CN102680692A - Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker - Google Patents
Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker Download PDFInfo
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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 detection by quantitative reagent based on the near-infrared fluorescent label.The preparation method who comprises near-infrared fluorescent test strip and corresponding reagent.The present invention adopts the near-infrared fluorescent molecule to serve as a mark; Adopt immunochromatography technique; Preparation near-infrared fluorescent immuno-chromatographic test paper strip constitutes the test card that comprises sample pad/glass fibre membrane/nitrocellulose filter and thieving paper then, wherein is fixed with detection line and nature controlling line on the nitrocellulose filter.In testing process; Adopt near infrared light to scan nature controlling line and sample wire respectively; The fluorescence excitation mulecular luminescence, the fluorescence of launching converts digital signal through optical filter to through photomultiplier; With the typical curve in the substitution fluorescence analyser after the nature controlling line fluorescence intensity correct detection line fluorescence intensity, get final product the concentration of the determinand in the analyzing and testing sample.This system can use in microorganism detection, food safety detection, drugs detection and hazardous chemical fast detecting.Highly sensitive, quantitatively accurate convenience operation that the present invention has.
Background technology
Immunochromatographic method has obtained in fields such as disease quick diagnosis, micromolecule detections using widely.Its principle is a certain district band that special antigen or antibody is fixed in nitrocellulose membrane earlier; After this dry nitrocellulose filter one end immerses sample, because capillarity, sample will move forward along this film; When moving to when being fixed with antibody regional; Corresponding antigen promptly combines with this antibody generation specificity in the sample, if can make this zone show certain color with immune colloid gold, thereby realizes specific immunodiagnosis.Label commonly used is colloid gold particle, enzyme and painted microballoon.Colloid gold particle and painted microballoon be through Electrostatic Absorption with antigen or antibody labeling at particle surface, judge testing result through observing colloid gold grain or the gathering colour developing of microballoon on detection line.Enzyme labeling shows testing result through the catalytic substrate colour developing.Above labelling technique exists sensitivity low, shortcoming that can not accurate quantification.Limited the immunochromatography range of application.
Characteristics such as the fluorescence labeling technology has highly sensitive, and is simple to operate are widely used in field of biological detection.Like determined dna sequence, protein expression analysis, clinical diagnosis etc.Fluorescent marker spectral range commonly used is mostly at visible region (400-750nm).Because the albumen of biosome, nucleic acid etc. can produce fluorescence under the exciting of ultraviolet light, therefore, use the fluorescent marker of visible region can produce stronger background fluorescence, the Interference Detection signal has reduced the sensitivity that detects.The emission wavelength ranges of near-infrared fluorescent material is at 650-1000nm, in this scope, the biosome autofluorescence a little less than; Therefore; Use the near-infrared fluorescent material as detecting fluorescent marker, have low background fluorescence, detection signal-to-noise ratio height, the characteristics 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 detection by quantitative reagent based on the near-infrared fluorescent label; And new detection method, the preparation method of said detection by quantitative pack test strip and test strips.Its principle of work is: on the sample pad of immuno-chromatographic test paper strip, be fixed with the object of reference of near-infrared fluorescent mark and the antigen or the antibody that can combine with the detected material specificity; Be added drop-wise on the 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, through detection line and nature controlling line the time, be fixed on two molecules on the line respectively and catch.Read the fluorescence intensity of line of reference and detection line respectively with the fluorescent scanning appearance,, can obtain the concentration of thing to be detected in the sample both fluorescence intensity substitution formula.
The method that the present invention adopted is:
When using sandwich method to detect target molecule (can be that double antibody sandwich method detects antigen, also can be two Detection of antigen antibody); Fixing on the detection line of immune chromatography test paper is that (double antibody sandwich method is an antibody for the antigen that can specificity combines with target molecule or antibody; Dual-antigen sandwich method is an antigen); As object of reference on nature controlling line is the antibody with the fixing irrelevant molecule of target molecule and detection line, normally goat anti chicken IgY polyclonal antibody.The antibody or the antigen of two or more near-infrared fluorescent marks have been fixed on the pad respectively with spraying process; A kind ofly be and detect antigen or the antibody that the target specificity combines; A kind of in addition is the antigen or the antibody that can combine with object of reference specificity on the nature controlling line, generally is chicken IgY.When the sample drop that contains target molecule was added on the sample pad, solution dissociated two kinds of compositions on the 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 the test strips upper end with solution.When antigen antibody complex moved to detection line, the target molecule specific binding molecules that is fixed on the detection line was caught.The Quality Control molecule continues to move with solution, and when moving to nature controlling line, the object of reference antibody that is fixed on the nature controlling line is caught.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with the fluorescent scanning appearance.Because the amount of object of reference is a definite value on the pad, simultaneously the object of reference monoclonal antibody not can with detect target molecule and antibodies, therefore, the fluorescence intensity of object of reference can be used as the confidential reference items of detectable.Through calculating the ratio of detection line and nature controlling line fluorescence intensity, and calculate, just can draw the concentration of target molecule in the sample with standard working curve;
When using indirect method to detect target molecule: what fix on the detection line at immune chromatography test paper is to detect antigen, and what on nature controlling line, fix is object of reference antibody, generally is goat anti chicken IgY polyclonal antibody.Having fixed the biomolecule of two kinds of near-infrared fluorescent marks on the pad respectively with spraying process, is respectively the SA and the chicken IgY of mouse anti human antibody.After the sample drop that contains target molecule is added on the sample pad; Drip dilution simultaneously; Solution dissociates the biomacromolecule on the pad, and the SA of near-infrared fluorescent mark and people's antibodies form antibody complex; Under the promotion of solution, antibody complex and chicken IgY move to the test strips upper end with solution.When antibody complex moved to detection line, the antigen that antibody complex is fixed on the detection line was caught.Chicken IgY continues to move with solution, and when moving to nature controlling line, the goat anti chicken IgY antibody that is fixed on the nature controlling line is caught.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with the fluorescent scanning appearance.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with the fluorescent scanning appearance.Through calculating the ratio of detection line and nature controlling line fluorescence intensity, and compare, whether contain HIV antibody in just can drawing in the sample with pre-set threshold.
When using competition law to detect micromolecule: what fix on the detection line at immune chromatography test paper is micromolecule-BSA conjugate, and what on nature controlling line, fix is the goat anti chicken IgY polyclonal antibody as reference.Having fixed the monoclonal antibody of two kinds of IR fluorescence marks on the pad respectively with spraying process, is respectively anti-micromolecule monoclonal antibody and chicken IgY.During detection, at first drip sample, drip dilution then to sample pad.Solution dissociates the biomacromolecule on the pad, if when there is certain density micromolecule in sample, whole fluorescently-labeled mouse monoclonal antibodies are combined, and the compound of micromolecule and antibodies is mobile to the test strips upper end under the promotion of solution.When antigen antibody complex moved to detection line, the BSA-micromolecule compound that anti-micromolecule monoclonal antibody can not be fixed on the detection line was caught.Chicken IgY continues to move with solution, and when moving to nature controlling line, the goat anti chicken IgY antibody that is fixed on the nature controlling line is caught.After reaction finishes, measure the fluorescence intensity of detection line and nature controlling line with the fluorescent scanning appearance.Through calculating the ratio of detection line and nature controlling line fluorescence intensity, and compare, just can draw whether contain micromolecule to be detected in the sample with pre-set threshold.
Employed fluorescent scanning instrument is conventional experiment scanner among the application, for example Beijing profit Bo Fude development in science and technology company limited portable high sensitivity near infrared point fluorescent scanning appearance;
Employed near-infrared fluorescent marker material is the dyestuff of emission wavelength at 650-1000nm among the application, is preferably the near infrared fluorescent dye Dylight800 of NHS activation.
This detection method is compared with other detection methods has following advantage:
Biological background signal is low, and detection sensitivity is high.Compare with colloidal gold immunochromatographimethod reagent, the sensitivity of molecule to be detected is improved about 10 times based on the near-infrared fluorescent system.Like the haemoglobin minimum detectability is 10ng/ml, and is generally 100ng/ml based on the haemoglobin detectable minimum detectability of collaurum.When using indirect method to detect the HIV specific antibody, will detect the 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, and few to reading the report operation steps from application of sample, operating personnel are easy to grasp.
Can detection by quantitative.Traditional immune chromatography method is a buildup effect sentence read result of leaning on gold grain.There are certain quantitative relation in the gathering and the concentration in the sample of gold grain, but can only carry out sxemiquantitative through the painted depth of observation gold grain.This method is marked at fluorescence molecule on the biomacromolecule, and the fluorescence intensity on the detection line has been reacted the amount of the sample that combines with capture molecules.Through the detection of scanner to detection line and control line fluorescent value, can be through calculating the two fluorescence intensity ratio and relatively drawing the concentration of detected material with typical curve.
Description of drawings
Fig. 1 is for detecting the side schematic view of test card;
1: adsorptive pads;
2: nitrocellulose membrane;
3: contain near-infrared fluorescent label glass fibre membrane;
4: the reaction holder.
5: detection line;
6: nature controlling line;
7: sample pad
Fig. 2 detects figure for the haemoglobin near-infrared fluorescent
Fig. 3 is the haemoglobin typical curve
Fig. 4 is that HIV antibody sandwich method near-infrared fluorescent detects figure
Fig. 5 is that HIV antibody indirect method near-infrared fluorescent detects figure
Fig. 6 should detect figure for the morphine near infrared
Fig. 7 is a variable concentrations morphine detected value
Embodiment
Embodiment 1: sandwich method detects human hemoglobin
1) IR fluorescence mark haemoglobin monoclonal antibody and chicken IgY.
Haemoglobin monoclonal antibody (Britain Abcam company) and chicken IgY antibody (Britain Abcam company) are dialysed with PBS; Ratio according to 7 μ lNHS activation Dylight800 near infrared fluorescent dye (U.S. power & light company) mark 1mg haemoglobin monoclonal antibodies is mixed antibody with fluorescent dye, mix back room temperature lucifuge reaction 1 hour.After reaction finished, it was in the 10K bag filter that marked product is put into the aperture, with 4 ℃ of dialysed overnight of PBS, removed free fluorescent dye.Adding final concentration in the solution is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 ℃ of preservations.
2) make pad
Choose the plain band of spun glass as the pad solid phase material, the antibody of two kinds of near infrared fluorescent dye marks of spraying on band, the air-dry band of room temperature.
3) make sample pad
Choose the cellulose membrane band, (5%BSA, 0.1%Tween 20, PBS) are sprayed on the film band, and is subsequent use after room temperature is air-dry with confining liquid.
4) make immuno-chromatographic test paper strip
Choose nitrocellulose filter; Haemoglobin is caught monoclonal antibody (Britain Abcam company) and goat-anti chicken IgY polyclonal antibody (Britain Abcam company) to be processed 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 detects test card
Sample pad, pad, nitrocellulose filter and adsorptive pads such as Fig. 1 mode are installed in order, be cut into test strips with cutting machine then, put into the plastics draw-in groove, assembling finished product test card.
6) formulate typical curve
Get the pure article of haemoglobin (Britain Abcam company) of 1mg/ml, (5%BSA, 0.1%Tween20 PBS) carry out the 1:10 serial dilution with standard items, process 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml, 0.01 μ g/ml sample with dilution.Draw the above sample of 50 μ l with micro sample adding appliance and be added drop-wise on the sample pad, treat to drip 100 μ l sample diluting liquids with dropper after the absorption of sample.Room temperature left standstill 5 minutes, and sample card is put into reading in the portable highly sensitive near infrared point fluorescent scanning appearance (Beijing profit Bo Fude development in science and technology company limited).As sample, measure detection line and nature controlling line fluorescent value with the haemoglobin standard article of 50 μ l serial dilutions respectively, use the detection line value to be measured value divided by nature controlling line.Each sample concentration measures twice, after averaging, with measured value sample concentration is figure.The result is as shown in Figure 2.
With the detected peaks fluorescent value divided by Quality Control peak fluorescent value as testing result.Each concentration is done revision test twice, gets two times result mean value, with mean value sample concentration is done standard working curve, and is as shown in Figure 3.
Table 1: the corresponding testing result of variable concentrations haemoglobin standard article.
| 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, (5%BSA, 0.1%Tween20 PBS) fully stir sample, draw the above sample of 50 μ with micro sample adding appliance and are added drop-wise on the sample pad, treat to drip 100 μ l sample diluting liquids with dropper after the absorption of sample with the 1ml dilution.Room temperature left standstill 5 minutes, and sample card is put into reading in the portable highly sensitive near infrared point fluorescent scanning appearance (Beijing profit Bo Fude development in science and technology company limited), and the result is as shown in the table.According to content of hemoglobin in the typical curve calculation sample.
| Sample number into spectrum | Detected value | HC (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 |
[0048]?
| 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
Genetic engineering Recombinant HIV-1gp41 (Immune Technology company) and HIV-2gp36 (Immune Technology company) antigen are dialysed with PBS; Peace is mixed the two according to the ratio of near infrared fluorescent dye Dylight 800 (U.S. power & light company) the mark 1mg recombinant antigen of 7 μ lNHS activation, mixes back room temperature lucifuge reaction 1 hour.After reaction finished, it was in the 10K bag filter that marked product is put into the aperture, with 4 ℃ of dialysed overnight of PBS, removed free fluorescent dye.Adding final concentration in the solution is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 ℃ of preservations.Choose the plain band of spun glass as the pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
2) make pad
Choose the plain band of spun glass as the pad solid phase material, the antibody of two kinds of near infrared fluorescent dye marks of spraying on band, the air-dry band of room temperature.
3) make sample pad
Choose the cellulose membrane band, (5%BSA, 0.1%Tween 20, PBS) are sprayed on the film band, and is subsequent use after room temperature is air-dry with confining liquid.
4) make immuno-chromatographic test paper strip
Choose nitrocellulose filter,, on film, process with pen machine with goat-anti chicken IgY polyclonal antibody then and detect band and quality control band, detect band and quality control band spacing 0.5cm, the air-dry film band of room temperature HIV-1gp41 antigen and HIV-2gp36 mixed antigen.
5) assembling detects test card
Sample pad, pad, nitrocellulose filter and adsorptive pads are installed according to Fig. 1 mode, be cut into test strips with cutting machine then, put into the plastics draw-in groove, assembling finished product test card.
6) confirm the Cutoff value
Carrying out fluorescence with portable highly sensitive near infrared point fluorescent scanning appearance (Beijing profit Bo Fude development in science and technology company limited) behind the HIV antibody near-infrared fluorescent immunochromatography reagent application of sample judges; What at first read is the nature controlling line fluorescent value; Be the detection line fluorescence intensity then; Be figure with the corresponding time of fluorescence intensity, obtain fluorescence intensity-time changing curve.As shown in Figure 4.
Detect 100 parts of HIV negative serums and 50 parts of positive serums with HIV-1 near infrared immunochromatographiassay assay reagent; Calculate the two ratio after reading its detection line and nature controlling line fluorescent value; All the HIV negative result equal < 0.13; Its mean value is 0.124, and the coefficient of variation is 0.006 ,+2 times of coefficient of variation=0.136 of the negative value of cut-off=.
7) detect not key sample
Choose HIV the infected and each 20 serum specimen 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 on the sample pad, treat to drip 100 μ l sample diluting liquids with dropper after the absorption of sample.Room temperature left standstill 5 minutes, and sample card is put into reading in the portable highly sensitive near infrared point fluorescent scanning appearance (Beijing profit Bo Fude development in science and technology company limited).Fluorescence reading and cutoff value according to T line (detection line) and C line (reference line) compare, and judge testing result.Testing result and ELISA result compare.The 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 marker detection antigen and Quality Control antibody
Goat anti-human igg antibody (available 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 back room temperature lucifuge reaction 1 hour.After reaction finished, it was in the 10K bag filter that marked product is put into the aperture, with 4 ℃ of dialysed overnight of PBS, removed free fluorescent dye.Adding final concentration in the solution is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 ℃ of preservations.
2) make pad
Choose the plain band of spun glass as the pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
3) system sample pad
Choose the cellulose membrane band, (5%BSA, 0.1%Tween 20, PBS) are sprayed on the film band, and is subsequent use after room temperature is air-dry with confining liquid.
4) make immuno-chromatographic test paper strip
Choose nitrocellulose filter; At first with HIV-1 gp41 and HIV-1 gp36 mixed antigen; Then hybrid antigen and goat anti chicken IgY polyclonal antibody (commodity article No. or source) are processed detection band and quality control band with pen machine on film; Detect band and quality control band spacing 0.5cm, the air-dry film band of room temperature.
5) assembling detects test card
Sample pad, pad, nitrocellulose filter and adsorptive pads are installed in order, be cut into test strips with cutting machine then, put into the plastics draw-in groove, assemble the 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 on the sample pad, treats to drip 100 μ l sample diluting liquids with dropper after the absorption of sample.Room temperature left standstill 5 minutes, and sample card is put into reading in the portable highly sensitive near infrared point fluorescent scanning appearance (Beijing profit Bo Fude company limited).Carry out fluorescence with scanner behind the HIV antibody near-infrared fluorescent immunochromatography reagent application of sample and judge that what at first read is the nature controlling line fluorescent value, is the detection line fluorescence intensity then, be figure with the fluorescence intensity correspondence time, obtain fluorescence intensity-time changing curve.As shown in Figure 5
7) confirm the Cutoff value of detectable
Detect 100 parts of HIV negative serums and 50 parts of positive serums with HIV-1 near infrared immunochromatographiassay assay reagent; Calculate the two ratio after reading its detection line and nature controlling line fluorescent value; Add up all negative and positive detection values, all < 0.13, its mean value is 0.122 to the HIV negative result; The coefficient of variation is 0.006 ,+2 times of coefficient of variation=0.134 of the negative value of cutoff=.
Detect not key sample:
Sample comprises 20 parts of normal persons and 20 parts of HIV infected person anteserums, and all serum specimen compares test with HIV quantitative fluorescent PCR reagent simultaneously.The result is: 20 parts of HIV positive sample testing results are all positive, and 20 parts of negative sample results of HIV are all negative, conform to fluorescent quantitative PCR result 100% with ELISA result.
Embodiment 4: competition law detects morphine near infrared fluorescent dye labelled antibody
Morphine monoclonal antibody (rich U.S. biotechnology company) is dialysed with PBS; Peace is mixed the two according to the ratio of the near infrared fluorescent dye of 7 μ l NHS activation (rich U.S. biotechnology company) mark 1mg monoclonal antibody, mixes back room temperature lucifuge reaction 1 hour.After reaction finished, it was in the 10K bag filter that marked product is put into the aperture, with 4 ℃ of dialysed overnight of PBS, removed free fluorescent dye.Adding final concentration in the solution is 1%BSA and 0.1%Tween20, sodium azide 0.1 ‰, 4 ℃ of preservations.Choose the plain band of spun glass as the pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
1) makes pad
Choose the plain band of spun glass as the pad solid phase material, on band, spray near infrared fluorescent dye-biomolecule solution, the air-dry band of room temperature.
2) preparation sample pad
Choose the cellulose membrane band, (5%BSA, 0.1%Tween 20, PBS) are sprayed on the film band, and is subsequent use after room temperature is air-dry with confining liquid.
3) make immuno-chromatographic test paper strip
Choose nitrocellulose filter, BSA-morphine (rich U.S. biotechnology company) is processed on film with pen machine with goat anti chicken IgY polyclonal antibody (Abcam company) detected band and quality control band, detection is with and quality control band spacing 0.5cm, the air-dry film band of room temperature.
4) assembling detects test card
Sample pad, pad, nitrocellulose filter and adsorptive pads are installed in order, be cut into test strips with cutting machine then, put into the plastics draw-in groove, assemble the 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 on the sample pad, treat to drip 100 μ l sample diluting liquids with dropper after the absorption of sample.Room temperature left standstill 5 minutes; Sample card is put into reading in the portable highly sensitive near infrared point fluorescent scanning appearance (Beijing profit Bo Fude development in science and technology company limited); What at first read is the nature controlling line fluorescent value; Be the detection line fluorescence intensity then, 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 figure, as shown in Figure 7.
Get the positive urines of 20 portions of morphines and detect with near-infrared fluorescent, do contrast test with colloid gold test paper simultaneously with 20 parts of morphine feminine gender urines.With 1.0 as the 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 (9)
1. near-infrared fluorescent molecular immune chromatographic test paper is characterized in that: by forming having on the reaction holder of bonding agent each other sample pad, pad, carrier film, the adsorptive pads of overlap joint successively, wherein,
The antibody or the antigen of two or more near-infrared fluorescent marker material marks have been fixed on the described pad respectively with spraying process; A kind ofly be and detect antigen or the antibody that the target specificity combines, a kind of in addition be with nature controlling line on object of reference the specificity antigen or the antibody that combine;
Described carrier film is nitrocellulose filter or nylon membrane, is respectively on the carrier film to be connected with antigen or the antibody that can specificity combines with target molecule and to be connected with the nature controlling line with the antibody of the fixing irrelevant molecule of target molecule and detection line;
Described detection line is corresponding with the typical curve of standard antigen concentration respectively in detection with the fluorescence intensity in nature controlling line zone, accomplishes detection by quantitative, can obtain the concentration of thing to be detected in the sample.
2. the near-infrared fluorescent molecular immune chromatographic test paper of claim 1, described near-infrared fluorescent marker material is the dyestuff of emission wavelength at 650-1000nm, is preferably the near infrared fluorescent dye Dylight800 of NHS activation.
3. claim 1 or 2 near-infrared fluorescent molecular immune chromatographic test paper, pad the above with nature controlling line on object of reference the specificity antigen or the antibody that combine be chicken IgY; The above the antibody with the fixing irrelevant molecule of target molecule and detection line of nature controlling line is goat anti chicken IgY polyclonal antibody.
4. the near-infrared fluorescent molecular immune chromatographic test paper of one of claim 1-3; Said test paper is applied to competition law and detects; What fix on the said detection line is micromolecule-BSA conjugate, and what fix on the said nature controlling line is the goat anti chicken IgY polyclonal antibody as reference.Having fixed the monoclonal antibody of two kinds of IR fluorescence marks on the said pad respectively with spraying process, is respectively anti-micromolecule monoclonal antibody and chicken IgY.
5. the near-infrared fluorescent molecular immune chromatographic test paper of one of claim 1-3, said test paper are applied to indirect method and detect, and fixing on the said detection line is to detect antigen, and fixing on nature controlling line is object of reference antibody, for example is goat anti chicken IgY polyclonal antibody.Having fixed the biomolecule of two kinds of near-infrared fluorescent marks on the pad respectively with spraying process, is respectively mouse or goat anti-human antibody's SA and chicken IgY.
6. the near-infrared fluorescent molecular immune chromatographic test paper of one of claim 1-3; Said test paper is applied to dual-antigen sandwich method or double antibody sandwich method detects; Fixing on the said detection line is antigen or the antibody that can specificity combines with detected material; What on nature controlling line, fix is object of reference antibody, for example is goat anti chicken IgY polyclonal antibody.Having fixed the biomolecule of two kinds of near-infrared fluorescent marks on the pad respectively with spraying process, is respectively antigen or antibody and the chicken IgY that can specificity combines with detected material.
7. the preparation method of the described near-infrared fluorescent molecular immune of claim 1-6 chromatographic test paper, its key step is:
1) IR fluorescence labeled monoclonal antibody and chicken IgY;
2) make pad;
3) make sample pad;
4) make immuno-chromatographic test paper strip.
8. method of utilizing the described near-infrared fluorescent molecular immune of one of claim 1-6 chromatographic test paper detection by quantitative antigen or antibody in turn includes the following steps:
I. antigen or antibody standard substance are mixed with the concentration series standard items of 2～10 times of gradients;
Ii. above-mentioned concentration series standard items are carried out immunochromatography with the described test paper of one of claim 1-5 respectively, utilize the fluorescent quantitation spectrometer to detect detection line and the regional fluorescence intensity of quality inspection line respectively, make the typical curve of antigen or AC;
Iii. determined antigen or antibody carry out immunochromatography with the described test paper of one of claim 1-5, utilize above-mentioned typical curve to try to achieve antigen or AC.
9. the method for fluorescence immune chromatography test paper detection by quantitative antigen according to claim 8 is characterized in that, described antigen or antibody are haemoglobin, HIV-1 gp41, HIV-2 gp36 or morphine monoclonal antibody.
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Cited By (27)
| Publication number | Priority date | Publication date | Assignee | Title |
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