CN107782898B - Fluorescence immunochromatography detection kit and detection method thereof - Google Patents
Fluorescence immunochromatography detection kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to a fluorescence immunochromatography detection kit and a detection method thereof, the kit comprises a chromatography test paper and a detection solution, the chromatography test paper comprises a substrate and a bearing body attached to the substrate, the bearing body comprises a first area, a second area and a third area, the first area and the third area are respectively positioned at two sides of the second area, a detection zone is formed at the intersection of the first area and the second area, a quality control zone is formed at the intersection of the second area and the third area, the chromatography test paper also comprises a combination pad attached to the first area of the bearing body, a sample pad attached to the combination pad and water absorption paper attached to the third area; the detection solution comprises a monoclonal antibody T1, a monoclonal antibody T2 and a diluent. The detection kit has high sensitivity, good stability and strong universality, and can be applied to the detection of fluorescence immunochromatography methods of PCT, cTnI, CK-MB, BNP and the like.
Description
Technical Field
The invention relates to a kit, in particular to a fluorescence immunochromatography detection kit and a detection method thereof, and belongs to the technical field of fluorescence immunochromatography in medical immunology.
Background
The immunochromatography is a new type immunity detection technology established on the basis of chromatography technology and antigen-antibody specificity immunoreaction, and its principle is that the specific antibody is firstly fixed in a certain zone of strip-shaped fibre chromatography material, after one end of said dried strip-shaped fibre chromatography material is soaked in the test sample, the tested substance is moved on the chromatography strip due to capillary action, when the tested substance is moved to the zone in which the antibody is fixed, the correspondent antigen in the tested substance can be specifically combined with said antibody, if the immune colloidal gold or immune enzyme is used for dyeing, said zone can display a certain colour so as to implement specific immunity diagnosis.
The fluorescence immunochromatography is based on immunochromatography, adopts a fluorescence labeled antibody and is matched with a fluorescence detector, so that the intelligent operation of a detection result is realized, the misjudgment of manual detection can be reduced, and the detection sensitivity is improved. And the fluorescence immunochromatography method is simple to operate and short in time consumption, and the detection result can be intelligently read only by adding samples. Therefore, the technology has been rapidly developed in recent years and has been widely used for clinical examination, analysis and diagnosis in small medical institutions.
Disclosure of Invention
The invention aims to provide a fluorescence immunochromatography detection kit which is high in sensitivity, good in stability and high in resource utilization rate.
The purpose of the invention can be realized by the following technical scheme: a fluorescence immunochromatography assay kit comprises
Chromatography test paper: the chromatographic test paper comprises a substrate and a bearing body attached to the substrate, wherein the bearing body comprises a first area, a second area and a third area, the first area and the third area are respectively positioned at two sides of the second area, a detection zone is formed at the intersection of the first area and the second area, a quality control zone is formed at the intersection of the second area and the third area, the chromatographic test paper further comprises a combination pad attached to the first area of the bearing body, a sample pad attached to the combination pad, and absorbent paper attached to the third area;
detection liquid: the detection solution comprises a monoclonal antibody T1, a monoclonal antibody T2 and a diluent.
In the above-mentioned fluorescence immunochromatography detection kit, the carrier includes but is not limited to nitrocellulose membrane, and the substrate includes but is not limited to PVC plate.
In the above fluorescence immunochromatography detection kit, the absorbent paper extends a predetermined length from the edge of the third area to the direction of the second area.
In the above fluorescence immunochromatography detection kit, the binding pad extends a predetermined length from the edge of the first region toward the second region, and the sample pad extends a predetermined length from the edge of the binding pad toward the first region. The conjugate pad includes, but is not limited to, polyester fibers and the sample pad includes, but is not limited to, a fiberglass membrane.
In the above fluorescence immunochromatography detection kit, the monoclonal antibody T1 and the monoclonal antibody T2 are different monoclonal antibodies.
In the above fluorescence immunochromatography detection kit, any one of the monoclonal antibody T1 and the monoclonal antibody T2 is a biotin-crosslinked monoclonal antibody, and the crosslinking of the antibody and biotin can ensure high specificity and high affinity of the antibody.
Biotin needs to be activated, the surface of the activated biotin contains stable active ester, then an antibody needing to be crosslinked is added, and the active ester can be condensed with amino on the antibody to form stable amido bond, so that the biotin-labeled antibody is obtained. Due to the space folding mode of the antibody and the strong hydrophobicity of the Fc end, most of the coupling reaction occurs at the Fc end, and the combination of the antibody and the antigen is not greatly influenced; and the biotin is a small molecule, and cannot influence the combination of the antigen and the antibody due to steric hindrance.
In the cross-linking process of biotin and an antibody, when active groups exist on the surface of biotin, the biotin can directly react with the antibody without using a chemical cross-linking agent; on the contrary, the chemical cross-linking agent is adopted to couple the antibody and the biotin. Among them, the chemical crosslinking agent includes, but is not limited to, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), succinic anhydride, and glutaraldehyde, and more preferably, EDC, which is a zero-bond bridge crosslinking agent. EDC is the smallest available reagent system in biological coupling, which mediates biological coupling between two molecules by forming a bond free of other atoms, one of which is covalently attached to one of which is not intervening any cross-linking agent or spacer.
In one of the above-mentioned fluorescence immunochromatography detection kits, an avidin-crosslinked tracer material is formed on the conjugate pad, and the tracer material includes, but is not limited to, fluorescent latex microspheres. The interaction between biotin and avidin is the most strongly known non-covalent interaction at present, and is at least 1 ten thousand times greater than the affinity between antigen and antibody. And the combination stability of the two is good, the specificity is strong, and the two are not influenced by organic solvents such as reagent concentration, pH environment, protein denaturant and the like. Since each avidin can bind 4 molecules of biotin, this feature can be used to construct a multi-level signal amplification system. Therefore, the biotin-avidin system can be used for quantitative and qualitative detection and positioning observation research of trace antigens, antibodies and receptors, and can also be prepared into an affinity medium for separation and purification of reactants in the various reaction systems. The invention is based on the high affinity between biotin and avidin, so that the reaction has higher binding efficiency even in the release of the binding pad and the chromatographic flow, thereby improving the detection sensitivity.
The tracer material of the present invention includes, but is not limited to, fluorescent latex microspheres, and the fluorescent latex microspheres are further surface-modified fluorescent latex microspheres, such as carboxyl-modified fluorescent latex microspheres. The fluorescent latex microspheres need to be activated, preferably, EDC/NHS cross-linking method is adopted to activate the fluorescent latex microspheres, the surfaces of the activated fluorescent latex microspheres contain stable active ester, then avidin needing to be cross-linked is added, and the active ester can be condensed with amino on the avidin to form stable amido bond, so that the avidin cross-linked fluorescent latex microspheres are obtained. Because the fluorescent latex microspheres contain a plurality of fluorescein molecules to amplify reaction signals, and the surface of each fluorescent latex microsphere can be crosslinked with a plurality of avidin molecules and the high affinity between biotin and avidin, the reaction of the invention has higher binding efficiency even in the release of the binding pad and the chromatographic flow, thereby improving the detection sensitivity.
In the above-mentioned fluorescence immunochromatography detection kit, the second antibody T3 is formed on the detection zone.
In the above fluorescence immunochromatography detection kit, the second antibody T3 is goat anti-rabbit IgG.
The second antibody T3 of the present invention is a corresponding polyclonal antibody recognizing the class of the T1 or T2 antibody not labeled with biotin, and the monoclonal antibody T1 or T2 of the present invention, which is not crosslinked with biotin, is immunoreactive at the second antibody T3. When the detection liquid contains the antigen to be detected, the formed avidin-crosslinked tracer substance-biotin-crosslinked monoclonal antibody-antigen to be detected-non-crosslinked biotin monoclonal antibody compound is bound on the detection belt and can be identified by a detection instrument, and the signal intensity is in direct proportion to the content of the antigen to be detected in the mixed liquid. The coating concentration of the T3 is far excessive, and different detection items can use different signal intensities generated by different calibration conversions to obtain the detection concentration value of the application object due to different ID cards.
In the above-mentioned fluorescence immunochromatography detection kit, the second antibody C1 is formed on the control band.
In the above-mentioned fluorescence immunochromatography detection kit, the second antibody C1 is goat anti-mouse IgG.
The optimal scheme of the quality control band is to select a secondary antibody corresponding to the biotin-labeled category in T1 or T2 in the system, so that the invention selects goat anti-mouse IgG, and can combine with the biotin-crosslinked monoclonal antibody T1 or T2 which is not crosslinked with the avidin of the complex formed with the antigen to be detected. The secondary antibody can capture a mouse anti-human antibody marked with biotin in a detection liquid, and indirectly combines with avidin-marked fluorescent latex particles to obtain a fluorescent signal, so that whether the quality control feedback detection liquid is suitable for the system or not is judged.
In the above fluorescence immunochromatography detection kit, the kit further comprises an ID card, and the ID card stores a calibration curve and also stores quality control and batch number information.
In one of the above-described fluorescence immunochromatography detection kits, the kit includes, but is not limited to, a kit for fluorescence immunochromatography detection of PCT (procalcitonin), cTnI (cardiac troponin I), CK-MB (creatine kinase MB isozyme), or BNP (B-type brain natriuretic peptide).
In the above fluorescence immunochromatography detection kit, when the kit is used for fluorescence immunochromatography detection of PCT detection, the monoclonal antibody T1 is a biotin-crosslinked mouse anti-human PCT monoclonal antibody, the monoclonal antibody T2 is a rabbit anti-human PCT monoclonal antibody, and a PCT calibration curve is stored in the ID card.
In the above-mentioned fluorescence immunochromatography detection kit, when the kit is used for fluorescence immunochromatography detection of cTnI, the monoclonal antibody T1 is a biotin-crosslinked mouse-anti-human cTnI monoclonal antibody, the monoclonal antibody T2 is a rabbit-anti-human cTnI monoclonal antibody, and the ID card stores therein a cTnI calibration curve.
In the above fluorescence immunochromatography detection kit, when the kit is used for detection of CK-MB by fluorescence immunochromatography, the monoclonal antibody T1 is a biotin-crosslinked mouse anti-human CK-MB monoclonal antibody, the monoclonal antibody T2 is a rabbit anti-human CK-MB monoclonal antibody, and the ID card stores a CK-MB calibration curve.
In the above fluorescence immunochromatography detection kit, when the kit is used for detecting BNP by fluorescence immunochromatography, the monoclonal antibody T1 is a biotin-crosslinked mouse anti-human BNP monoclonal antibody, the monoclonal antibody T2 is a rabbit anti-human BNP monoclonal antibody, and a BNP calibration curve is stored in the ID card.
The invention also aims to provide a detection method of the fluorescence immunochromatography detection kit, which comprises the following steps:
(1) and the volume ratio of 1: (1-50) adding the sample into the detection solution, and fully and uniformly mixing to obtain a mixed solution;
(2) adding 50-100 mu L of mixed solution into a test area of the chromatographic test paper, and reacting for 3-20 minutes;
(3) and putting the chromatographic test paper into a fluorescence detector, inserting an ID card, automatically detecting by the instrument and outputting a result report.
Compared with the prior art, the invention has the following advantages:
1. the invention uses micromolecular biotin to couple to the amino group on the surface of the monoclonal antibody, does not influence the combination of the antigen and the antibody, ensures that the reaction of two antibodies and the antigen of the double-antibody sandwich method is carried out in a liquid phase, does not influence the affinity of the antibody, and can avoid that after the antibody is directly marked on a tracer substance, a macromolecular tracer substance can influence the combination of the antigen and the antibody because the macromolecular tracer substance physically blocks the space near the Fab section of the antibody, thereby greatly enhancing the sensitivity. Meanwhile, the problem of poor stability caused by the fact that the tracer substance is contained in the detection liquid can be avoided.
2. The chromatographic test paper only contains an avidin crosslinked tracer substance and two high-affinity second antibodies, and the second antibodies T3 and C1 directly identify the first antibodies (T3 identifies the antibodies of unlabelled biotin class and C1 identifies the antibodies of biotin class) in chromatography, so that the problems of insufficient affinity and insufficient binding force of a detection line due to the fact that the high-specificity antibodies are relied on during detection of the antigen to be detected (the high-specificity monoclonal antibodies have relatively small affinity) are solved, and the sensitivity is further improved.
3. The chromatographic test paper only contains an avidin crosslinked tracer substance, two high-affinity second antibodies T3 and C1, can be used universally in all fluorescence immunochromatography detection projects, different detection projects only need to replace the monoclonal antibody T1 and the monoclonal antibody T2 in detection liquid, and repeated development work of different detection projects is avoided. In addition, the waste of resources caused by the fact that detection cannot be carried out due to the fact that detection liquid is not matched with the chromatography test paper in the actual use of a doctor is reduced.
4. The fluorescence immunochromatography detection kit disclosed by the invention has strong universality and can be applied to the detection of fluorescence immunochromatography of PCT, cTnI, CK-MB, BNP and the like.
Drawings
FIG. 1 is a schematic structural diagram of the chromatographic test paper of the present invention;
FIG. 2 is a PCT calibration curve for example 1 of the present invention;
FIG. 3 is a graph comparing example 1 of the present invention with a plasma sample measured by luminescence;
FIG. 4 is a comparison of the conventional method for preparing a PCT reagent and the luminescence method for detecting a plasma sample;
FIG. 5 is a graph comparing example 1 of the present invention with luminescence detection of low value plasma samples;
FIG. 6 is a comparison of PCT reagent preparation using conventional methods and luminescence detection of low value plasma samples;
FIG. 7 is a cTnI calibration curve for example 2 of the present invention;
FIG. 8 is a graph comparing example 2 of the present invention with a plasma sample measured by luminescence;
FIG. 9 is a graph comparing the preparation of cTnI reagents using conventional methods with the detection of plasma samples by luminescence;
FIG. 10 is a graph comparing example 2 of the present invention with luminescence detection of low value plasma samples;
FIG. 11 is a graph comparing the preparation of cTnI reagent using conventional methods with the detection of low value plasma samples by luminescence;
FIG. 12 is a CK-MB calibration curve according to example 3 of the present invention;
FIG. 13 is a graph comparing example 3 of the present invention with a plasma sample detected by luminescence;
FIG. 14 is a graph comparing the preparation of CK-MB reagent using a conventional method with the detection of plasma samples by luminescence;
FIG. 15 is a graph comparing example 3 of the present invention with luminescence detection of low value plasma samples;
FIG. 16 is a graph comparing the preparation of CK-MB reagent using conventional methods with the detection of low-value plasma samples by luminescence;
FIG. 17 is a BNP calibration curve of example 4 of the invention;
FIG. 18 is a graph comparing example 4 of the present invention with a plasma sample detected by luminescence;
FIG. 19 is a comparison of BNP reagent preparation using conventional methods and plasma sample detection using luminescence;
FIG. 20 is a graph comparing example 4 of the present invention with luminescence detection of low value plasma samples;
FIG. 21 is a graph comparing the preparation of BNP reagents using conventional methods with the detection of low-value partial plasma samples by luminescence;
in the figure: 1. a substrate; 2. a carrier; 3 a bonding pad; 4. a sample pad; 5. absorbent paper; 21. detecting a belt; 22. a quality control band; 201. a first region; 202. a second region; 203. and a third region.
Detailed Description
The following are specific embodiments of the present invention and are further described with reference to the accompanying drawings, but the present invention is not limited to these embodiments.
The kit is used for detecting the PCT by a fluorescence immunochromatography method and comprises a chromatography test paper, a detection solution and an ID card.
As shown in fig. 1, the chromatography test paper comprises a substrate 1, a carrier 2, a binding pad 3, a sample pad 4 and absorbent paper 5, wherein the carrier 2 is preferably nitrocellulose membrane, the substrate 1 is preferably PVC plate, the sample pad 4 is preferably glass fiber membrane, and the binding pad 3 is preferably polyester fiber. The carrier 2 is attached to the PVC substrate 1 through the nitrocellulose membrane, and comprises a first area 201, a second area 202 and a third area 203, wherein the first area 201 and the third area 203 are respectively positioned at two sides of the second area 202, a detection band 21 is formed at the intersection of the first area 201 and the second area 202, and a quality control band 22 is formed at the intersection of the second area 202 and the third area 203. The bonding pad 3 is attached to the first region 201 of the carrier 2 and extends a predetermined length from the edge of the first region 201 toward the second region 202, the sample pad 4 is attached to the bonding pad 3 and extends a predetermined length from the edge of the bonding pad 3 toward the first region 201, and the absorbent paper 5 is attached to the third region 203 of the carrier 2 and extends a predetermined length from the edge of the third region 203 toward the second region 202.
The conjugate pad 3 has formed thereon an avidin-crosslinked tracer material, preferably fluorescent latex microspheres. The preparation method comprises the following steps:
mixing the fluorescent latex microspheres with EDC and NHS, taking MES buffer as a reaction medium, and incubating for 10-120 minutes at 37 ℃ to obtain the activated fluorescent latex microspheres. The surface of the activated fluorescent latex microsphere contains stable active ester, then avidin needing to be crosslinked is added, and the active ester can be condensed with amino on the avidin to form stable amido bond, so that the avidin crosslinked fluorescent latex microsphere is obtained. The mass ratio of the avidin to the fluorescent latex microspheres in crosslinking is 1: 5-1: 50. then, macromolecules such as BSA, casein, PEG and the like are selected for blocking. And (3) cleaning the avidin crosslinked fluorescent latex microspheres in a centrifugal mode, and fixing the volume to a proper concentration by using a diluent. The diluent comprises the following components: PBS: 10m-100mM/L, CASEIN: 0.1% -1%, TW-20: 0.1% -2%, NaCl: 0.9%, trehalose: 0.1% -10%, NaN 3: 0.01-1%, and the pH value of the diluent is 6.8-8.0.
The bonding pad 3 is treated with a bonding pad pretreatment solution, which can accelerate the release of the sprayed fluorescent latex microspheres and improve the fluidity. The components of the pretreatment liquid are as follows: Tris-HCl: 20-50Mm, PVP: 0.1-2%, TW-20: 0.1-2%, Casein: 0.1-1%, pH 6-10, drying in a dehumidifier.
Then, the diluted avidin cross-linked fluorescent latex microspheres are sprayed on the dried bonding pad 3 by using an instrument at the concentration of 0.02mg/mL-0.2mg/mL, and dried before being placed in a dehumidifier.
A secondary goat anti-rabbit IgG antibody (T3) was used as the detection antibody, and the coating concentration was 1-10 mg/mL. A goat anti-mouse IgG secondary antibody (C1) was used as a quality control antibody, and the coating concentration was 1-10 mg/mL. The coating technology comprises the following steps: dissolving the antibody to be coated with 10-50mM phosphate buffer solution containing 0.9-3% NaCl, 0.1-5% trehalose and 0.1-2% surfactant, and having pH of 6-9. Wherein, the surfactant includes, but is not limited to, Tween 20, Tween 80, Triton X-100, polyethylene glycol and polyvinylpyrrolidone.
The preparation method of the chromatography test paper comprises the following steps: taking a nitrocellulose membrane (NC membrane) as a carrier 2, loading the prepared T3 liquid into a film cutting machine, setting the cutting amount to be 1 muL/cm, and cutting a line at the intersection of the first area 201 and the second area 202 to be used as a detection strip 21; the obtained C1 liquid was charged into a film cutter, and the amount of cutting was set to 1 μ L/cm, and a cut was made at the intersection of the second region 202 and the third region 203 to obtain a quality control band 22. The lined nitrocellulose membrane (NC membrane) was dried at 55 degrees for at least 2 hours with a constant humidity of 20% or less in the drying environment.
Then, the non-spotting surface of the NC film 2 is pasted on the PVC substrate 1, the bonding pad 3 is pasted on the PVC substrate 1, the edge of the first area 201 of the NC film 2 is covered and extends to a preset length in the direction of the second area 202, the sample pad 4 is pasted on the PVC substrate 1, the edge of the bonding pad 3 is covered and extends to a preset length in the direction of the first area 201, the absorbent paper 5 is pasted on the PVC substrate 1, the edge of the third area 203 of the NC film 2 is covered and extends to a preset length in the direction of the second area 202. And lightly pressing the adhered chromatographic test paper by using a rolling brush, then using a shearing machine to arrange the chromatographic test paper with the preset width of the adhered chromatographic test paper into a shell, putting the shell and a drying agent into an aluminum foil bag together, vacuumizing and sealing to obtain the final chromatographic test paper.
The detection solution comprises biotin cross-linked mouse anti-human PCT monoclonal antibody T1, rabbit anti-human PCT monoclonal antibody T2 and a diluent. The concentration of the biotin-labeled mouse anti-human PCT monoclonal antibody T1 in the detection solution is 0.001-0.05mg/mL, the concentration of the rabbit anti-human PCT monoclonal antibody T2 is 0.001-0.05mg/mL, and the components and the concentrations of the dilution solution are as follows: TRIS-HCl: 5-200mM/L, BSA: 0.1-3%, TW-20: 0.1% -2%, NaCl: 0.9% of NaN3: 0.01-1%, and the pH value of the diluent is 6.0-9.0.
The preparation method of the biotin-labeled mouse anti-human PCT monoclonal antibody T1 comprises the following steps: the biotin solution was dissolved in MES (pH6.0) and the resulting solution was adjusted to a concentration of 5-10mg/mL to obtain a biotin stock solution. And mixing the biotin storage solution with EDC and NHS, taking MES buffer solution as a reaction medium, and incubating for 10-120 minutes at 37 ℃ to obtain activated biotin. The surface of the activated biotin contains stable active ester, then the antibody needing to be crosslinked is added, and the active ester can be condensed with amino on the antibody to form stable amido bond, so that the biotin-labeled antibody is obtained. The mass ratio of biotin to antibody when crosslinked is 1: 5-1: 50. then, free biotin is removed by dialysis or ultrafiltration, and the volume is adjusted to a proper concentration by using a diluent. The diluent is as follows: TRIS-HCl: 5-200mM/L, BSA: 0.1% -3%, TW-20: 0.1% -2%, NaCl: 0.9% of NaN3: 0.01 to-1 percent, and the pH value of the diluent is 6.0 to 9.0.
In the above fluorescence immunochromatography detection kit, the kit further comprises an ID card, and the PCT calibration curve and the quality control and lot number information are stored in the ID card. The preparation method comprises the following steps: and writing the PCT calibration curve, the quality control information and the batch number information into the ID card by using a reader-writer through reading and writing software.
Example 1:
preparation of detection liquid:
and (3) activating the avidin by using an EDC/NHS crosslinking method. Biotin was dissolved in MES (pH6.0) and the solution was made to a concentration of 10mg/mL to obtain a biotin stock solution. The biotin stock solution was mixed with EDC and NHS and incubated for 60 minutes at 37 ℃ with 50mM MES buffer pH6.0 as the reaction medium to obtain activated biotin. 1mg of the activated biotin was added to 20mg of mouse anti-human PCT antibody (T1) and incubated at 37 ℃ for 60 minutes. Free biotin was then dialyzed out using TRIS-HCl 50mM/L pH7.4, and the dialysate was changed every 6 hours for a total of three times. Collecting the liquid in the dialysis bag, ultrafiltering and concentrating, and adding TRIS-HCl 50mM/L, 1% BSA, 0.6% Tween-20, 0.9% NaCl, 0.05% NaN3And diluting the biotin antibody diluent with the pH value of 7.4 to 10mg/ml for storage.
TRIS-HCl 50mM/L, 1% BSA, 0.6% Tween-20, 0.9% NaCl, 0.05% NaN was used3And diluting the diluent with the pH value of 7.4 to obtain a detection solution. The concentration of the biotin-labeled mouse anti-human PCT monoclonal antibody T1 in the detection solution is 0.01mg/mL, and the concentration of the rabbit anti-human PCT monoclonal antibody T2 in the detection solution is 0.01 mg/mL.
Preparation of the chromatography test paper:
preparing avidin cross-linked fluorescent latex microspheres: selecting carboxyl modified fluorescent latex microspheres, and activating the fluorescent latex by adopting an EDC/NHS crosslinking method. 10mg of fluorescent latex microspheres, 0.01mg of EDC and 0.01mg of NHS are mixed, MES buffer is used as a reaction medium, and the mixture is incubated at 37 ℃ for 20 minutes to obtain the activated fluorescent latex microspheres. To 10mg of activated fluorescent latex microspheres, 1mg of avidin to be crosslinked was added and incubated at 37 ℃ for 2 hours. Centrifuging to remove supernatant, adding 1% casein water solution for sealing, and ultrasonically shaking to disperse precipitate. And then cleaning the avidin crosslinked fluorescent latex microspheres in a centrifugal and ultrasonic mode, and fixing the volume to a proper concentration by using a diluent. The diluent comprises the following components: PBS 20mM/L, 0.5% CASEIN, 0.2% TW-20, 0.9% NaCl, 5% trehalose, 0.1% NaN3The pH was 7.4.
And (3) bonding pad treatment: the conjugate pad pretreatment solution was 20mM Tris-HCl pH 10, and the conjugate pad containing 2% PVP, 2% TW-20, 0.2% Casein, 30cm by 0.8cm polyester fiber was treated with 1.5mL of the sample pad pretreatment solution and dried before being placed in a dehumidifier. And then spraying the diluted avidin crosslinked fluorescent latex microspheres onto the dried bonding pads by using an instrument at the concentration of 0.1mg/mL, and drying before a dehumidifier.
Coating antibody: a goat anti-rabbit IgG secondary antibody (T3) subjected to affinity chromatography was used as a detection antibody, and the coating concentration was 1 mg/mL. A goat anti-mouse IgG secondary antibody (C1) was used as a quality control antibody, and the coating concentration was 1 mg/mL. The technical key of the coating is as follows: the antibody to be coated was dissolved using 20mM/L phosphate buffer containing 0.9% NaCl, 0.5% trehalose and 0.2% TW-20 surfactant at pH 7.4.
Then cutting the nitrocellulose membrane (NC membrane) into strips with the width of 2.5cm, loading the prepared T3 liquid into a film cutting machine, setting the cutting amount to be 1 mu L/cm, and cutting lines at the intersection of the first area and the second area to be used as detection strips; and (3) loading the prepared C1 liquid into a film cutting machine, setting the cutting amount to be 1 mu L/cm, cutting a line at the intersection of the second area and the third area to be used as a quality control band, wherein the length of the first area is 1cm, and the distance between the detection band and the quality control band is 0.4 cm. The lined nitrocellulose membrane (NC membrane) was dried at 55 degrees for at least 2 hours with a constant humidity of 20% or less in the drying environment.
Then the non-spotting surface of the NC film is stuck on a PVC base plate. The polyester fiber bonding pad was adhered to the PVC base plate covering the first zone edge of the NC film by 3 mm. The glass fiber sample pad is cut into a strip with the width of 1.6cm, and the strip is attached to a PVC base plate and covers the edge of the bonding pad for 3 mm. The absorbent paper is cut into a long strip with the width of 1.9cm, is adhered to the PVC bottom plate, and covers the edge of the third area of the NC film by 3 mm. And lightly pressing the adhered chromatographic test paper by using a rolling brush, cutting the adhered chromatographic test paper into 4mm wide chromatographic test paper by using a shearing machine, filling the chromatographic test paper into a shell, putting the shell and a drying agent into an aluminum foil bag, vacuumizing and sealing to obtain the final chromatographic test paper.
Preparation of an ID card:
and writing the PCT calibration curve, the quality control information and the batch number information into the purchased ID card by a reader-writer through reading and writing software to obtain the PCT ID card. The concentrations of the standard points in the calibration curve stored in the prepared PCT ID card are 0mg/L, 0.3mg/L, 1.5mg/L, 7.5mg/L, 25mg/L and 54mg/L respectively.
Carrying out a detection test on the PCT fluorescent immunochromatography detection kit obtained by the method: adding 25 mu L of sample into 110 mu L of detection solution, fully and uniformly mixing, adding 70 mu L of mixed solution into a test area of the chromatographic test paper, placing the chromatographic test paper into a fluorescence detector after reacting for 15 minutes, inserting an ID card, automatically detecting by an instrument and outputting a result report.
Meanwhile, the same sample is detected by adopting a luminescence method, and a reagent is prepared by using a direct labeling/coating monoclonal antibody method and is detected by adopting the luminescence method.
The detection result is as follows:
table 1:
| concentration value | Magnification of RLU | sd | cv |
| 54 | 3.787385 | 0.125877 | 3.32％ |
| 25 | 1.981016 | 0.024334 | 1.23％ |
| 7.5 | 0.695425 | 0.017449 | 2.51％ |
| 1.5 | 0.223694 | 0.013055 | 5.84％ |
| 0.3 | 0.067028 | 0.006493 | 9.69％ |
| 0 | 0.001159 | 0.001061 | - |
Table 1 and fig. 2 show PCT calibration curves of example 1 of the present invention, and as can be seen from table 1 and fig. 2, the linear range interval of the calibration curve meets the requirements of clinical examination.
FIG. 3 is a comparison graph of plasma samples detected by the luminescence method in example 1 of the present invention, and it can be seen from FIG. 3 that the immunochromatographic reagent prepared in example 1 and the chemiluminescence kit have good conformity in the aspects of correlation of sample measurement results and the like.
FIG. 4 is a graph showing the comparison between the conventional method for preparing a PCT reagent and the luminescence method for measuring a plasma sample, and FIG. 5 is a graph showing the comparison between example 1 of the present invention and the luminescence method for measuring a low-value plasma sample; FIG. 6 is a comparison of PCT reagent preparation using conventional methods and luminescence detection of low value plasma samples. As can be seen from fig. 4, 5 and 6, the immunochromatography reagent prepared by the conventional method has better conformity in the correlation between the sample measurement result and the chemiluminescence method kit, but the conformity in the low value part is inferior to the correlation between example 1 and the chemiluminescence method kit. The superiority of example 1 over the conventional method is demonstrated.
When the kit is used for detecting cTnI, CK-MB and BNP by a fluorescence immunochromatography, the kit comprises chromatography test paper, detection liquid and an ID card as when detecting PCT by the fluorescence immunochromatography, and the structure of the chromatography test paper is also shown in figure 1. The difference is that when the kit is used for detecting cTnI by a fluorescence immunochromatography method, the corresponding monoclonal antibody T1 in the detection solution is a biotin-crosslinked mouse anti-human cTnI monoclonal antibody, the monoclonal antibody T2 is a rabbit anti-human cTnI monoclonal antibody, and a cTnI calibration curve is stored in the ID card. When the kit is used for detecting CK-MB by a fluorescence immunochromatography method, the corresponding monoclonal antibody T1 in the detection solution is a biotin-crosslinked mouse anti-human CK-MB monoclonal antibody, the monoclonal antibody T2 is a rabbit anti-human CK-MB monoclonal antibody, and a CK-MB calibration curve is stored in the ID card. When the kit is used for detecting BNP by a fluorescence immunochromatographic assay, the corresponding monoclonal antibody T1 in the detection solution is a biotin-crosslinked mouse anti-human BNP monoclonal antibody, the monoclonal antibody T2 is a rabbit anti-human BNP monoclonal antibody, and a BNP calibration curve is stored in the ID card. Different detection items only need to replace the monoclonal antibody T1 and the monoclonal antibody T2 in the detection solution, so that repeated development work of different detection items is avoided.
Example 2:
example 2 differs from example 1 only in that the kit of example 2 was used for the detection of cTnI by fluorescence immunochromatography, the corresponding monoclonal antibody T1 in the detection solution was a biotin-crosslinked murine anti-human cTnI monoclonal antibody, the monoclonal antibody T2 was a rabbit anti-human cTnI monoclonal antibody, a cTnI calibration curve was stored in the ID card, and the concentrations of the standard points in the calibration curve were 0, 0.5, 2, 8, 32, and 54ng/mL, respectively.
And (3) carrying out a detection test on the cTnI fluorescence immunochromatography detection kit obtained by the method: and adding 75 mu L of sample into 75 mu L of detection solution, fully and uniformly mixing, adding 75 mu L of mixed solution into a test area of the chromatographic test paper, reacting for 12 minutes, putting the chromatographic test paper into a fluorescence detector, inserting an ID card, automatically detecting by an instrument, and outputting a result report.
Meanwhile, the same sample is detected by adopting a luminescence method, and a reagent is prepared by using a direct labeling/coating monoclonal antibody method and is detected by adopting the luminescence method.
The detection result is as follows:
table 2:
table 2 and fig. 7 are cTnI calibration curves of example 2 of the present invention, and it can be seen from table 2 and fig. 7 that the linear range interval of the calibration curve meets the clinical examination requirements.
FIG. 8 is a comparison graph of plasma samples detected by the luminescence method in example 2 of the present invention, and it can be seen from FIG. 8 that the immunochromatographic reagent prepared in example 2 and the chemiluminescence kit have good conformity in the correlation of the measurement results of the samples.
FIG. 9 is a graph showing the comparison between the preparation of cTnI reagent using a conventional method and the detection of plasma samples by luminescence, and FIG. 10 is a graph showing the comparison between example 2 of the present invention and the detection of low-value plasma samples by luminescence; fig. 11 is a graph comparing the preparation of cTnI reagent using conventional methods with the detection of low value plasma samples by luminescence. As is clear from fig. 9, 10, and 11, the immunochromatographic reagent prepared by the conventional method has good conformity in the correlation between the sample measurement result and the chemiluminescence method kit, but the conformity in the low value part is inferior to the correlation between example 2 and the chemiluminescence method kit. The superiority of example 2 over the conventional method is shown.
Example 3:
example 3 differs from example 1 only in that the kit of example 3 was used for the detection of CK-MB by fluorescence immunochromatography, the corresponding monoclonal antibody T1 in the detection solution was a biotin-crosslinked murine anti-human CK-MB monoclonal antibody, the monoclonal antibody T2 was a rabbit anti-human CK-MB monoclonal antibody, a CK-MB calibration curve was stored in the ID card, and the concentrations of the standard points in the calibration curve were 0, 6.25, 12.5, 25, 50, and 100ng/mL, respectively.
And (3) carrying out a detection test on the CK-MB fluorescence immunochromatography detection kit obtained by the method: and adding 75 mu L of sample into 75 mu L of detection solution, fully and uniformly mixing, adding 75 mu L of mixed solution into a test area of the chromatographic test paper, reacting for 12 minutes, putting the chromatographic test paper into a fluorescence detector, inserting an ID card, automatically detecting by an instrument, and outputting a result report.
Meanwhile, the same sample is detected by adopting a luminescence method, and a reagent is prepared by using a direct labeling/coating monoclonal antibody method and is detected by adopting the luminescence method.
The detection result is as follows:
table 3:
| concentration value | Magnification of | sd | cv | |
| 100 | 1.6653 | 0.1042 | 6.26％ | |
| 50 | 0.8216 | 0.0623 | 7.59％ | |
| 25 | 0.4425 | 0.0119 | 2.69％ | |
| 12.5 | 0.2169 | 0.0122 | 5.60％ | |
| 6.25 | 0.0921 | 0.0081 | 8.75％ | |
| 0 | 0.0079 | 0.0029 | - |
Table 3 and fig. 12 show CK-MB calibration curves of example 3 of the present invention, and it can be seen from table 3 and fig. 12 that the linear range interval of the calibration curve meets the clinical examination requirements.
FIG. 13 is a comparison graph of plasma samples detected by the luminescence method in example 3 of the present invention, and it can be seen from FIG. 13 that the immunochromatographic reagent prepared in example 3 and the chemiluminescence kit have good conformity in the correlation of the measurement results of the samples.
FIG. 14 is a graph showing comparison between the CK-MB reagent prepared by the conventional method and a plasma sample measured by luminescence, and FIG. 15 is a graph showing comparison between example 3 of the present invention and a plasma sample measured by luminescence in a low-value portion; FIG. 16 is a graph comparing the preparation of CK-MB reagent using conventional methods with the detection of low-value plasma samples by luminescence. As is clear from fig. 14, 15, and 16, the immunochromatographic reagent prepared by the conventional method has good conformity in the correlation between the sample measurement result and the chemiluminescence method kit, but the conformity in the low value part is inferior to the correlation between example 3 and the chemiluminescence method kit. The superiority of example 3 over the conventional method is demonstrated.
Example 4:
example 4 differs from example 1 only in that the kit of example 4 is used for detecting BNP by fluorescence immunochromatography, the corresponding monoclonal antibody T1 in the detection solution is a biotin-crosslinked murine anti-human BNP monoclonal antibody, the monoclonal antibody T2 is a rabbit anti-human BNP monoclonal antibody, a BNP calibration curve is stored in the ID card, and the concentrations of the standard points in the calibration curve are 0, 200, 1000, 5000, 15000, and 30000pg/mL, respectively.
Carrying out detection tests on the BNP fluorescence immunochromatography detection kit obtained by the method: and adding 75 mu L of sample into 75 mu L of detection solution, fully and uniformly mixing, adding 75 mu L of mixed solution into a test area of the chromatographic test paper, reacting for 12 minutes, putting the chromatographic test paper into a fluorescence detector, inserting an ID card, automatically detecting by an instrument, and outputting a result report.
Meanwhile, the same sample is detected by adopting a luminescence method, and a reagent is prepared by using a direct labeling/coating monoclonal antibody method and is detected by adopting the luminescence method.
The detection result is as follows:
table 4:
| concentration value | Magnification of | sd | cv | |
| 30000 | 3.6091 | 0.1878 | 5.20％ | |
| 15000 | 2.3643 | 0.0708 | 2.99％ | |
| 5000 | 0.8005 | 0.0165 | 2.06％ | |
| 1000 | 0.1688 | 0.0055 | 3.28％ | |
| 200 | 0.0406 | 0.0029 | 7.04％ | |
| 0 | 0.0079 | 0.0029 | - |
Table 4 and fig. 17 show BNP calibration curves of example 4 of the present invention, and it can be seen from table 4 and fig. 17 that the linear range interval of the calibration curve meets the requirements of clinical examination.
FIG. 18 is a comparison graph of plasma samples detected by the luminescence method in example 4 of the present invention, and it can be seen from FIG. 18 that the immunochromatographic reagent prepared in example 4 and the chemiluminescence kit have good compatibility in the aspects of correlation of sample measurement results and the like.
FIG. 19 is a graph comparing the preparation of BNP reagents using a conventional method with the detection of plasma samples by luminescence, and FIG. 20 is a graph comparing example 4 of the present invention with the detection of low-value partial plasma samples by luminescence; FIG. 21 is a graph comparing the preparation of BNP reagents using conventional methods with the detection of low-value partial plasma samples by luminescence. As is clear from fig. 19, 20, and 21, the immunochromatographic reagent prepared by the conventional method has good conformity in the correlation between the sample measurement result and the chemiluminescence method kit, but the conformity in the low value part is inferior to the correlation between example 4 and the chemiluminescence method kit. The superiority of example 4 over the conventional method is demonstrated.
In view of the numerous embodiments of the present invention, the experimental data of each embodiment is huge and is not suitable for being listed and explained herein one by one, but the contents to be verified and the final conclusions obtained by each embodiment are close. Therefore, the contents of the verification of each example are not described herein one by one, and only examples 1 to 4 are used as representatives to describe the excellent points of the present invention.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Claims (3)
1. A fluorescence immunochromatography detection kit is characterized by comprising
Chromatography test paper: the chromatographic test paper comprises a substrate and a bearing body attached to the substrate, wherein the bearing body comprises a first area, a second area and a third area, the first area and the third area are respectively positioned at two sides of the second area, a detection zone is formed at the intersection of the first area and the second area, a quality control zone is formed at the intersection of the second area and the third area, the chromatographic test paper further comprises a combination pad attached to the first area of the bearing body, a sample pad attached to the combination pad, and absorbent paper attached to the third area;
detection liquid: the detection solution comprises a monoclonal antibody T1, a monoclonal antibody T2 and a diluent; the monoclonal antibody T1 is a mouse anti-human monoclonal antibody, and the monoclonal antibody T2 is a rabbit anti-human monoclonal antibody; the mouse anti-human monoclonal antibody is a biotin cross-linked monoclonal antibody; an avidin-crosslinked tracer material is formed on the conjugate pad; a second antibody T3 is formed on the detection belt, and the second antibody T3 is goat anti-rabbit IgG;
the quality control band is provided with a second antibody C1, and the second antibody C1 is goat anti-mouse IgG.
2. The fluorescence immunochromatography detection kit according to claim 1, further comprising an ID card having a calibration curve stored therein.
3. The method of detecting the fluorescent immunochromatographic detection kit according to any one of claims 1 to 2, comprising the steps of:
(1) and the volume ratio of 1: (1-50) adding the sample into the detection solution, and fully and uniformly mixing to obtain a mixed solution;
(2) adding 50-100 mu L of mixed solution into a test area of the chromatographic test paper, and reacting for 3-20 minutes;
(3) and putting the chromatographic test paper into a fluorescence detector, inserting an ID card, automatically detecting by the instrument and outputting a result report.
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| WO2011005357A2 (en) * | 2009-07-08 | 2011-01-13 | Anp Technologies, Inc. | Immunogenicity assay |
| CN103163294A (en) * | 2013-03-21 | 2013-06-19 | 厦门市波生生物技术有限公司 | Chemiluminiscence detection kit for 5 items of hepatitis B and preparation method thereof |
| CN204188616U (en) * | 2014-10-28 | 2015-03-04 | 广州天宝颂原生物科技开发有限公司 | Procalcitonin immunochromatographiassay assay quantitative detection test paper |
| CN106771168A (en) * | 2016-12-30 | 2017-05-31 | 武汉纽康度生物科技股份有限公司 | Fluorescence immune chromatography detection card and its preparation method and application |
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| WO2011005357A2 (en) * | 2009-07-08 | 2011-01-13 | Anp Technologies, Inc. | Immunogenicity assay |
| CN103163294A (en) * | 2013-03-21 | 2013-06-19 | 厦门市波生生物技术有限公司 | Chemiluminiscence detection kit for 5 items of hepatitis B and preparation method thereof |
| CN204188616U (en) * | 2014-10-28 | 2015-03-04 | 广州天宝颂原生物科技开发有限公司 | Procalcitonin immunochromatographiassay assay quantitative detection test paper |
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