Chemiluminescence immunoassay test strip and preparation method and application thereof
阅读说明：本技术 一种化学发光免疫检测试纸条及其制备方法与应用 (Chemiluminescence immunoassay test strip and preparation method and application thereof ) 是由 王永彬 于 2019-10-25 设计创作，主要内容包括：本发明提供了一种化学发光免疫检测试纸条及其制备方法与应用,试纸条的制备方法为：将羧基活化的富勒烯与检测抗体、酶偶联,得到抗体-富勒烯-酶复合材料,喷在载体上制成标记片；羧基活化的碳纳米管与检测抗体偶联,喷在载体上制成捕捉片；将玻璃纤维素膜、捕捉片、标记片、样品片、吸水片贴在PVC塑料底板上,并喷涂羊抗鼠多克隆抗体溶液作为质控线。本发明利用羧基化富勒烯将酶与抗体偶联,使二者牢固结合,并且不浪费抗体的结合位点,未结合的抗体和酶通过洗涤即可分离,同时,检测抗体偶联在羧基化碳纳米管上,再通过碳纳米管与纤维素膜的作用,将检测抗体固定在试纸条上,不会造成待测抗原的流失,降低检测误差。(The invention provides a chemiluminescent immunoassay test strip and a preparation method and application thereof, wherein the preparation method of the test strip comprises the following steps: coupling the fullerene activated by carboxyl with a detection antibody and enzyme to obtain an antibody-fullerene-enzyme composite material, and spraying the antibody-fullerene-enzyme composite material on a carrier to prepare a marking sheet; coupling the carboxyl activated carbon nano tube with a detection antibody, and spraying the carbon nano tube on a carrier to prepare a capture sheet; the glass cellulose membrane, the capture sheet, the marker sheet, the sample sheet and the water absorption sheet are pasted on a PVC plastic bottom plate, and the goat anti-mouse polyclonal antibody solution is sprayed to serve as a quality control line. The enzyme and the antibody are coupled by using the carboxylated fullerene, so that the enzyme and the antibody are firmly combined, the binding sites of the antibody are not wasted, the antibody and the enzyme which are not combined can be separated by washing, meanwhile, the detection antibody is coupled on the carboxylated carbon nano tube, and the detection antibody is fixed on the test strip under the action of the carbon nano tube and the cellulose membrane, so that the loss of the antigen to be detected is avoided, and the detection error is reduced.)
1. A preparation method of a chemiluminescent immunoassay test strip is characterized by comprising the following steps:
(1) preparation of a marking sheet: performing carboxyl activation on the carboxylated fullerene, mixing the carboxyl activated fullerene with a detection antibody and an enzyme, and performing a coupling reaction to obtain an antibody-fullerene-enzyme composite material; spraying the antibody-fullerene-enzyme composite material on a marking material carrier to prepare a marking sheet for later use;
(2) preparing a catching sheet: performing carboxyl activation on the carboxylated carbon nanotubes, mixing the carboxyl activated carbon nanotubes with a detection antibody, and performing a coupling reaction to obtain a capture material; the catching material is sprayed on the catching material carrier to prepare a catching sheet for standby;
(3) assembling the test strip: get PVC plastic bottom plate (6), paste glass cellulose membrane, catching piece, mark piece, sample piece, the piece that absorbs water on PVC plastic bottom plate in proper order, from left to right position order on PVC plastic bottom plate is in proper order: the kit comprises a sample sheet (1), a marking sheet (2), a capture sheet (3), a glass cellulose membrane (4) and a water absorption sheet (5), wherein a goat anti-mouse polyclonal antibody solution is sprayed on the glass cellulose membrane and between the capture sheet and the water absorption sheet to serve as a quality control line (7).
2. The method for preparing a chemiluminescent immunoassay test strip according to claim 1, wherein the carboxylated fullerene of step (1) comprises one or more of carboxylated fullerene of mono-organic carboxylic acid with surface grafted C1-C3, carboxylated fullerene of di-organic carboxylic acid with surface grafted C2-C6; the C1-C3 monocarboxylic organic carboxylic acid is carboxylic acid with only one carboxyl, wherein the carbon number of the carbon chain is 1-3; the C2-C6 dicarboxylic acid is carboxylic acid with two carboxyl groups and 2-6 carbon atoms in the carbon chain.
3. The method for preparing a chemiluminescent immunoassay test strip of claim 1, wherein the capture material carrier of step (2) is a nitrocellulose membrane, and the capture material dispersion is 4-4.5 μ g/cm 2The amount of the compound is sprayed on a nitrocellulose membrane, and the capture sheet is obtained after drying.
4. The method for preparing a chemiluminescent immunoassay test strip according to claim 1, wherein in step (3), the PVC plastic base plate at one end of the water absorbing sheet of the test strip is sprayed with fluorinated polyurethane, and the mixture is left to stand and dry to form a film, so as to form the protective tape (8).
5. The method for preparing a chemiluminescent immunoassay test strip according to any one of claims 1 to 4, wherein after the antibody-fullerene-enzyme composite solution is obtained in the step (1), the carboxyl-activated fluorescent microspheres are added into the solution, the solution is stirred at room temperature to perform a coupling reaction, the supernatant is centrifuged and removed, the supernatant is resuspended in PBS buffer solution, the supernatant is sealed with a sealing solution, the supernatant is washed with the PBS buffer solution and then dispersed in the PBS buffer solution to prepare a fluorescent microsphere-antibody-fullerene-enzyme composite dispersion solution, and the fluorescent microsphere-antibody-fullerene-enzyme composite material is sprayed on a marking material carrier to prepare a marking sheet for later use.
6. A chemiluminescent immunoassay test strip prepared by the preparation method of any one of claims 1 to 4.
7. A chemiluminescent immunoassay test strip prepared by the preparation method of claim 5.
8. The application of the chemiluminescent immunoassay test strip of claim 6 in immune monitoring is characterized in that an antigen concentration gradient solution to be detected is respectively dripped on a sample plate, the test strip is horizontally placed, standing chromatography reaction is carried out for 10-15min, a chemiluminescent substrate corresponding to enzyme is added in a capture area, a chemiluminescent signal is collected by a chemiluminescent instrument after reaction, and a chemiluminescent percent luminous rate-antigen concentration semilog linear regression standard curve to be detected is prepared; dripping a sample to be detected on a sample sheet, horizontally placing a test strip, standing for chromatographic reaction for 10-15min, adding a chemiluminescent substrate corresponding to an enzyme in a capture area, collecting a chemiluminescent signal by using a chemiluminescent instrument after the reaction, wherein the chemiluminescent intensity is in positive correlation with the quantity of enzyme molecules, the quantity of the enzyme molecules is in positive correlation with the quantity of an antigen to be detected, and substituting the chemiluminescent percentage luminescence rate into a standard curve to calculate the concentration of the antigen to be detected in a sample liquid to be detected.
9. The chemiluminescent immunoassay test strip of claim 7, which is applied to immunoassay monitoring, wherein when performing rapid qualitative detection, a sample to be detected is dripped onto a sample plate, the test strip is horizontally placed, and the standing chromatography reaction lasts for 10-15 min; irradiating the capture area by using an ultraviolet lamp, wherein if fluorescence exists, the sample liquid to be detected contains the antigen to be detected, and if no fluorescence exists, the sample liquid to be detected does not contain the antigen to be detected;
when quantitative chemiluminescence detection is carried out, the antigen concentration gradient solution to be detected is respectively dripped onto a sample sheet, the test strip is horizontally placed, standing chromatography reaction is carried out for 10-15min, a chemiluminescence substrate corresponding to enzyme is added into a capture area, a chemiluminescence instrument is used for collecting chemiluminescence signals after reaction, and a chemiluminescence percent luminosity factor-antigen concentration semilogarithmic regression standard curve to be detected is manufactured; dripping a sample to be detected on a sample sheet, horizontally placing a test strip, standing for chromatographic reaction for 10-15min, adding a chemiluminescent substrate corresponding to an enzyme in a capture area, collecting a chemiluminescent signal by using a chemiluminescent instrument after the reaction, wherein the chemiluminescent intensity is in positive correlation with the quantity of enzyme molecules, the quantity of the enzyme molecules is in positive correlation with the quantity of an antigen to be detected, and substituting the chemiluminescent percentage luminescence rate into a standard curve to calculate the concentration of the antigen to be detected in a sample liquid to be detected;
when the fluorescence quantitative detection is carried out, the antigen concentration gradient solution to be detected is respectively dripped on a sample slice, the test strip is horizontally placed, the standing chromatography reaction is carried out for 10-15min, the fluorescence intensity of a capture area and a quality control line is detected, and a linear regression standard curve of the fluorescence intensity of the capture area/the fluorescence intensity of the quality control line-the antigen concentration to be detected in a standard solution is manufactured; dripping a sample to be detected on a sample sheet, horizontally placing the test strip, standing for chromatographic reaction for 10-15min, detecting the fluorescence intensity of the capture area and the quality control line, substituting the fluorescence intensity of the capture area/the fluorescence intensity of the quality control line into a standard curve equation, and calculating to obtain the concentration of the antigen to be detected in the sample liquid to be detected.
10. The use of the chemiluminescent immunoassay test strip of claim 8 or 9 in immunoassay monitoring, wherein a result test is further required for the quantitative detection of chemiluminescence, and when the result test fails, the result test is performed again by using fluorescent quantitative detection, wherein the result test comprises the following steps:
a1, obtaining the standard deviation of the sample liquid drop to be detected in the historical inspection, and determining the repeated detection times of the sample to be detected;
wherein N is the repeated detection times, Car is the standard deviation of the historical examination,
step A2, carrying out N times of repeated tests on the sample liquid drop to be tested, and storing the concentration of the antigen to be tested in the sample liquid to be tested obtained by the tests into a concentration vector X;
step A3, determining the fluctuation frequency of the concentration vector X;
wherein S is the fluctuation frequency, X iIn order to test the sample liquid drop to be tested for the ith time, the concentration of the antigen to be tested in the sample liquid to be tested is obtained, wherein i is 1, 2 and 3L N;
step A3, determining the qualification rate of the concentration vector X;
wherein rt is the percent of pass;
and A4, when the qualification rate rt is larger than a preset value, taking the average value of the concentration vector X as the concentration of the antigen to be detected, otherwise, detecting the concentration of the antigen to be detected by using fluorescent quantitative detection.
Technical Field
The invention relates to the technical field of immunoassay, in particular to a chemiluminescent immunoassay test strip and a preparation method and application thereof.
Background
A chemiluminescence immunoassay test strip is a method for calculating the concentration of an antigen to be detected by combining an enzyme-labeled antibody with the antigen to be detected, capturing a product after the combination by using a detection antibody, and measuring the luminescence percentage after the reaction of a chemiluminescence agent and an enzyme.
In addition, the current method for fixing the detection antibody on the test strip is usually only coating, which may cause the detection antibody coated on the test strip to have low density and possibly cause detection errors, and meanwhile, the loosely coated detection antibody may move along with the chromatography along with the combined product, which may also cause larger detection errors.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for preparing a chemiluminescent immunoassay test strip, which is characterized by comprising the following steps:
(1) preparation of a marking sheet: performing carboxyl activation on the carboxylated fullerene, mixing the carboxyl activated fullerene with a detection antibody and an enzyme, and performing a coupling reaction to obtain an antibody-fullerene-enzyme composite material; spraying the antibody-fullerene-enzyme composite material on a marking material carrier to prepare a marking sheet for later use;
(2) preparing a catching sheet: performing carboxyl activation on the carboxylated carbon nanotubes, mixing the carboxyl activated carbon nanotubes with a detection antibody, and performing a coupling reaction to obtain a capture material; the catching material is sprayed on the catching material carrier to prepare a catching sheet for standby;
(3) assembling the test paper: get PVC
The carboxylated fullerene in the step (1) comprises one or more of carboxylated fullerene of mono-organic carboxylic acid with C1-C3 grafted on the surface and carboxylated fullerene of di-organic carboxylic acid with C2-C6 grafted on the surface.
The C1-C3 monocarboxylic organic carboxylic acid is carboxylic acid with only one carboxyl, wherein the carbon number of the carbon chain is 1-3; the C2-C6 dicarboxylic acid is carboxylic acid with two carboxyl groups and 2-6 carbon atoms in the carbon chain.
The carboxyl activation of the carboxylated fullerene specifically comprises the following steps: dissolving the carboxylated fullerene in deionized water, adding EDC and NHS, stirring for reaction at normal temperature, centrifuging to remove supernatant, washing with PBS buffer solution to remove unreacted EDC and NHS, and performing ultrasonic dispersion with PBS buffer solution to obtain carboxyl activated fullerene aqueous solution.
The carboxylated fullerene is dissolved in deionized water, the concentration of the prepared solution is 1mg/mL calculated by the fullerene, the final concentration of the added EDC is 20mg/mL, the final concentration of the added NHS is 10mg/mL, the reaction temperature is normal temperature, the time is 1h, and the specification of the PBS buffer solution is 1.0mM and the pH is 7.4; the concentration of the aqueous carboxyl group-activated fullerene solution obtained by dispersion was 1mg/mL in terms of fullerene.
The coupling reaction of the carboxyl activated fullerene, a detection antibody and an enzyme specifically comprises the following steps: adding a detection antibody solution and an enzyme into the obtained carboxyl activated fullerene aqueous solution, stirring to perform a coupling reaction, centrifuging to remove the supernatant, washing with a PBS buffer solution to remove unreacted free detection antibody and enzyme, and redispersing with the PBS buffer solution to prepare an antibody-fullerene-enzyme composite material solution. .
The concentration of the detection antibody solution is 4 mu g/mL, and the volume ratio of the carboxyl activated fullerene aqueous solution to the detection antibody solution to the enzyme is 100:15: 1; the coupling reaction temperature is 4 ℃ and the time is 10-24 h; the specification of the PBS buffer solution is 1.0mM, pH7.4; the concentration of the antibody-fullerene-enzyme composite material solution obtained by dispersion is 1mg/mL calculated by fullerene.
Preferably, after the antibody-fullerene-enzyme composite material solution is obtained in the step (1), adding the carboxyl activated fluorescent microspheres into the solution, stirring at room temperature to perform a coupling reaction, centrifuging to remove the supernatant, resuspending with a PBS buffer solution, blocking with a blocking solution, washing with the PBS buffer solution, and dispersing with the PBS buffer solution to prepare the fluorescent microsphere-antibody-fullerene-enzyme composite material dispersion solution.
The final concentration of the added carboxyl activated fluorescent microspheres is 5.0mg/mL, the coupling reaction temperature is 25-30 ℃, the time is 2-3h, and the specification of the PBS buffer solution is 1.0mM and the pH value is 7.4; the concentration of the dispersed fluorescent microsphere-antibody-fullerene-enzyme composite material dispersion liquid is 1mg/mL calculated by fullerene.
The dispersion of the fluorescent microsphere-antibody-fullerene-enzyme composite material is 4-4.5 mu g/cm 2The dosage of the marking agent is sprayed on a marking material carrier, and the marking piece is obtained after drying.
The preparation method of the carboxyl activated fluorescent microsphere comprises the following steps: placing the carboxyl fluorescent microspheres in a boric acid buffer solution, adding EDC and NHS, stirring for reaction, centrifuging to remove a supernatant, and washing by using a PBS buffer solution to remove unreacted EDC and NHS.
The concentration of the boric acid buffer solution is 0.05M, the final concentration of the added EDC is 0.01M, the final concentration of the added NHS is 0.02M, the reaction temperature is room temperature, and the reaction time is 1-1.5 h; the PBS buffer specification is 1.0mM, pH7.4.
The above-mentioned marking material carrier is glass cellulose membrane, and the antibody-fullerene-enzyme composite material solution is added in the concentration of 1-1.5 microgram/cm 2The amount of the compound is sprayed on a glass cellulose membrane, and the marking piece is obtained after drying. Preferably, the glass cellulose membrane as a carrier of the marking material is treated with a treatment liquid a: soaking the glass cellulose membrane in the treatment solution A for 2h, and drying at the constant temperature of 37 ℃ for 2 h.
The treatment fluid A is 0.1MPH8.0Tris-Hcl buffer solution, which comprises 20 mass percent of Tween 20, 0.5-1 mass percent of BSA, 3 mass percent of sucrose and 0.05 mass percent of NaN 3。
The carboxyl activation of the carboxylated carbon nanotube in the step (2) specifically comprises the following steps: dispersing the carboxylated carbon nanotubes in deionized water, adding EDC and NHS, stirring and reacting at normal temperature, centrifuging to remove supernatant, washing with PBS buffer solution to remove unreacted EDC and NHS, and performing ultrasonic dispersion with PBS buffer solution to obtain carboxyl activated carbon nanotube dispersion liquid.
Dispersing the carboxylated carbon nanotubes in deionized water to prepare a dispersion liquid with the concentration of 4mg/mL calculated by the carbon nanotubes, the final concentration of added EDC of 20mg/mL and the final concentration of added NHS of 10mg/mL, the reaction temperature is normal temperature, the reaction time is 1h, and the specification of the PBS buffer solution is 1.0mM and the pH value is 7.4; the concentration of the carboxyl-activated carbon nanotube dispersion obtained by dispersion was 4mg/mL in terms of carbon nanotubes.
The coupling reaction of the carboxyl activated carbon nanotube and the detection antibody specifically comprises the following steps: adding a detection antibody solution into the obtained carboxyl activated carbon nano tube dispersion liquid, stirring to perform coupling reaction, centrifuging to remove supernatant, washing by using PBS buffer solution to remove unreacted free detection antibody, and re-dispersing by using the PBS buffer solution to prepare a capture material dispersion liquid.
Wherein the concentration of the detection antibody solution is 4 mug/mL, and the volume ratio of the carboxyl activated carbon nano tube dispersion liquid to the detection antibody solution is 20: 3; the coupling reaction temperature is 4 ℃ and the time is 10-24 h; the specification of the PBS buffer solution is 1.0mM, pH7.4; the concentration of the capturing material dispersion obtained by dispersion was 4mg/mL in terms of carbon nanotubes.
The carrier of the capture material is a nitrocellulose membrane, and the dispersion liquid of the capture material is 4-4.5 mu g/cm 2The amount of the compound is sprayed on a nitrocellulose membrane, and the capture sheet is obtained after drying.
Preferably, the nitrocellulose membrane as a capture material carrier is treated with a treatment liquid a: soaking the nitrocellulose membrane in the treatment solution A for 2h, and drying at the constant temperature of 37 ℃ for 2 h.
The treatment fluid A is 0.1MPH8.0Tris-Hcl buffer solution, which comprises 20 mass percent of Tween 20, 0.5-1 mass percent of BSA, 3 mass percent of sucrose and 0.05 mass percent of NaN 3。
The left side edge of the marking sheet extends to a position between the sample sheet and the PVC plastic bottom plate, the width of the area of the marking sheet covered by the sample sheet is 1-3mm, and the left side of the sample sheet extends out of the left side edge of the marking sheet; the left edge of the catching sheet extends to the position between the marking sheet and the PVC plastic bottom plate, the width of the area of the catching sheet covered by the marking sheet is 1-3mm, and the left side of the marking sheet extends out of the left edge of the catching sheet; the left edge of the glass cellulose membrane extends to a position between the capture sheet and the PVC plastic bottom plate, the width of the area of the glass cellulose membrane covered by the capture sheet is 2-4mm, and the left side of the capture sheet extends out of the left edge of the glass cellulose membrane; the right side edge of the glass cellulose membrane extends to a position between the water absorption sheet and the PVC plastic bottom plate, the area of the glass cellulose membrane covered by the water absorption sheet is 3-5mm wide, and the right side of the water absorption sheet extends out of the right side edge of the glass cellulose membrane; the left and right materials which are not adjacent in sequence are not overlapped in the direction vertical to the PVC plastic bottom plate; the concentration of the goat anti-mouse polyclonal antibody solution is 1mg/mL, and the goat anti-mouse polyclonal antibody solution is diluted by 0.02MpH7.2PBS buffer solution.
The preparation method of the sample sheet comprises the following steps: soaking the glass cellulose membrane in 1.0mM PBS solution with pH7.4 for 1-3h, and drying.
The water absorption sheet is water absorption filter paper, a water absorption sponge sheet or water absorption fiber.
The PVC plastic bottom plate, the glass cellulose membrane, the sample sheet, the marking sheet, the capturing sheet and the water absorbing sheet are all cut into rectangles with required specifications. Wherein the PVC plastic bottom plate is cut according to the shape and the size required by the test strip.
Preferably, in the step (3), the PVC plastic base plate at one end of the water absorption sheet of the test strip is sprayed with fluorine-containing polyurethane, and the test strip is kept still and dried to form a film, so that the
Preferably, a result check is also required to be carried out on the result of the chemiluminescence quantitative detection when the quantitative chemiluminescence detection is carried out, and when the result check fails, the quantitative fluorescence detection is used for re-detection, wherein the result check comprises the following steps:
a1, obtaining the standard deviation of the sample liquid drop to be detected in the historical inspection, and determining the repeated detection times of the sample to be detected;
wherein N is the repeated detection times, Car is the standard deviation of the historical examination, is a t test value corresponding to the preset value a, Ts is a preset tolerance rate,
step A2, carrying out N times of repeated tests on the sample liquid drop to be tested, and storing the concentration of the antigen to be tested in the sample liquid to be tested obtained by the tests into a concentration vector X;
step A3, determining the fluctuation frequency of the concentration vector X;
wherein S is the fluctuation frequency, X iIn order to test the sample liquid drop to be tested for the ith time, the concentration of the antigen to be tested in the sample liquid to be tested is obtained, wherein i is 1, 2 and 3L N;
step A3, determining the qualification rate of the concentration vector X;
wherein rt is the percent of pass;
and A4, when the qualification rate rt is larger than a preset value, taking the average value of the concentration vector X as the concentration of the antigen to be detected, otherwise, detecting the concentration of the antigen to be detected by using fluorescent quantitative detection.
Advantageous effects
The preparation method of the test strip provided by the invention has the advantages that the carboxylated fullerene is utilized to couple the antibody, so that the antibody and the enzyme are firmly combined without wasting sites, the unbound antibody and the enzyme can be separated by washing, meanwhile, the detection antibody is coupled on the carboxylated carbon nano tube, and the detection antibody is fixed on the test strip under the action of the carbon nano tube and the cellulose membrane, so that the loss of the antigen to be detected can not be caused, and the detection error is reduced.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure and methods particularly pointed out in the written description and claims hereof as well as the appended drawings.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
fig. 1 is a schematic structural diagram of a chemiluminescent immunoassay test strip prepared in
fig. 2 is a schematic structural diagram of the chemiluminescent immunoassay test strip prepared in
FIG. 3 is a standard curve prepared in example 4;
FIG. 4 is a calibration curve prepared in example 6.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.