stronger background fluorescence, Interference Detection signal, has reduced the sensitivity detecting.The emission wavelength ranges of near-infrared fluorescent material is at 650-1000nm, within the scope of this, biosome autofluorescence a little less than, therefore, use near-infrared fluorescent material as detecting fluorescent marker, there is low background fluorescence, detection signal-to-noise ratio is high, the feature that detection sensitivity is high.
Summary of the invention
The present invention has overcome with colloid gold particle low, the quantitative inaccurate problem of thing immunochromatography reagent sensitivity that serves as a mark, set up a kind of immunochromatography quantitative detecting reagent based on near-infrared fluorescent label, and new detection method, the preparation method of described quantitative detecting reagent bag test strip and test strips.Its principle of work is: in the sample pad of immuno-chromatographic test paper strip, be fixed with near-infrared fluorescent mark object of reference and can with the antigen of detected material specific binding or antibody, be added drop-wise in sample pad when detecting sample, the antigen of near-infrared fluorescent mark or antibody and detected material form compound, upwards spring up under capillary action with object of reference, when through detection line and nature controlling line, be fixed on respectively two molecules on line and catch.Read respectively the fluorescence intensity of line of reference and detection line with fluorescent scanning instrument, by both fluorescence intensity substitution formula, can obtain the concentration of thing to be detected in sample.
The method applied in the present invention is:
In the time using sandwich method to detect target molecule (can be double antibody sandwich method detectable antigens, can be also dual anti-former detection antibody); On the detection line of immune chromatography test paper fixing be with target molecule can specific binding antigen or antibody (double antibody sandwich method is antibody, dual-antigen sandwich method is antigen), on nature controlling line as object of reference be and the antibody of target molecule and the fixing irrelevant molecule of detection line, the normally anti-chicken IgY of goat polyclonal antibody.Antibody or the antigen of two or more near-infrared fluorescent marks on pad, have been fixed respectively with spraying process, a kind of antigen or antibody that is and detects target specific binding, another be can with antigen or the antibody of object of reference specific binding on nature controlling line, be generally chicken IgY.In the time that the sample drop that contains target molecule is added in sample pad, solution dissociates in connection with two kinds of compositions on pad, and target molecule forms antigen antibody complex with the specific binding molecules that dissociates out.Antigen antibody complex and object of reference move to test strips upper end with solution.In the time that antigen antibody complex moves to detection line, the target molecule specific binding molecules being fixed on detection line is caught.Quality Control molecule continues mobile with solution, in the time moving to nature controlling line, the object of reference antibody being fixed on nature controlling line is caught.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with fluorescent scanning instrument.Because the amount of object of reference on pad is a definite value, object of reference monoclonal antibody can be combined with detecting target molecule and antibody simultaneously, and therefore, the fluorescence intensity of object of reference can be used as the internal reference that detects reagent.By calculating the ratio of detection line and nature controlling line fluorescence intensity, and calculate with standard working curve, just can draw the concentration of target molecule in sample;
In the time using indirect method to detect target molecule: what fix on the detection line at immune chromatography test paper is detectable antigens, what on nature controlling line, fix is object of reference antibody, is generally the anti-chicken IgY of goat polyclonal antibody.On pad, having fixed respectively with spraying process the biomolecule of two kinds of near-infrared fluorescent marks, is respectively second antibody and the chicken IgY of mouse anti human antibody.After the sample drop that contains target molecule is added in sample pad, drip dilution simultaneously, solution dissociates in connection with the biomacromolecule on pad, the second antibody of near-infrared fluorescent mark is combined with people's antibody, form antibody complex, under the promotion of solution, antibody complex and chicken IgY move to test strips upper end with solution.In the time that antibody complex moves to detection line, the antigen that antibody complex is fixed on detection line is caught.Chicken IgY continues mobile with solution, in the time moving to nature controlling line, the anti-chicken IgY of the goat antibody being fixed on nature controlling line is caught.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with fluorescent scanning instrument.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with fluorescent scanning instrument.By calculating the ratio of detection line and nature controlling line fluorescence intensity, and compare with predefined threshold value, whether contain HIV antibody in just can drawing in sample.
When using competition law to detect the little point of period of the day from 11 p.m. to 1 a.m: what fix on the detection line at immune chromatography test paper is little molecule-BSA conjugate, and what fix on nature controlling line is the anti-chicken IgY of the goat polyclonal antibody as reference.On pad, having fixed respectively with spraying process the monoclonal antibody of two kinds of Infrared fluorescence marks, is respectively anti-little molecule monoclonal antibody and chicken IgY.When detection, first drip sample to sample pad, then drip dilution.Solution dissociates in connection with the biomacromolecule on pad, if sample exists the certain density little point of period of the day from 11 p.m. to 1 a.m, whole fluorescently-labeled mouse monoclonal antibodies are combined, and the compound that little molecule is combined with antibody moves to test strips upper end under the promotion of solution.In the time that antigen antibody complex moves to detection line, the little molecular complex of BSA-that anti-little molecule monoclonal antibody can not be fixed on detection line is caught.Chicken IgY continues mobile with solution, in the time moving to nature controlling line, the anti-chicken IgY of the goat antibody being fixed on nature controlling line is caught.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with fluorescent scanning instrument.By calculating the ratio of detection line and nature controlling line fluorescence intensity, and compare with predefined threshold value, just can draw in sample, whether to contain little molecule to be detected.
The fluorescent scanning instrument using in the application is conventional experiment scanner, for example Beijing profit Bo Fu get development in science and technology company limited portable high sensitivity near infrared point fluorescent scanning instrument;
The near-infrared fluorescent marker material using in the application, for emission wavelength is at the dyestuff of 650-1000nm, is preferably the near infrared fluorescent dye Dylight800 of NHS activation.
This detection method tool compared with other detection methods has the following advantages:
Biological background signal is low, and detection sensitivity is high.Compared with colloidal gold immunochromatographimethod reagent, based on near-infrared fluorescent system, the sensitivity of detected molecule is improved to approximately 10 times.If haemoglobin minimum detectability is 10ng/ml, be generally 100ng/ml and detect reagent minimum detectability based on the haemoglobin of collaurum.While using indirect method to detect HIV specific antibody, will detect sample serial dilution, near-infrared fluorescent system detection sensitivity is higher 10 times than colloidal gold immunochromatographimethod equally.
Simple to operate.This method running program and traditional immune chromatography method are similar, report that from being loaded onto to read operation steps is few, and operating personnel are easy to grasp.
Can quantitatively detect.Traditional immune chromatography method is the buildup effect sentence read result by gold grain.There is certain quantitative relation in the concentration in gathering and the sample of gold grain, but can only carry out sxemiquantitative by the painted depth of observation gold grain.This method is marked at fluorescence molecule on biomacromolecule, and the fluorescence intensity on detection line has been reacted the amount of the sample combining with capture molecules.Detection by scanner to detection line and control line fluorescent value, can be by calculating the two fluorescence intensity ratio and relatively drawing the concentration of detected material with typical curve.
Brief description of the drawings
Fig. 1 is the side schematic view of Test paper card;
1: adsorptive pads;
2: nitrocellulose membrane;
3: contain near-infrared fluorescent label glass fibre membrane;
4: reaction holder.
5: detection line;
6: nature controlling line;
7: sample pad
Fig. 2 is haemoglobin near-infrared fluorescent detection figure
Fig. 3 is haemoglobin typical curve
Fig. 4 is HIV antibody sandwich method near-infrared fluorescent detection figure
Fig. 5 is HIV antibody indirect method near-infrared fluorescent detection figure
Fig. 6 is that morphine near infrared should detection figure
Fig. 7 is variable concentrations morphine detected value
Embodiment
Embodiment 1: sandwich method detects human hemoglobin
1) Infrared fluorescence mark haemoglobin monoclonal antibody and chicken IgY.
Haemoglobin monoclonal antibody (Abcam company of Britain) and chicken IgY antibody (Abcam company of Britain) are dialysed with PBS, ratio according to 7 μ lNHS activation Dylight800 near infrared fluorescent dye (power & light company of the U.S.) mark 1mg haemoglobin monoclonal antibodies is mixed antibody with fluorescent dye, mix rear room temperature lucifuge reaction 1 hour.After reaction finishes, it is in 10K bag filter that marked product is put into aperture, by 4 DEG C of dialysed overnight of PBS, removes free fluorescent dye.In solution, adding final concentration is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 DEG C of preservations.
2) make pad
Choose glass fibre element band as pad solid phase material, on band, spray the antibody of two kinds of near infrared fluorescent dye marks, the air-dry band of room temperature.
3) make sample pad
Choose cellulose membrane band, confining liquid (5%BSA, 0.1%Tween 20, PBS) is sprayed on film band, room temperature is air-dry rear for subsequent use.
4) make immuno-chromatographic test paper strip
Choose nitrocellulose filter, haemoglobin is caught to monoclonal antibody (Abcam company of Britain) and goat-anti chicken IgY polyclonal antibody (Abcam company of Britain) to be made on film with pen machine and detects band and quality control band, detect band and quality control band spacing 0.5cm, the air-dry film band of room temperature.
5) assembling Test paper card
Sample pad, pad, nitrocellulose filter and adsorptive pads, as Fig. 1 mode installs in order, are then cut into test strips with cutting machine, put into plastics draw-in groove, assembling finished product test card.
6) formulate typical curve
Get the haemoglobin sterling (Abcam company of Britain) of 1mg/ml, use dilution (5%BSA, 0.1%Tween20, PBS) that standard items are carried out to 1:10 serial dilution, make 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml 0.01 μ g/ml sample.Draw the above sample of 50 μ l with micro sample adding appliance and be added drop-wise in sample pad, after absorption of sample, drip 100 μ l sample diluting liquids with dropper.Room temperature leaves standstill 5 minutes, and sample card is put into reading in portable highly sensitive near infrared point fluorescent scanning instrument (Beijing profit Bo Fu get development in science and technology company limited).Using the haemoglobin standard product of 50 μ l serial dilutions as sample, measure respectively detection line and nature controlling line fluorescent value, with detection line value be measured value divided by nature controlling line.Each sample concentration measures twice, after averaging, with measured value, sample concentration is done to figure.Result as shown in Figure 2.
With detected peaks fluorescent value divided by Quality Control peak fluorescent value as testing result.Each concentration is done double repeated experiment, gets two times result mean value, with mean value, sample concentration is done to standard working curve, as shown in Figure 3.
Table 1: the corresponding testing result of variable concentrations haemoglobin standard product.
| Concentration (μ g/ml) | 10000 | 7500 | 5000 | 2500 | 1000 | 100 | 10 |
| Measure for the first time (T/C) | 1.399058 | 1.065468 | 0.71044 | 0.374832 | 0.22438 | 0.02288 | 0.008268 |
| Measure for the second time (T/C) | 1.399058 | 0.96568 | 0.714093 | 0.374063 | 0.200634 | 0.023265 | 0.008268 |
| Mean value | 1.399058 | 1.015574 | 0.712267 | 0.374447 | 0.212507 | 0.023072 | 0.008268 |
7) detect clinical samples
Get clinical samples, use 1ml dilution (5%BSA, 0.1%Tween20, PBS) that sample is fully stirred evenly, draw the above sample of 50 μ with micro sample adding appliance and be added drop-wise in sample pad, after absorption of sample, drip 100 μ l sample diluting liquids with dropper.Room temperature leaves standstill 5 minutes, and sample card is put into reading in portable highly sensitive near infrared point fluorescent scanning instrument (Beijing profit Bo Fu get development in science and technology company limited), and result is as shown in the table.According to content of hemoglobin in typical curve calculation sample.
| Sample number into spectrum | Detected value | Hemoglobin concentration (mg/ml) |
| 1 | 0.1213 | 0.683704 |
| 2 | 0.0039 | 0 |
| 3 | 0.2465 | 1.611111 |
| 4 | 0.0915 | 0.462963 |
| 5 | 0.0903 | 0.454074 |
| 6 | 0.0128 | 0 |
| 7 | 0.2376 | 1.545185 |
| 8 | 0.0373 | 0.061481 |
| 9 | 0.3234 | 2.180741 |
| 10 | 0.0198 | 0 |
| 11 | 0.2964 | 1.980741 |
| 12 | 0.0091 | 0 |
| 13 | 0.2703 | 1.787407 |
| 14 | 0.01385 | 0 |
| 15 | 0.02846 | 0 |
| 16 | 0.0368 | 0.057778 |
| 17 | 0.0137 | 0 |
| 18 | 0.3729 | 2.547407 |
| 19 | 0.6835 | 4.848148 |
| 20 | 0.1249 | 0.71037 |
Embodiment 2: sandwich method detects HIV antibody
1) biomacromolecule near-infrared fluorescent mark
By genetic engineering Recombinant HIV-1gp41(Immune Technology company) and HIV-2gp36(Immune Technology company) antigen dialyses with PBS, peace is according to the power & light company of the near infrared fluorescent dye Dylight 800(U.S. of 7 μ lNHS activation) ratio of mark 1mg recombinant antigen mixes the two, mixes rear room temperature lucifuge reaction 1 hour.After reaction finishes, it is in 10K bag filter that marked product is put into aperture, by 4 DEG C of dialysed overnight of PBS, removes free fluorescent dye.In solution, adding final concentration is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 DEG C of preservations.Choose glass fibre element band as pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
2) make pad
Choose glass fibre element band as pad solid phase material, on band, spray the antibody of two kinds of near infrared fluorescent dye marks, the air-dry band of room temperature.
3) make sample pad
Choose cellulose membrane band, confining liquid (5%BSA, 0.1%Tween 20, PBS) is sprayed on film band, room temperature is air-dry rear for subsequent use.
4) make immuno-chromatographic test paper strip
Choose nitrocellulose filter, HIV-1gp41 antigen and HIV-2gp36 antigen are mixed, then make on film with pen machine and detect band and quality control band with goat-anti chicken IgY polyclonal antibody, detect band and quality control band spacing 0.5cm, the air-dry film band of room temperature.
5) assembling Test paper card
Sample pad, pad, nitrocellulose filter and adsorptive pads are installed according to Fig. 1 mode, be then cut into test strips with cutting machine, put into plastics draw-in groove, assembling finished product test card.
6) determine Cutoff value
After HIV antibody near-infrared fluorescent immunochromatography reagent application of sample, carrying out fluorescence with portable highly sensitive near infrared point fluorescent scanning instrument (Beijing profit Bo Fu get development in science and technology company limited) judges, what first read is nature controlling line fluorescent value, then be detection line fluorescence intensity, be figure with the corresponding time of fluorescence intensity, obtain fluorescence intensity-time changing curve.As shown in Figure 4.
With HIV-1 near infrared immunochromatographiassay assay reagent 100 parts of HIV negative serums of detection and 50 parts of positive serums, after reading its detection line and nature controlling line fluorescent value, calculate the two ratio, all equal <0.13 of HIV negative result, its mean value is 0.124, the coefficient of variation is+2 times of coefficient of variation=0.136 of the negative value of 0.006, cut-off=.
7) detect not key sample
Choose HIV the infected and each 20 serum specimens of the infected not, all samples are confirmed with ELISA and Western-Blot.Draw 50 μ l serum with micro sample adding appliance and be added drop-wise in sample pad, after absorption of sample, drip 100 μ l sample diluting liquids with dropper.Room temperature leaves standstill 5 minutes, and sample card is put into reading in portable highly sensitive near infrared point fluorescent scanning instrument (Beijing profit Bo Fu get development in science and technology company limited).Compare according to fluorescence reading and the cutoff value of T line (detection line) and C line (reference line), judge testing result.Testing result and ELISA result compare.Result is shown as:
20 parts of HIV negative samples, near-infrared fluorescent detects all negative, and specificity is 100%.
20 parts of HIV sun samples, near-infrared fluorescent detects all positive, and sensitivity is 100%.
Embodiment 3: indirect method detects HIV antibody
1) Dylight800 mark detectable antigens and Quality Control antibody
Goat anti-human igg antibody (purchased from Abcam company), with PBS dialysis, is mixed the two according to the ratio of the Dylight800 mark 1mg antibody of 7 μ lNHS activation, mix rear room temperature lucifuge reaction 1 hour.After reaction finishes, it is in 10K bag filter that marked product is put into aperture, by 4 DEG C of dialysed overnight of PBS, removes free fluorescent dye.In solution, adding final concentration is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 DEG C of preservations.
2) make pad
Choose glass fibre element band as pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
3) sample pad processed
Choose cellulose membrane band, confining liquid (5%BSA, 0.1%Tween 20, PBS) is sprayed on film band, room temperature is air-dry rear for subsequent use.
4) make immuno-chromatographic test paper strip
Choose nitrocellulose filter, first HIV-1 gp41 and HIV-1 gp36 antigen are mixed, then hybrid antigen and the anti-chicken IgY of goat polyclonal antibody (commodity article No. or source) are made to detection band and quality control band on film with pen machine, detect band and quality control band spacing 0.5cm, the air-dry film band of room temperature.
5) assembling Test paper card
Sample pad, pad, nitrocellulose filter and adsorptive pads are installed in order, be then cut into test strips with cutting machine, put into plastics draw-in groove, assemble finished product test card according to Fig. 1 mode.
6) testing process:
Micro sample adding appliance is drawn 50 μ l serum and is added drop-wise in sample pad, after absorption of sample, drips 100 μ l sample diluting liquids with dropper.Room temperature leaves standstill 5 minutes, and sample card is put into reading in portable highly sensitive near infrared point fluorescent scanning instrument (Beijing profit Bo Fu get company limited).After HIV antibody near-infrared fluorescent immunochromatography reagent application of sample, carry out fluorescence with scanner and judge, what first read is nature controlling line fluorescent value, is then detection line fluorescence intensity, is figure with the corresponding time of fluorescence intensity, obtains fluorescence intensity-time changing curve.As shown in Figure 5
7) determine the Cutoff value that detects reagent
With HIV-1 near infrared immunochromatographiassay assay reagent 100 parts of HIV negative serums of detection and 50 parts of positive serums, after reading its detection line and nature controlling line fluorescent value, calculate the two ratio, add up all feminine genders and positive detection value, all equal <0.13 of HIV negative result, its mean value is 0.122, the coefficient of variation is+2 times of coefficient of variation=0.134 of the negative value of 0.006, cutoff=.
Detect not key sample:
Sample comprises 20 parts of normal persons and 20 parts of HIV infected person anteserums, and all serum specimen carries out contrast test with HIV quantitative fluorescent PCR reagent simultaneously.Result is: 20 parts of HIV positive sample testing results are all positive, and 20 parts of HIV ' negative ' specimens results are all negative, conforms to fluorescent quantitative PCR result 100% with ELISA result.
Embodiment 4: competition law detects morphine near infrared fluorescent dye labelled antibody
Morphine monoclonal antibody (Bo Mei biotechnology company) is dialysed with PBS, peace is mixed the two according to the ratio of near infrared fluorescent dye (Bo Mei biotechnology company) the mark 1mg monoclonal antibody of 7 μ l NHS activation, mixes rear room temperature lucifuge reaction 1 hour.After reaction finishes, it is in 10K bag filter that marked product is put into aperture, by 4 DEG C of dialysed overnight of PBS, removes free fluorescent dye.In solution, adding final concentration is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 DEG C of preservations.Choose glass fibre element band as pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
1) make pad
Choose glass fibre element band as pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
2) prepare sample pad
Choose cellulose membrane band, confining liquid (5%BSA, 0.1%Tween 20, PBS) is sprayed on film band, room temperature is air-dry rear for subsequent use.
3) make immuno-chromatographic test paper strip
Choose nitrocellulose filter, BSA-morphine (Bo Mei biotechnology company) and the anti-chicken IgY of goat polyclonal antibody (Abcam company) are made to detection band and quality control band on film with pen machine, detect band and quality control band spacing 0.5cm, the air-dry film band of room temperature.
4) assembling Test paper card
Sample pad, pad, nitrocellulose filter and adsorptive pads are installed in order, be then cut into test strips with cutting machine, put into plastics draw-in groove, assemble finished product test card according to Fig. 1 mode.
5) testing process
Get 1 μ g/ml morphine standard items, become 1200ng/ml, 600ng/ml, 300ng/ml, 200ng/ml, 100ng/ml concentration sample with normal person's urine serial dilution.Draw 50 μ l samples with micro sample adding appliance and be added drop-wise in sample pad, after absorption of sample, drip 100 μ l sample diluting liquids with dropper.Room temperature leaves standstill 5 minutes, sample card is put into reading in portable highly sensitive near infrared point fluorescent scanning instrument (Beijing profit Bo Fu get development in science and technology company limited), what first read is nature controlling line fluorescent value, then be detection line fluorescence intensity, be figure with the corresponding time of fluorescence intensity, obtain fluorescence intensity-time changing curve.As shown in Figure 6.
1200ng/ml, 600ng/ml, 300ng/ml, 200ng/ml, 100ng/ml concentration detection result of specimen see the following form.
With morphine concentration, detected value (T/C) is done to figure, as shown in Figure 7.
Get the positive urines of 20 portions of morphines and detect with 20 parts of morphine feminine gender urine near-infrared fluorescents, do contrast test with colloid gold test paper simultaneously.Using 1.0 as cotoff value, the 20 parts of positive urine specimen near-infrared fluorescent of morphine testing results are all positive; 20 parts of negative urines of morphine are all negative.
Claims (6)
1. a near-infrared fluorescent molecular immune chromatographic test paper, is characterized in that: by supporting in the reaction with bonding agent successively
On thing, the sample pad of overlap joint, pad, carrier film, adsorptive pads form mutually, wherein,
Antibody or the antigen of two or more near-infrared fluorescent marker material marks on described pad, have been fixed respectively with spraying process, be and detect antigen or the antibody of target specific binding, another be with nature controlling line on antigen or the antibody of object of reference specific binding;
Described carrier film is nitrocellulose filter or nylon membrane, on carrier film, be respectively be connected with can specific binding with target molecule antigen or antibody, and be connected with and the nature controlling line of the antibody of target molecule and the fixing irrelevant molecule of detection line;
The fluorescence intensity in described detection line and nature controlling line region is corresponding with the typical curve of standard antigen concentration respectively in detection, completes quantitative detection, can obtain the concentration of thing to be detected in sample;
Described near-infrared fluorescent marker material is the dyestuff of emission wavelength at 650-1000nm;
Pad the above with nature controlling line on antigen or the antibody of object of reference specific binding be chicken IgY; The above the antibody with target molecule and the fixing irrelevant molecule of detection line of nature controlling line is the anti-chicken IgY of goat polyclonal antibody.
2. the near-infrared fluorescent molecular immune chromatographic test paper of claim 1, described test paper is applied to competition law and detects, what on described detection line, fix is little molecule-BSA conjugate, what on described nature controlling line, fix is the anti-chicken IgY of the goat polyclonal antibody as reference, on described pad, having fixed respectively with spraying process the monoclonal antibody of two kinds of near-infrared fluorescent marks, is respectively anti-little molecule monoclonal antibody and chicken IgY.
3. the near-infrared fluorescent molecular immune chromatographic test paper of claim 1, described test paper is applied to indirect method and detects, and what on described detection line, fix is detectable antigens, and what on nature controlling line, fix is object of reference antibody, the anti-chicken IgY of goat polyclonal antibody; On pad, having fixed respectively with spraying process the biomolecule of two kinds of near-infrared fluorescent marks, is respectively mouse or goat anti-human antibody's second antibody and chicken IgY.
4. the near-infrared fluorescent molecular immune chromatographic test paper of claim 1, described test paper is applied to dual-antigen sandwich method or double antibody sandwich method detects, on described detection line fixing be with detected material can specific binding antigen or antibody, what on nature controlling line, fix is object of reference antibody, the anti-chicken IgY of goat polyclonal antibody; On pad, fixed respectively the biomolecule of two kinds of near-infrared fluorescent marks with spraying process, be respectively with detected material can specific binding antigen or antibody and chicken IgY.
5. the preparation method of the near-infrared fluorescent molecular immune chromatographic test paper described in one of claim 1-4, its key step is:
1) near-infrared fluorescent labeled monoclonal antibody and chicken IgY;
2) make pad;
3) make sample pad;
4) make immuno-chromatographic test paper strip.
6. utilize the described quantitative detectable antigens of near-infrared fluorescent molecular immune chromatographic test paper of one of claim 1-4 or a method for antibody, in turn include the following steps:
I. antigen or antibody standard substance are mixed with to the concentration series standard items of 2～10 times of gradients;
Ii. above-mentioned concentration series standard items are carried out to immunochromatography with the test paper one of claim 1-4 Suo Shu respectively, utilize fluorescent quantitation spectrometer to detect respectively the fluorescence intensity in detection line and quality inspection line region, make the typical curve of antigen or antibody concentration;
Iii. determined antigen or antibody carry out immunochromatography with the test paper one of claim 1-4 Suo Shu, utilize above-mentioned typical curve to try to achieve antigen or antibody concentration;
Described antigen or antibody are haemoglobin, HIV-1gp41, HIV-2gp36 or morphine antigen and antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210152948.XA CN102680692B (en) | 2012-05-16 | 2012-05-16 | Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210152948.XA CN102680692B (en) | 2012-05-16 | 2012-05-16 | Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102680692A CN102680692A (en) | 2012-09-19 |
| CN102680692B true CN102680692B (en) | 2014-10-29 |
Family
ID=46812935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210152948.XA Active CN102680692B (en) | 2012-05-16 | 2012-05-16 | Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102680692B (en) |
Families Citing this family (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197074B (en) * | 2013-04-22 | 2015-11-25 | 北京润博福得生物科技发展有限公司 | Based on the immunochromatography quantitative detecting reagent of near-infrared fluorescent Nano microsphere label |
| CN104502608A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Group A rotavirus chromatography test paper strip based on low-noise excitation fluorescent label |
| CN104502590A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Hepatitis A virus chromatography test paper strip based on low-noise excitation fluorescent label |
| CN104483459A (en) * | 2014-12-23 | 2015-04-01 | 北京出入境检验检疫局检验检疫技术中心 | Low-noise excitation type fluorescent marker based chromatography test strip for listeria monocytogenes |
| CN104502598A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Fluorescent mark-based immunochromatographic test strip for vibrio cholerae O1 serotype ogawa |
| CN104459128A (en) * | 2014-12-23 | 2015-03-25 | 北京出入境检验检疫局检验检疫技术中心 | Salmonella immunochromatography test strip based on low-noise excitation type fluorescent mark |
| CN104483480A (en) * | 2014-12-23 | 2015-04-01 | 北京出入境检验检疫局检验检疫技术中心 | Low-noise excitation type fluorescent marker based immunochromatography test strip for vibrio parahaemolyticus |
| CN104502597A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Norovirus immunochromatographic strip based on low-noise laser-type fluorescence marker |
| CN104698181A (en) * | 2015-03-23 | 2015-06-10 | 武汉真福医药股份有限公司 | Near infrared fluorescence molecular immunochromatography test paper and preparing method thereof |
| CN105137072B (en) * | 2015-04-30 | 2017-03-01 | 广东国际旅行卫生保健中心（广东出入境检验检疫局口岸门诊部） | A kind of mycobacterium tuberculosis LAM detection kit, preparation method and using method |
| CN106290881A (en) * | 2015-06-01 | 2017-01-04 | 上海凯创生物技术有限公司 | Salmonella near-infrared fluorescent method detection kit |
| CN108139395A (en) * | 2015-10-16 | 2018-06-08 | 东洋纺株式会社 | Immunochromatographytest test piece |
| KR20170062742A (en) * | 2015-11-30 | 2017-06-08 | 주식회사 이뮨메드 | Diagnostic kit for tsutsugamushi disease |
| CN106053827A (en) * | 2016-05-17 | 2016-10-26 | 上海凯璟生物科技有限公司 | Irritable bowel syndrome marker Vinculin antibody detection kit and preparation method thereof |
| CN105784989A (en) * | 2016-05-17 | 2016-07-20 | 上海凯璟生物科技有限公司 | Irritable bowel syndrome marker CdtB antibody detection kit and preparation method thereof |
| CN105974110A (en) * | 2016-07-06 | 2016-09-28 | 北京康思润业生物技术有限公司 | Immune lateral chromatographic detection system as well as preparation method and application thereof |
| CN106290832A (en) * | 2016-08-12 | 2017-01-04 | 上海铭源数康生物芯片有限公司 | A kind of immunity lateral chromatography quantitative detecting reagent and preparation method thereof, detection method |
| CN106198966A (en) * | 2016-09-06 | 2016-12-07 | 中国农业大学 | A kind of near-infrared fluorescent immune chromatography reagent kit detecting aflatoxin and application thereof |
| CN107478642B (en) * | 2017-07-27 | 2021-02-23 | 山东师范大学 | Quantitative detection device and method for colloidal gold test strip based on photothermal effect detection |
| CN107525937A (en) * | 2017-08-25 | 2017-12-29 | 苏州优函信息科技有限公司 | Based on up-conversion luminescence label, protein chip and detection method |
| CN108535482A (en) * | 2018-04-11 | 2018-09-14 | 上海萨迦生物科技有限公司 | A kind of antibody chip kit for tumour early screening and diagnosis |
| CN110702900A (en) * | 2019-10-10 | 2020-01-17 | 南京欧凯生物科技有限公司 | Immunofluorescence chromatography detection card for serum amyloid A |
| CN110567930A (en) * | 2019-10-22 | 2019-12-13 | 中国人民解放军军事科学院军事医学研究院 | Quantum dot fluorescence detection device and quantum dot fluorescence monitor |
| CN110987882B (en) * | 2019-11-15 | 2022-04-12 | 上海大学 | Fluorescence-quenched colloidal gold immunochromatographic test strip, preparation method and application thereof |
| CN111340091B (en) * | 2020-02-21 | 2022-08-23 | 上海艾瑞德生物科技有限公司 | Training method of CNN (CNN) model for classifying immune data and application of CNN model |
| WO2022114941A1 (en) | 2020-11-24 | 2022-06-02 | Autonomous Organization Of Education "Nazarbayev University" | Method for detecting hpv16 and in vitro diagnostic device for detection |
| CN113985032A (en) * | 2021-09-14 | 2022-01-28 | 广州万孚生物技术股份有限公司 | Double-label immunodetection method containing internal reference and application thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6770487B2 (en) * | 2001-05-01 | 2004-08-03 | Ischemia Technologies, Inc. | Bar code readable diagnostic strip test |
| CN1690711B (en) * | 2004-04-23 | 2010-04-14 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatographic test paper bar based on up conversion luminescence technology |
| DK3613338T3 (en) * | 2007-08-30 | 2021-07-12 | Siemens Healthcare Diagnostics Inc | NON-VISIBLE DETECTIVE MARKING FOR MEDICAL DIAGNOSTICS |
| ES2461992T3 (en) * | 2009-10-09 | 2014-05-22 | Invisible Sentinel, Inc. | Device for the detection of antigens and their uses |
| US20130052668A1 (en) * | 2010-04-30 | 2013-02-28 | Amanda G. Paulovich | Identification and use of biomarkers for detection and quantification of the level of radiation exposure in a biological sample |
-
2012
- 2012-05-16 CN CN201210152948.XA patent/CN102680692B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102680692A (en) | 2012-09-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102680692B (en) | Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker | |
| CN103197074B (en) | Based on the immunochromatography quantitative detecting reagent of near-infrared fluorescent Nano microsphere label | |
| CN106872420B (en) | Kit and method for time-resolved fluorescence quantitative detection of microalbuminuria | |
| CN103940798B (en) | A kind of entity fluorescent nanometer microsphere and its preparation method and application | |
| CN207248894U (en) | Bladder chalone C time resolution detection card and kit | |
| CN105403698A (en) | Diagnostic test kits employing an internal calibration system | |
| CN101000343A (en) | Immunological test element with improved control zone | |
| WO2017138946A1 (en) | Multiplexed lateral flow assay | |
| KR101718485B1 (en) | Device for Detecting Colored Reaction or Fluorescence Reaction of Immunochromatography | |
| CN114107019B (en) | Microfluidic chip for simultaneously detecting nucleic acid and protein, detection method and application | |
| CN102539751A (en) | Immunofluorescence test paper strip and quantitative detection method thereof | |
| CN107957495A (en) | A kind of CK-MB detection kits and its application method | |
| CN202049158U (en) | Immunofluorescence test paper strip | |
| KR20190117350A (en) | Lateral flow assay strip | |
| Fan et al. | Lateral flow immunoassay for 5-hydroxyflunixin based on near-infrared fluorescence molecule as an alternative label to gold nanoparticles | |
| CN205484371U (en) | Tumour mark detect reagent box | |
| KR20160120675A (en) | Rapid Quantitative Diagnostic Kit | |
| CN104698181A (en) | Near infrared fluorescence molecular immunochromatography test paper and preparing method thereof | |
| CN108918865A (en) | Fluorescence immune chromatography test paper bar and reagent card | |
| US20220308049A1 (en) | Lateral flow test strips with competitive assay control | |
| CN102087215A (en) | Fluorescent quantitative detection instrument | |
| CN107505459A (en) | Quantitatively detect people H FABP time-resolved fluoroimmunoassay chromatograph test strip, kit and preparation method thereof | |
| CN105738616A (en) | Preparation method and application of dual-amplifying fluorescent immune labeling probe and method for preparing fluorescent immune chromatography reagent strip from probe | |
| JP5562508B1 (en) | Additives for measuring diluted samples in undiluted immunochromatographic reagents | |
| WO2007016665A2 (en) | Single use fluorescent assays for determination of analytes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20181119 Address after: 102600 Beijing Economic and Technological Development Zone 518, 5th floor, No. 4 Building, 109 Jinghai Third Road Patentee after: CIIA Zhilian (Beijing) Technology Co., Ltd. Address before: 101200 269 Da Huashan street, Dahuashan Town, Pinggu District, Beijing. Patentee before: Beijing Runbo Fude Biological Technology Development Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190724 Address after: 101207 Beijing City Dahua Town, Pinggu District, Huashan street, No. 269 Patentee after: Beijing Runbo Fude Biological Technology Development Co., Ltd. Address before: 102600 Beijing Economic and Technological Development Zone 518, 5th floor, No. 4 Building, 109 Jinghai Third Road Patentee before: CIIA Zhilian (Beijing) Technology Co., Ltd. |
|
| TR01 | Transfer of patent right |