CN116298252A - Preparation method and application of novel PET (polyethylene terephthalate) substituted sample pad and binding pad test strip - Google Patents
Preparation method and application of novel PET (polyethylene terephthalate) substituted sample pad and binding pad test strip Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the technical field of test strip preparation methods, and provides a preparation method and application of a novel PET (polyethylene terephthalate) alternative sample pad and a test strip with a combination pad.
Description
Technical Field
The invention relates to the technical field of test strip preparation methods, in particular to a preparation method and application of a novel PET (polyethylene terephthalate) alternative sample pad and combination pad test strip.
Background
The immunochromatography test strip is used as a main stream product of POCT, and the traditional immunochromatography test strip comprises seven parts of a sample pad, a binding pad, an NC film (nitrocellulose film), a T line (detection line), a C line (quality control line), water absorbing paper and a PVC bottom plate. Common materials of the sample pad include glass fiber membrane, filter paper, non-woven fabrics and polyester materials, wherein the glass fiber membrane is widely used with the advantages of relatively tough and stable property, natural hydrophilicity, high chromatographic speed, large water absorption and good uniformity. The sample pad itself can filter impurities such as particulate matters and cells in the sample, the difference of the sample reaching the conjugate release and detection area can be reduced through chemical infiltration modification, the non-specific binding between the reaction membrane and the analyte or detection preparation can be reduced through adding the blocking substance, the chromatographic speed can be reduced through adding the substance which thickens the sample, and the reaction time can be increased.
At present, the material of the bonding pad is mainly glass fiber and polyester, and the bonding pad is generally composed of an antibody labeling compound marked with latex microspheres, colloidal gold particles or fluorescent microspheres. On one hand, the reagent is protected to be in an effective state in the shelf life of the product after being dried; on the other hand, after the sample reaches the binding pad, the antibody-labeled complex can be combined with the substance to be detected and completely released, so that the sample can be uniformly and reliably transferred and combined on an NC (nitrocellulose) membrane. Because traditional test paper strip sample pad and bond pad are through overlapping mode linking, overlap the part and probably lead to the chromatography in-process resistance to increase for the finished product is batched and is criticized the intra-batch difference and is showing, leads to the repeatability not good, if can use novel PET polyester film to replace sample pad and bond pad, not only simplified test paper strip pretreatment process and equipment process step, can also improved test paper strip's repeatability, specificity and sensitivity, make quantitative result more accurate.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects existing in the prior art, the invention provides a preparation method and application of a novel PET (polyethylene terephthalate) alternative sample pad and a binding pad test strip, and aims to prepare the test strip by using a PET polyester film to replace the sample pad and the binding pad, so that the repeatability, the specificity and the sensitivity of the test strip are improved.
The invention relates to a preparation method of a novel PET (polyethylene terephthalate) substitution sample pad and a bonding pad test strip, which comprises the following preparation steps:
step1, preparation of antibody protein solution to be marked: adding antibody protein to be marked into NaCl solution at 4 ℃ for dialysis overnight, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ for 1h at 10000r/min to obtain protein solution, and then adjusting the concentration of the protein solution to 1mg/ml by PBS with the concentration of 0.01mol/L to obtain the antibody protein solution to be marked;
step2, preparation of labeled antibody protein: the labeled antibody protein comprises a latex microsphere labeled antibody protein, a colloidal gold labeled antibody protein and a fluorescent microsphere labeled antibody protein, and the preparation processes of the three labeled antibody proteins are as follows:
1) Preparation of latex microsphere marked antibody protein: activating the latex microsphere by using NHS solution and EDC solution, and mixing the antibody protein solution to be marked with the activated latex microsphere according to the following steps of 3:1, mixing, stirring for 2-8 hours by a magnetic stirrer, centrifuging for 8-10 minutes under the condition of 10000r/min to obtain a precipitate, and diluting the precipitate by using an MES buffer solution to obtain a solution which is the latex microsphere marked antibody protein;
2) Preparation of colloidal gold-labeled antibody protein: using K 2 CO 3 The solution is adjusted to pH 9 of the colloidal gold solution and the antibody protein solution to be marked, then 100mL of the colloidal gold solution is poured into a magnetic stirrer to be stirred, 3mL of the antibody protein solution to be marked is added and then is continuously stirred for 10min, then 5mL of PEG20000 solution with concentration of 1% is added and is stirred for 5min again, and the precipitate is obtained by centrifugation at 4 ℃ and 10000r/min for 30 min; suspending the precipitate in MES buffer solution containing PEG20000, centrifuging again under the above conditions, recovering with MES buffer solution containing PEG20000, separating and purifying by gel chromatography with SephadexG-200 column, eluting with MES buffer solution containing BSA, and obtaining colloidal gold labeled antibody protein;
3) Preparation of fluorescent microsphere labeled antibody protein: 1mL of fluorescent microsphere with the concentration of 10mg/mL is measured and added into 3mL of MES buffer solution, 15 mu L of EDC solution is added, 200 mu L of antibody protein solution to be marked is added after uniform oscillation, the mixture is fully mixed, stirred and reacted for 2 hours at room temperature, 1/10 volume of BSA solution is added for sealing for 1 hour, centrifugation is carried out for 20 minutes at the temperature of 4 ℃ and the speed of 11000r/min, supernatant is removed, 3mL of MES buffer solution is used for recovery, and centrifugation is carried out for one time under the conditions to obtain precipitate, and the precipitate is re-dissolved to 3mL by using MES buffer solution, thus obtaining the fluorescent microsphere marked antibody protein;
Step3, preparation of PET to be assembled: spraying the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein on a PET polyester film by using a Bio-DotXYZ3050 three-dimensional spray point platform respectively, wherein the spraying amount is 3 mu L/cm, and drying the PET polyester film in an oven at 50 ℃ for 48 hours to obtain PET to be assembled;
step4, preparation of nitrocellulose membrane: firstly, screening a second specific antibody protein and a goat anti-mouse antibody which can be specifically combined with the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein; then, the concentrations of three second specific antibody proteins in Step5 are respectively regulated to 0.6mg/mL by using PB buffer solution (3% sucrose is added) with the concentration of 0.01-M, pH and 1.0mg/mL by using sheep anti-mouse antibody in Step5, then the three second specific antibody proteins and sheep anti-mouse antibody are coated on a nitrocellulose membrane by using a Bio-Dot XYZ3050 type three-dimensional spray point platform and are respectively used as a detection line and a quality control line, the two lines are separated by 5mm, the spray film amount is 1.0 mu L/cm, and the nitrocellulose membrane is obtained by drying in an oven at 50 ℃ for 48 hours, wherein the three detection lines of the nitrocellulose membrane are respectively selected second specific antibody proteins capable of being specifically combined with a latex microsphere marked antibody protein, a colloidal gold marked antibody protein and a fluorescent microsphere marked antibody protein, and the quality control line of the three nitrocellulose membrane is the mouse anti-mouse antibody;
Step5, assembling a test strip: sequentially and lapped and stuck with PET to be assembled in Step5, nitrocellulose membrane and absorbent paper in Step6 on an adhesive PVC bottom plate, then cut into rectangles of 60mm multiplied by 4.0mm by a slitter, and then put on a plastic clamping seat to obtain a latex microsphere marked antibody protein test strip, a colloidal gold marked antibody protein test strip and a fluorescent microsphere marked antibody protein test strip respectively. The latex microsphere marked antibody protein test strip, the colloidal gold marked antibody protein test strip and the fluorescent microsphere marked antibody protein test strip can be respectively arranged in the plastic shell of the existing test strip to form a kit.
Further, the preparation method of the NHS solution in Step2 comprises the following steps: 1g of 2- (N-morpholine) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by using a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of N-hydroxysuccinimide is weighed and fully dissolved in the mixed solution, and the NHS solution is obtained.
Further, the preparation method of the EDC solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and fully dissolved in the mixed solution, and the EDC solution is obtained.
Further, the method for activating the latex microspheres in Step2 comprises the following steps: according to 1:15-20 weight percent of latex microsphere is added into NHS solution, EDC solution with the same amount as the NHS solution is added after uniform mixing, after ultrasonic dispersion for 1min, centrifugation is carried out for 10min under the condition of 10000-12000r/min, and supernatant is discarded, thus obtaining the activated latex microsphere.
Further, the preparation method of the MES buffer solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is weighed and fully dissolved in 90mL of deionized water, the pH value is adjusted to 5.5 by sodium hydroxide solution, and deionized water is added to fix the volume to 100mL.
Further, K in Step2 2 CO 3 The concentration of the solution was 0.1mol/L.
Further, the ratio of the PEG20000 in the MES buffer solution containing PEG20000 in Step3 to the MES buffer solution is 1:2 (mg/mL), and the BSA concentration in Step3 in the MES buffer containing BSA was 0.1%.
Further, the preparation method of the colloidal gold solution in Step2 comprises the following steps: measuring 99mL of triple distilled water filtered by a 0.22um filter membrane, adding 1mL of chloroauric acid solution with the concentration of 1% into a conical flask, heating and boiling in an electric furnace, adding 1.5mL of triple distilled water with the concentration of 1% filtered by the 0.22um filter membrane under the stirring condition, continuously heating until the pale yellow chloroauric acid water gradually turns from yellow to black and finally turns to red or reddish wine, continuously heating and stirring for 2-3min after the solution turns to clear red or reddish wine, naturally cooling to room temperature, and adding triple distilled water to a constant volume of 100mL to obtain the colloidal gold solution.
Further, the preparation method of the BSA solution in Step2 comprises the following steps: 0.1g of bovine serum albumin was weighed and sufficiently dissolved in 10mL of ultrapure water.
An application of a novel PET substitution sample pad and conjugate pad test strip, the application comprising the following two modes:
(1) The latex microsphere marked antibody protein test strip and the colloidal gold marked antibody protein test strip are used as qualitative products, a sample and a sample preservation solution are fully mixed, a mixed solution of the sample and the sample preservation solution is added into a sample hole of the test strip, and negative and positive can be judged by naked eyes after reacting for 5-10 min;
(2) The fluorescent microsphere marked antibody protein test strip is used as a quantitative product, a mixed solution of a sample and a sample preservation solution is added into a sample hole of the test strip, after the reaction is carried out for 5-10min, the ratio of the fluorescence intensity of a detection line to the fluorescence intensity of a quality control line is manufactured by using a standard substance at the position of 365nm of the wavelength of an excitation light source, and the content of a substance to be detected in the sample is read.
Advantageous effects
The invention provides a preparation method and application of a novel PET (polyethylene terephthalate) alternative sample pad and a combination pad test strip, and compared with the prior art, the preparation method has the following beneficial effects:
the method for preparing the test strip by using the PET polyester film to replace the sample pad and the bonding pad not only simplifies the pretreatment process and the assembly process steps of the test strip, but also improves the consistency of fluid in the chromatography process, reduces the adsorption of the fluid, and ensures quick and smooth release, thereby reducing the difference between finished products and in batches, remarkably improving the color development effect of the test strip, greatly improving the repeatability, the specificity and the sensitivity of the test strip, ensuring that the distribution of the points of different concentration standard substances in the standard curve of the double-antibody sandwich quantitative product is more uniform, the R2 value is closer to 1, and the quantitative result is more accurate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is evident that the drawings in the following description are only some embodiments of the present invention and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a schematic diagram of a test strip according to the present invention;
FIG. 2 is a flowchart of a method for preparing a test strip according to the present invention;
FIGS. 3-10 are graphs showing the results of the performance test of the present invention;
reference numerals in the drawings represent respectively: 1-a PVC bottom plate; 2-PET to be assembled; 3-nitrocellulose membrane; 4-water absorbing paper; 5-detecting lines; and 6, a quality control line.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation method of the novel PET substitution sample pad and the binding pad test strip comprises the following preparation steps:
step1, preparation of antibody protein solution to be marked: adding antibody protein to be marked into NaCl solution at 4 ℃ for dialysis overnight, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ for 1h at 10000r/min to obtain protein solution, and then adjusting the concentration of the protein solution to 1mg/ml by PBS with the concentration of 0.01mol/L to obtain the antibody protein solution to be marked;
step2, preparation of labeled antibody protein: the labeled antibody protein comprises a latex microsphere labeled antibody protein, a colloidal gold labeled antibody protein and a fluorescent microsphere labeled antibody protein, and the preparation processes of the three labeled antibody proteins are as follows:
1) Preparation of latex microsphere marked antibody protein: activating the latex microsphere by using NHS solution and EDC solution, and mixing the antibody protein solution to be marked with the activated latex microsphere according to the following steps of 3:1, mixing, stirring for 2 hours by a magnetic stirrer, centrifuging for 8 minutes under the condition of 10000r/min to obtain a precipitate, and diluting the precipitate by using an MES buffer solution to obtain a solution which is the latex microsphere marked antibody protein;
2) Preparation of colloidal gold-labeled antibody protein: using K 2 CO 3 The solution is adjusted to pH 9 of the colloidal gold solution and the antibody protein solution to be marked, then 100mL of the colloidal gold solution is poured into a magnetic stirrer to be stirred, 3mL of the antibody protein solution to be marked is added and then is continuously stirred for 10min, then 5mL of PEG20000 solution with concentration of 1% is added and is stirred for 5min again, and the precipitate is obtained by centrifugation at 4 ℃ and 10000r/min for 30 min; suspending the precipitate in MES buffer solution containing PEG20000, centrifuging again under the above conditions, recovering with MES buffer solution containing PEG20000, separating and purifying by gel chromatography with SephadexG-200 column, eluting with MES buffer solution containing BSA, and collecting colloidal gold labeled antibody protein;
3) Preparation of fluorescent microsphere labeled antibody protein: 1mL of fluorescent microsphere with the concentration of 10mg/mL is measured and added into 3mL of MES buffer solution, 15 mu L of EDC solution is added, 200 mu L of antibody protein solution to be marked is added after shaking uniformly, the mixture is stirred and reacted for 2 hours at room temperature after fully mixing, 1/10 volume of BSA solution is added for sealing for 1 hour, centrifugation is carried out for 20 minutes at the temperature of 4 ℃ and the speed of 11000r/min, supernatant is removed, 3mL of MES buffer solution is used for recovery, and then the sediment is obtained after one centrifugation under the conditions, and is redissolved into 3mL by using MES buffer solution, thus obtaining the fluorescent microsphere marked antibody protein;
Step3, preparation of PET to be assembled: spraying the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein on a PET polyester film by using a Bio-DotXYZ3050 three-dimensional spray point platform respectively, wherein the spraying amount is 3 mu L/cm, and drying the PET polyester film in an oven at 50 ℃ for 48 hours to obtain PET to be assembled;
step4, preparation of nitrocellulose membrane: firstly, screening a second specific antibody protein and a goat anti-mouse antibody which can be specifically combined with the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein; then, the concentrations of three second specific antibody proteins in Step5 are respectively regulated to 0.6mg/mL by using PB buffer solution (3% sucrose is added) with the concentration of 0.01-M, pH and 1.0mg/mL by using sheep anti-mouse antibody in Step5, then three second specific antibody proteins and sheep anti-mouse antibody are coated on a nitrocellulose membrane by using a Bio-Dot XYZ3050 type three-dimensional spray point platform, the two specific antibody proteins are respectively used as a detection line and a quality control line, the two lines are separated by 5mm, the spray film amounts are 1.0 mu L/cm, and the nitrocellulose membrane is dried for 48 hours at 50 ℃, so that the nitrocellulose membrane is obtained, the three detection lines of the nitrocellulose membrane are respectively selected second specific antibody proteins which can be specifically combined with a latex microsphere marked antibody protein, a colloidal gold marked antibody protein and a fluorescent microsphere marked antibody protein, and the quality control line of the three nitrocellulose membrane is an anti-mouse antibody;
Step5, assembling a test strip: sequentially and lapped and stuck with PET to be assembled in Step5, nitrocellulose membrane and absorbent paper in Step6 on an adhesive PVC bottom plate, then cut into rectangles of 60mm multiplied by 4.0mm by a slitter, and then put on a plastic clamping seat to obtain a latex microsphere marked antibody protein test strip, a colloidal gold marked antibody protein test strip and a fluorescent microsphere marked antibody protein test strip respectively. The latex microsphere marked antibody protein test strip, the colloidal gold marked antibody protein test strip and the fluorescent microsphere marked antibody protein test strip can be respectively arranged in the plastic shell of the existing test strip to form a kit.
The preparation method of the NHS solution in Step2 comprises the following steps: 1g of 2- (N-morpholine) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by using a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of N-hydroxysuccinimide is weighed and fully dissolved in the mixed solution, and the NHS solution is obtained.
The preparation method of EDC solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and fully dissolved in the mixed solution, and the EDC solution is obtained.
The method for activating the latex microspheres in Step2 comprises the following steps: according to 1:15, adding the latex microspheres into NHS solution, uniformly mixing, adding EDC solution with the same amount as the NHS solution, ultrasonically dispersing for 1min, centrifuging for 10min under the condition of 10000r/min, and discarding the supernatant to obtain the activated latex microspheres.
The preparation method of the MES buffer solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is weighed and fully dissolved in 90mL of deionized water, the pH value is adjusted to 5.5 by sodium hydroxide solution, and deionized water is added to fix the volume to 100mL.
K in Step2 2 CO 3 The concentration of the solution was 0.1mol/L.
The ratio of PEG20000 in MES buffer solution containing PEG20000 in Step3 to MES buffer solution is 1:2 (mg/mL), and the BSA concentration in Step3 in the MES buffer containing BSA was 0.1%.
The preparation method of the colloidal gold solution in Step2 comprises the following steps: measuring 99mL of triple distilled water filtered by a 0.22um filter membrane, adding 1mL of chloroauric acid solution with the concentration of 1% into a conical flask, then placing the conical flask on an electric furnace for heating and boiling, adding 1.5mL of triple nano water solution with the concentration of 1% filtered by the 0.22um filter membrane under the stirring condition, continuing heating until the pale yellow chloroauric acid water solution gradually turns from yellow to black and finally turns to red or reddish wine, continuing heating and stirring for 2min after the solution turns to clear red or reddish wine, and adding triple distilled water to a volume of 100mL after naturally cooling to room temperature, thus obtaining the colloidal gold solution.
The preparation method of the BSA solution in Step2 comprises the following steps: 0.1g of bovine serum albumin was weighed and sufficiently dissolved in 10mL of ultrapure water.
The application of the novel PET substitution sample pad and the binding pad test strip comprises the following two modes:
(1) The latex microsphere marked antibody protein test strip and the colloidal gold marked antibody protein test strip are used as qualitative products, a sample and a sample preservation solution are fully mixed, a mixed solution of the sample and the sample preservation solution is added into a sample hole of the test strip, and negative and positive can be judged by naked eyes after reacting for 5 min;
(2) The fluorescent microsphere marked antibody protein test strip is used as a quantitative product, a mixed solution of a sample and a sample preservation solution is added into a sample hole of the test strip, after reacting for 5min, the ratio of the fluorescence intensity of a detection line to the fluorescence intensity of a quality control line is manufactured by using a standard product at the position of 365nm of the wavelength of an excitation light source, and the content of a substance to be detected in the sample is read.
Example 2
The preparation method of the novel PET substitution sample pad and the binding pad test strip comprises the following preparation steps:
step1, preparation of antibody protein solution to be marked: adding antibody protein to be marked into NaCl solution at 4 ℃ for dialysis overnight, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ for 1h at 10000r/min to obtain protein solution, and then adjusting the concentration of the protein solution to 1mg/ml by PBS with the concentration of 0.01mol/L to obtain the antibody protein solution to be marked;
Step2, preparation of labeled antibody protein: the labeled antibody protein comprises a latex microsphere labeled antibody protein, a colloidal gold labeled antibody protein and a fluorescent microsphere labeled antibody protein, and the preparation processes of the three labeled antibody proteins are as follows:
1) Preparation of latex microsphere marked antibody protein: activating the latex microsphere by using NHS solution and EDC solution, and mixing the antibody protein solution to be marked with the activated latex microsphere according to the following steps of 3:1, mixing, stirring for 8 hours by a magnetic stirrer, centrifuging for 10 minutes under the condition of 10000r/min to obtain a precipitate, and diluting the precipitate by using an MES buffer solution to obtain a solution which is the latex microsphere marked antibody protein;
2) Preparation of colloidal gold-labeled antibody protein: using K 2 CO 3 The solution the pH of the colloidal gold solution and the antibody protein solution to be labeled were adjusted to 9, followed byPouring 100mL of colloidal gold solution into a magnetic stirrer for stirring, adding 3mL of antibody protein solution to be marked, continuing stirring for 10min, then adding 5mL of PEG20000 solution with concentration of 1%, stirring for 5min again, and centrifuging for 30min at 4 ℃ and 10000r/min to obtain a precipitate; suspending the precipitate in MES buffer solution containing PEG20000, centrifuging again under the above conditions, recovering with MES buffer solution containing PEG20000, separating and purifying by gel chromatography with SephadexG-200 column, eluting with MES buffer solution containing BSA, and collecting colloidal gold labeled antibody protein;
3) Preparation of fluorescent microsphere labeled antibody protein: 1mL of fluorescent microsphere with the concentration of 10mg/mL is measured and added into 3mL of MES buffer solution, 15 mu L of EDC solution is added, 200 mu L of antibody protein solution to be marked is added after shaking uniformly, the mixture is stirred and reacted for 2 hours at room temperature after fully mixing, 1/10 volume of BSA solution is added for sealing for 1 hour, centrifugation is carried out for 20 minutes at the temperature of 4 ℃ and the speed of 11000r/min, supernatant is removed, 3mL of MES buffer solution is used for recovery, and then the sediment is obtained after one centrifugation under the conditions, and is redissolved into 3mL by using MES buffer solution, thus obtaining the fluorescent microsphere marked antibody protein;
step3, preparation of PET to be assembled: spraying the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein on a PET polyester film by using a Bio-DotXYZ3050 three-dimensional spray point platform respectively, wherein the spraying amount is 3 mu L/cm, and drying the PET polyester film in an oven at 50 ℃ for 48 hours to obtain PET to be assembled;
step4, preparation of nitrocellulose membrane: firstly, screening a second specific antibody protein and a goat anti-mouse antibody which can be specifically combined with the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein; then, the concentrations of three second specific antibody proteins in Step5 are respectively regulated to 0.6mg/mL by using PB buffer solution (3% sucrose is added) with the concentration of 0.01-M, pH and 1.0mg/mL by using sheep anti-mouse antibody in Step5, then three second specific antibody proteins and sheep anti-mouse antibody are coated on a nitrocellulose membrane by using a Bio-Dot XYZ3050 type three-dimensional spray point platform, the two specific antibody proteins are respectively used as a detection line and a quality control line, the two lines are separated by 5mm, the spray film amounts are 1.0 mu L/cm, and the nitrocellulose membrane is dried for 48 hours at 50 ℃, so that the nitrocellulose membrane is obtained, the three detection lines of the nitrocellulose membrane are respectively selected second specific antibody proteins which can be specifically combined with a latex microsphere marked antibody protein, a colloidal gold marked antibody protein and a fluorescent microsphere marked antibody protein, and the quality control line of the three nitrocellulose membrane is an anti-mouse antibody;
Step5, assembling a test strip: sequentially and lapped and stuck with PET to be assembled in Step5, nitrocellulose membrane and absorbent paper in Step6 on an adhesive PVC bottom plate, then cut into rectangles of 60mm multiplied by 4.0mm by a slitter, and then put on a plastic clamping seat to obtain a latex microsphere marked antibody protein test strip, a colloidal gold marked antibody protein test strip and a fluorescent microsphere marked antibody protein test strip respectively. The latex microsphere marked antibody protein test strip, the colloidal gold marked antibody protein test strip and the fluorescent microsphere marked antibody protein test strip can be respectively arranged in the plastic shell of the existing test strip to form a kit.
The preparation method of the NHS solution in Step2 comprises the following steps: 1g of 2- (N-morpholine) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by using a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of N-hydroxysuccinimide is weighed and fully dissolved in the mixed solution, and the NHS solution is obtained.
The preparation method of EDC solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and fully dissolved in the mixed solution, and the EDC solution is obtained.
The method for activating the latex microspheres in Step2 comprises the following steps: according to 1:20, adding the latex microspheres into NHS solution according to the weight ratio, uniformly mixing, adding EDC solution equivalent to the NHS solution, ultrasonically dispersing for 1min, centrifuging for 10min under the condition of 12000r/min, and discarding the supernatant to obtain the activated latex microspheres.
The preparation method of the MES buffer solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is weighed and fully dissolved in 90mL of deionized water, the pH value is adjusted to 5.5 by sodium hydroxide solution, and deionized water is added to fix the volume to 100mL.
K in Step2 2 CO 3 The concentration of the solution was 0.1mol/L.
The ratio of PEG20000 in MES buffer solution containing PEG20000 in Step3 to MES buffer solution is 1:2 (mg/mL), and the BSA concentration in Step3 in the MES buffer containing BSA was 0.1%.
The preparation method of the colloidal gold solution in Step2 comprises the following steps: measuring 99mL of triple distilled water filtered by a 0.22um filter membrane, adding 1mL of chloroauric acid solution with the concentration of 1% into a conical flask, then placing the conical flask on an electric furnace for heating and boiling, adding 1.5mL of triple nano water solution with the concentration of 1% filtered by the 0.22um filter membrane under the stirring condition, continuing heating until the pale yellow chloroauric acid water solution gradually turns from yellow to black and finally turns to red or reddish wine, continuing heating and stirring for 2min after the solution turns to clear red or reddish wine, and adding triple distilled water to a volume of 100mL after naturally cooling to room temperature, thus obtaining the colloidal gold solution.
The preparation method of the BSA solution in Step2 comprises the following steps: 0.1g of bovine serum albumin was weighed and sufficiently dissolved in 10mL of ultrapure water.
The application of the novel PET substitution sample pad and the binding pad test strip comprises the following two modes:
(1) The latex microsphere marked antibody protein test strip and the colloidal gold marked antibody protein test strip are used as qualitative products, a sample and a sample preservation solution are fully mixed, a mixed solution of the sample and the sample preservation solution is added into a sample hole of the test strip, and negative and positive can be judged by naked eyes after reacting for 10 min;
(2) The fluorescent microsphere marked antibody protein test strip is used as a quantitative product, a mixed solution of a sample and a sample preservation solution is added into a sample hole of the test strip, after reacting for 10min, the ratio of the fluorescence intensity of a detection line to the fluorescence intensity of a quality control line is manufactured by using a standard product at the position of 365nm of the wavelength of an excitation light source, and the content of a substance to be detected in the sample is read.
Example 3
The preparation method of the novel PET substitution sample pad and the binding pad test strip comprises the following preparation steps:
step1, preparation of antibody protein solution to be marked: adding antibody protein to be marked into NaCl solution at 4 ℃ for dialysis overnight, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ for 1h at 10000r/min to obtain protein solution, and then adjusting the concentration of the protein solution to 1mg/ml by PBS with the concentration of 0.01mol/L to obtain the antibody protein solution to be marked;
Step2, preparation of labeled antibody protein: the labeled antibody protein comprises a latex microsphere labeled antibody protein, a colloidal gold labeled antibody protein and a fluorescent microsphere labeled antibody protein, and the preparation processes of the three labeled antibody proteins are as follows:
1) Preparation of latex microsphere marked antibody protein: activating the latex microsphere by using NHS solution and EDC solution, and mixing the antibody protein solution to be marked with the activated latex microsphere according to the following steps of 3:1, mixing, stirring for 6 hours by a magnetic stirrer, centrifuging for 9 minutes under the condition of 10000r/min to obtain a precipitate, and diluting the precipitate by using an MES buffer solution to obtain a solution which is the latex microsphere marked antibody protein;
2) Preparation of colloidal gold-labeled antibody protein: using K 2 CO 3 The solution is adjusted to pH 9 of the colloidal gold solution and the antibody protein solution to be marked, then 100mL of the colloidal gold solution is poured into a magnetic stirrer to be stirred, 3mL of the antibody protein solution to be marked is added and then is continuously stirred for 10min, then 5mL of PEG20000 solution with concentration of 1% is added and is stirred for 5min again, and the precipitate is obtained by centrifugation at 4 ℃ and 10000r/min for 30 min; suspending the precipitate in MES buffer solution containing PEG20000, centrifuging again under the above conditions, recovering with MES buffer solution containing PEG20000, separating and purifying by gel chromatography with SephadexG-200 column, eluting with MES buffer solution containing BSA, and collecting colloidal gold labeled antibody protein;
3) Preparation of fluorescent microsphere labeled antibody protein: 1mL of fluorescent microsphere with the concentration of 10mg/mL is measured and added into 3mL of MES buffer solution, 15 mu L of EDC solution is added, 200 mu L of antibody protein solution to be marked is added after shaking uniformly, the mixture is stirred and reacted for 2 hours at room temperature after fully mixing, 1/10 volume of BSA solution is added for sealing for 1 hour, centrifugation is carried out for 20 minutes at the temperature of 4 ℃ and the speed of 11000r/min, supernatant is removed, 3mL of MES buffer solution is used for recovery, and then the sediment is obtained after one centrifugation under the conditions, and is redissolved into 3mL by using MES buffer solution, thus obtaining the fluorescent microsphere marked antibody protein;
step3, preparation of PET to be assembled: spraying the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein on a PET polyester film by using a Bio-DotXYZ3050 three-dimensional spray point platform respectively, wherein the spraying amount is 3 mu L/cm, and drying the PET polyester film in an oven at 50 ℃ for 48 hours to obtain PET to be assembled;
step4, preparation of nitrocellulose membrane: firstly, screening a second specific antibody protein and a goat anti-mouse antibody which can be specifically combined with the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein; then, the concentrations of three second specific antibody proteins in Step5 are respectively regulated to 0.6mg/mL by using PB buffer solution (3% sucrose is added) with the concentration of 0.01-M, pH and 1.0mg/mL by using sheep anti-mouse antibody in Step5, then three second specific antibody proteins and sheep anti-mouse antibody are coated on a nitrocellulose membrane by using a Bio-Dot XYZ3050 type three-dimensional spray point platform, the two specific antibody proteins are respectively used as a detection line and a quality control line, the two lines are separated by 5mm, the spray film amounts are 1.0 mu L/cm, and the nitrocellulose membrane is dried for 48 hours at 50 ℃, so that the nitrocellulose membrane is obtained, the three detection lines of the nitrocellulose membrane are respectively selected second specific antibody proteins which can be specifically combined with a latex microsphere marked antibody protein, a colloidal gold marked antibody protein and a fluorescent microsphere marked antibody protein, and the quality control line of the three nitrocellulose membrane is an anti-mouse antibody;
Step5, assembling a test strip: sequentially and lapped and stuck with PET to be assembled in Step5, nitrocellulose membrane and absorbent paper in Step6 on an adhesive PVC bottom plate, then cut into rectangles of 60mm multiplied by 4.0mm by a slitter, and then put on a plastic clamping seat to obtain a latex microsphere marked antibody protein test strip, a colloidal gold marked antibody protein test strip and a fluorescent microsphere marked antibody protein test strip respectively. The latex microsphere marked antibody protein test strip, the colloidal gold marked antibody protein test strip and the fluorescent microsphere marked antibody protein test strip can be respectively arranged in the plastic shell of the existing test strip to form a kit.
The preparation method of the NHS solution in Step2 comprises the following steps: 1g of 2- (N-morpholine) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by using a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of N-hydroxysuccinimide is weighed and fully dissolved in the mixed solution, and the NHS solution is obtained.
The preparation method of EDC solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and fully dissolved in the mixed solution, and the EDC solution is obtained.
The method for activating the latex microspheres in Step2 comprises the following steps: according to 1:18, adding the latex microspheres into NHS solution according to the weight ratio, uniformly mixing, adding EDC solution equivalent to the NHS solution, ultrasonically dispersing for 1min, centrifuging for 10min under the condition of 11000r/min, and discarding the supernatant to obtain the activated latex microspheres.
The preparation method of the MES buffer solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is weighed and fully dissolved in 90mL of deionized water, the pH value is adjusted to 5.5 by sodium hydroxide solution, and deionized water is added to fix the volume to 100mL.
K in Step2 2 CO 3 The concentration of the solution was 0.1mol/L.
The ratio of PEG20000 in MES buffer solution containing PEG20000 in Step3 to MES buffer solution is 1:2 (mg/mL), and the BSA concentration in Step3 in the MES buffer containing BSA was 0.1%.
The preparation method of the colloidal gold solution in Step2 comprises the following steps: measuring 99mL of triple distilled water filtered by a 0.22um filter membrane, adding 1mL of chloroauric acid solution with the concentration of 1% into a conical flask, then placing the conical flask on an electric furnace for heating and boiling, adding 1.5mL of triple nano water solution with the concentration of 1% filtered by the 0.22um filter membrane under the stirring condition, continuing heating until the pale yellow chloroauric acid water solution gradually turns from yellow to black and finally turns to red or reddish wine, continuing heating and stirring for 3min after the solution turns to clear red or reddish wine, and adding triple distilled water to a volume of 100mL after naturally cooling to room temperature, thus obtaining the colloidal gold solution.
The preparation method of the BSA solution in Step2 comprises the following steps: 0.1g of bovine serum albumin was weighed and sufficiently dissolved in 10mL of ultrapure water.
The application of the novel PET substitution sample pad and the binding pad test strip comprises the following two modes:
(1) The latex microsphere marked antibody protein test strip and the colloidal gold marked antibody protein test strip are used as qualitative products, a sample and a sample preservation solution are fully mixed, a mixed solution of the sample and the sample preservation solution is added into a sample hole of the test strip, and negative and positive can be judged by naked eyes after the reaction is carried out for 8 min;
(2) The fluorescent microsphere marked antibody protein test strip is used as a quantitative product, a mixed solution of a sample and a sample preservation solution is added into a sample hole of the test strip, after the reaction is carried out for 8min, the ratio of the fluorescence intensity of a detection line to the fluorescence intensity of a quality control line is manufactured by using a standard product at the position of 365nm of the wavelength of an excitation light source, and the content of a substance to be detected in the sample is read.
Performance testing
1. The same test sample liquid is tested by the HCG latex microsphere marked antibody protein test strip prepared by the preparation method in any one of the examples 1-3 and the common test strip with the sample pad and the binding pad, the common test strip with the sample pad and the binding pad is marked as a control 1, the HCG latex microsphere marked antibody protein test strip prepared by the preparation method in any one of the examples 1-3 is marked as a control 2, and a comparison chart of test results is recorded in figure 3 of the attached drawing of the specification.
2. The HCG latex microsphere labeled antibody protein test strip and the common test strip with the sample pad and the binding pad (namely NC test strip) prepared by the preparation method in any one of examples 1 to 3 are used for respectively detecting detection sample liquids with different concentrations, wherein the concentration of the detection sample liquid is respectively high (0.09 ng/ml), medium (0.05 ng/ml) and low (0.001 ng/ml), each concentration gradient detects two test strips, the concentration of the detection sample liquid of the NC test strip is medium (0.05 ng/ml), and the comparison chart of the detection results is recorded in fig. 4 of the attached drawing of the specification.
3. The test strips of three different production batches of HCG fluorescent microsphere labeled antibody protein test strips prepared by the preparation method in any of examples 1-3 were used for measuring three different concentration samples of high concentration (0.09 ng/ml), medium concentration (0.05 ng/ml) and low concentration (0.001 ng/ml), and the batch-to-batch and batch-to-batch differences of the three different production batches were analyzed, and the obtained detection results and data were recorded in the attached figures 5-10 of the specification.
According to the figures 3-10 of the drawings in the specification, the test strip prepared by the preparation method provided by the invention has higher detection sensitivity and better application prospect.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The preparation method of the novel PET substitution sample pad and the binding pad test strip is characterized by comprising the following preparation steps:
step1, preparation of antibody protein solution to be marked: adding antibody protein to be marked into NaCl solution at 4 ℃ for dialysis overnight, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ for 1h at 10000r/min to obtain protein solution, and then adjusting the concentration of the protein solution to 1mg/ml by PBS with the concentration of 0.01mol/L to obtain the antibody protein solution to be marked;
step2, preparation of labeled antibody protein: the labeled antibody protein comprises a latex microsphere labeled antibody protein, a colloidal gold labeled antibody protein and a fluorescent microsphere labeled antibody protein, and the preparation processes of the three labeled antibody proteins are as follows:
1) Preparation of latex microsphere marked antibody protein: activating the latex microsphere by using NHS solution and EDC solution, and mixing the antibody protein solution to be marked with the activated latex microsphere according to the following steps of 3:1, mixing, stirring for 2-8 hours by a magnetic stirrer, centrifuging for 8-10 minutes under the condition of 10000r/min to obtain a precipitate, and diluting the precipitate by using an MES buffer solution to obtain a solution which is the latex microsphere marked antibody protein;
2) Preparation of colloidal gold-labeled antibody protein: using K 2 CO 3 The solution is adjusted to pH 9 of the colloidal gold solution and the antibody protein solution to be marked, then 100mL of the colloidal gold solution is poured into a magnetic stirrer to be stirred, 3mL of the antibody protein solution to be marked is added and then is continuously stirred for 10min, then 5mL of PEG20000 solution with concentration of 1% is added and is stirred for 5min again, and the precipitate is obtained by centrifugation at 4 ℃ and 10000r/min for 30 min; suspending the precipitate in MES buffer solution containing PEG20000, centrifuging again under the above conditions, recovering with MES buffer solution containing PEG20000, separating and purifying by gel chromatography with SephadexG-200 column, eluting with MES buffer solution containing BSA, and obtaining colloidal gold labeled antibody protein;
3) Preparation of fluorescent microsphere labeled antibody protein: 1mL of fluorescent microsphere with the concentration of 10mg/mL is measured and added into 3mL of MES buffer solution, 15 mu L of EDC solution is added, 200 mu L of antibody protein solution to be marked is added after uniform oscillation, the mixture is fully mixed, stirred and reacted for 2 hours at room temperature, 1/10 volume of BSA solution is added for sealing for 1 hour, centrifugation is carried out for 20 minutes at the temperature of 4 ℃ and the speed of 11000r/min, supernatant is removed, 3mL of MES buffer solution is used for recovery, and centrifugation is carried out for one time under the conditions to obtain precipitate, and the precipitate is re-dissolved to 3mL by using MES buffer solution, thus obtaining the fluorescent microsphere marked antibody protein;
step3, preparation of PET to be assembled: spraying the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein on a PET polyester film by using a Bio-DotXYZ3050 three-dimensional spray point platform respectively, wherein the spraying amount is 3 mu L/cm, and drying the PET polyester film in an oven at 50 ℃ for 48 hours to obtain PET to be assembled;
step4, preparation of nitrocellulose membrane: firstly, screening a second specific antibody protein and a goat anti-mouse antibody which can be specifically combined with the latex microsphere marked antibody protein, the colloidal gold marked antibody protein and the fluorescent microsphere marked antibody protein; then, the concentrations of three second specific antibody proteins in Step5 are respectively regulated to 0.6mg/mL by using PB buffer solution (3% sucrose is added) with the concentration of 0.01-M, pH and 1.0mg/mL by using sheep anti-mouse antibody in Step5, then the three second specific antibody proteins and sheep anti-mouse antibody are coated on a nitrocellulose membrane by using a Bio-Dot XYZ3050 type three-dimensional spray point platform and are respectively used as a detection line and a quality control line, the two lines are separated by 5mm, the spray film amount is 1.0 mu L/cm, and the nitrocellulose membrane is obtained by drying in an oven at 50 ℃ for 48 hours, wherein the three detection lines of the nitrocellulose membrane are respectively selected second specific antibody proteins capable of being specifically combined with a latex microsphere marked antibody protein, a colloidal gold marked antibody protein and a fluorescent microsphere marked antibody protein, and the quality control line of the three nitrocellulose membrane is the mouse anti-mouse antibody;
Step5, assembling a test strip: sequentially and lapped and stuck with PET to be assembled in Step5, nitrocellulose membrane and absorbent paper in Step6 on an adhesive PVC bottom plate, then cut into rectangles of 60mm multiplied by 4.0mm by a slitter, and then put on a plastic clamping seat to obtain a latex microsphere marked antibody protein test strip, a colloidal gold marked antibody protein test strip and a fluorescent microsphere marked antibody protein test strip respectively.
2. The method for preparing the novel PET substitution sample pad and conjugate pad test strip according to claim 1, wherein the method for preparing the NHS solution in Step2 is as follows: 1g of 2- (N-morpholine) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by using a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of N-hydroxysuccinimide is weighed and fully dissolved in the mixed solution, and the NHS solution is obtained.
3. The method for preparing the novel PET substitution sample pad and conjugate pad test strip according to claim 1, wherein the method for preparing the EDC solution in Step2 comprises the following steps: 1g of 2- (N-morpholino) ethanesulfonic acid is firstly weighed and fully dissolved in 90mL of deionized water, the pH value is regulated to 6 by a sodium hydroxide solution, deionized water is added to fix the volume to 100mL, then 500mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and fully dissolved in the mixed solution, and the EDC solution is obtained.
4. The method for preparing the novel PET substitution sample pad and the binding pad test strip according to claim 1, wherein the method for activating the latex microspheres in Step2 is as follows: according to 1:15-20 weight percent of latex microsphere is added into NHS solution, EDC solution with the same amount as the NHS solution is added after uniform mixing, after ultrasonic dispersion for 1min, centrifugation is carried out for 10min under the condition of 10000-12000r/min, and supernatant is discarded, thus obtaining the activated latex microsphere.
5. The method for preparing the novel PET substitution sample pad and the binding pad test strip according to claim 1, wherein the method for preparing the MES buffer solution in Step2 is as follows: 1g of 2- (N-morpholino) ethanesulfonic acid is weighed and fully dissolved in 90mL of deionized water, the pH value is adjusted to 5.5 by sodium hydroxide solution, and deionized water is added to fix the volume to 100mL.
6. The method for preparing a novel PET-substituted sample pad and conjugate pad test strip as claimed in claim 1, which comprisesCharacterized in that K in Step2 2 CO 3 The concentration of the solution was 0.1mol/L.
7. The method for preparing the novel PET substitution sample pad and the binding pad test strip according to claim 1, wherein the ratio of the PEG20000 in the MES buffer solution containing PEG20000 in Step3 to the MES buffer solution is 1:2 (mg/mL), and the BSA concentration in Step3 in the MES buffer containing BSA was 0.1%.
8. The method for preparing the novel PET substitution sample pad and the binding pad test strip according to claim 1, wherein the method for preparing the colloidal gold solution in Step2 is as follows: measuring 99mL of triple distilled water filtered by a 0.22um filter membrane, adding 1mL of chloroauric acid solution with the concentration of 1% into a conical flask, heating and boiling in an electric furnace, adding 1.5mL of triple distilled water with the concentration of 1% filtered by the 0.22um filter membrane under the stirring condition, continuously heating until the pale yellow chloroauric acid water gradually turns from yellow to black and finally turns to red or reddish wine, continuously heating and stirring for 2-3min after the solution turns to clear red or reddish wine, naturally cooling to room temperature, and adding triple distilled water to a constant volume of 100mL to obtain the colloidal gold solution.
9. The method for preparing the novel PET substitution sample pad and the binding pad test strip according to claim 1, wherein the method for preparing the BSA solution in Step2 is as follows: 0.1g of bovine serum albumin was weighed and sufficiently dissolved in 10mL of ultrapure water.
10. Use of a novel PET replacement sample pad and conjugate pad strip according to any of claims 1-9, characterized in that the use comprises the following two ways:
(1) The latex microsphere marked antibody protein test strip and the colloidal gold marked antibody protein test strip are used as qualitative products, a sample and a sample preservation solution are fully mixed, a mixed solution of the sample and the sample preservation solution is added into a sample hole of the test strip, and negative and positive can be judged by naked eyes after reacting for 5-10 min;
(2) The fluorescent microsphere marked antibody protein test strip is used as a quantitative product, a mixed solution of a sample and a sample preservation solution is added into a sample hole of the test strip, after the reaction is carried out for 5-10min, the ratio of the fluorescence intensity of a detection line to the fluorescence intensity of a quality control line is manufactured by using a standard substance at the position of 365nm of the wavelength of an excitation light source, and the content of a substance to be detected in the sample is read.
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