Cortisol detection immune test paper and preparation method thereof
阅读说明：本技术 皮质醇检测免疫试纸及其制备方法 (Cortisol detection immune test paper and preparation method thereof ) 是由 黄晨珠 杨晶 邓艳珍 梁才弗 岑赞询 王伟 于 2019-11-22 设计创作，主要内容包括：本发明涉及一种皮质醇检测免疫试纸及其制备方法,该试纸包括样品垫、结合垫、硝酸纤维素膜、吸水垫和背衬,结合垫上包被有抗体微球偶联复合物,抗体微球偶联复合物是皮质醇单克隆抗体和第一质控抗体共同偶联于标记微球上的复合物,检测线包被有皮质醇抗原,质控线包被有能够与第一质控抗体特异性结合的第二质控抗体。发明人通过大量研究发现,将皮质醇单克隆抗体和第一质控抗体共同偶联在标记微球上形成抗体微球偶联复合物,能将反应体系的信号量放大的同时,还能有效解决抗体与标记微球结合不均匀的问题,从而大大提高了灵敏度、准确性和扩宽线性范围。(The invention relates to a cortisol detection immune test paper and a preparation method thereof, wherein the test paper comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a back lining, wherein an antibody microsphere coupling compound is coated on the combination pad, the antibody microsphere coupling compound is a compound in which a cortisol monoclonal antibody and a first quality control antibody are coupled on a marked microsphere together, a detection line is coated with a cortisol antigen, and a quality control line is coated with a second quality control antibody which can be specifically combined with the first quality control antibody. The inventor finds that the cortisol monoclonal antibody and the first quality control antibody are coupled on the marked microsphere together to form an antibody microsphere coupled compound, so that the signal quantity of a reaction system can be amplified, and the problem of uneven combination of the antibody and the marked microsphere can be effectively solved, thereby greatly improving the sensitivity, the accuracy and the widening the linear range.)
1. A cortisol detection immune test paper is characterized by comprising a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a back lining, the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are disposed on the backing, the two ends of the combination pad are respectively lapped with the sample pad and the nitrocellulose membrane, one end of the nitrocellulose membrane far away from the combination pad is lapped with the water absorption pad, the nitrocellulose membrane is provided with a detection line and a quality control line which are mutually spaced, the detection line is closer to the combination pad than the quality control line, the combination pad is coated with an antibody microsphere coupling compound which is a compound formed by coupling a cortisol monoclonal antibody and a first quality control antibody on a marked microsphere together, the detection line is coated with a cortisol antigen, and the quality control line is coated with a second quality control antibody capable of specifically binding with the first quality control antibody.
2. The cortisol detection immunoassay test strip according to claim 1, wherein the mass ratio of the cortisol monoclonal antibody to the first quality control antibody in the antibody-microsphere conjugate complex is (1-2): 1.
3. The cortisol test strip according to claim 1, wherein the labelled microspheres are selected from the group consisting of fluorescent latex microspheres, gold colloidal microspheres and magnetic beads.
4. The cortisol test strip according to claim 1, wherein one of said first and second control antibodies is selected from goat anti-rabbit IgG and the other is selected from rabbit anti-mouse IgG.
5. The cortisol test strip according to claim 4, wherein the first control antibody is selected from goat anti-rabbit IgG and the second control antibody is selected from rabbit anti-mouse IgG.
6. The cortisol-detecting dipstick according to any one of claims 1 to 5, wherein the cortisol-detecting dipstick is a canine/feline cortisol-detecting dipstick, the cortisol monoclonal antibody is a canine/feline cortisol monoclonal antibody, and the cortisol antigen is a canine/feline cortisol antigen.
7. A preparation method of the cortisol detection immune test paper is characterized by comprising the following steps:
coating an antibody microsphere coupling compound on the binding pad, wherein the antibody microsphere coupling compound is a compound formed by coupling a cortisol monoclonal antibody and a first quality control antibody on a labeled microsphere together;
coating a cortisol antigen on a nitrocellulose membrane to form a detection line;
coating a second quality control antibody on the nitrocellulose membrane to form a quality control line spaced from the detection line, wherein the second quality control antibody can be specifically combined with the first quality control antibody;
and arranging a sample pad, a water absorption pad, the combination pad coated with an antibody microsphere coupling compound and the nitrocellulose membrane provided with the detection line and the quality control line on a backing to obtain the cortisol detection immune test paper, wherein two ends of the combination pad are respectively lapped with the sample pad and the nitrocellulose membrane, one end of the nitrocellulose membrane, which is far away from the combination pad, is lapped with the water absorption pad, and the detection line is closer to the combination pad than the quality control line.
8. The method of claim 7, wherein the step of coating the antibody microsphere conjugate complex on the conjugate pad comprises:
mixing a cortisol monoclonal antibody, a first quality control antibody and a labeled microsphere to prepare a labeled solution containing the antibody microsphere coupling compound;
the labeling solution was sprayed on the conjugate pad and dried.
9. The method of claim 8, wherein the preparation of the labeling solution comprising the antibody microsphere conjugate complex comprises the steps of:
preparing a labeled microsphere solution from the labeled microsphere, mixing the labeled microsphere solution with the cortisol monoclonal antibody and the first quality control antibody, separating and taking a precipitate, and dissolving the precipitate by adopting PBS-TBN (phosphate buffer solution-tert-butyl ether) to prepare the labeled solution containing the antibody microsphere coupling compound.
10. The method according to claim 9, wherein the concentration of the labeled microspheres in the labeled microsphere solution is (800 to 1200) μ g/100 μ L; the dosage ratio of the total amount of the cortisol monoclonal antibody and the first quality control antibody to the labeled microsphere solution is (5-20) mu g/100 mu L.
Technical Field
The invention relates to the technical field of medical detection, in particular to cortisol detection immune test paper and a preparation method thereof.
Background
Cortisol (Cortisol), also called hydrocortisone, is an adrenocortical hormone extracted from the adrenal cortex and having the strongest effect on carbohydrate metabolism, i.e. one of the glucocorticoids. The Corisol detection method mainly comprises an enzyme-linked immunosorbent assay (ELISA) method and a chemiluminescence method. The ELISA method has the advantages of poor quantitative accuracy, long detection time, low automation degree and easiness in error occurrence; the chemiluminescence method has strong specificity and high sensitivity, but has high technical requirements. Therefore, the development of a Cortisol detection product which is simple in operation, high in sensitivity, strong in specificity and wide in linear range is an urgent problem to be solved.
Disclosure of Invention
Therefore, the need exists for a cortisol detection immunoassay test paper which is fast, convenient, high in sensitivity, strong in specificity and wide in linear range and a preparation method thereof.
A cortisol test immune test paper comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a back lining, the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are disposed on the backing, the two ends of the combination pad are respectively lapped with the sample pad and the nitrocellulose membrane, one end of the nitrocellulose membrane far away from the combination pad is lapped with the water absorption pad, the nitrocellulose membrane is provided with a detection line and a quality control line which are mutually spaced, the detection line is closer to the combination pad than the quality control line, the combination pad is coated with an antibody microsphere coupling compound which is a compound formed by coupling a cortisol monoclonal antibody and a first quality control antibody on a marked microsphere together, the detection line is coated with a cortisol antigen, and the quality control line is coated with a second quality control antibody capable of specifically binding with the first quality control antibody.
The inventor finds that a cortisol monoclonal antibody and a first quality control antibody are coupled on a marked microsphere together to form an antibody microsphere coupled complex, and then the antibody microsphere coupled complex is coated on a combination pad.
In one embodiment, in the antibody microsphere conjugate compound, the mass ratio of the cortisol monoclonal antibody to the first quality control antibody is (1-2): 1.
In one embodiment, the labeled microspheres are selected from fluorescent latex microspheres, colloidal gold microspheres, or magnetic bead microspheres.
In one embodiment, one of the first and second quality control antibodies is selected from a goat anti-rabbit IgG antibody and the other is selected from a rabbit anti-mouse IgG antibody.
In one embodiment, the first quality control antibody is selected from a goat anti-rabbit IgG antibody and the second quality control antibody is selected from a rabbit anti-mouse IgG antibody.
In one embodiment, the cortisol test strip is a canine/feline cortisol test strip, the cortisol monoclonal antibody is a canine/feline cortisol monoclonal antibody, and the cortisol antigen is a canine/feline cortisol antigen.
A preparation method of the cortisol detection immune test paper comprises the following steps:
coating an antibody microsphere coupling compound on the binding pad, wherein the antibody microsphere coupling compound is a compound formed by coupling a cortisol monoclonal antibody and a first quality control antibody on a labeled microsphere together;
coating a cortisol antigen on a nitrocellulose membrane to form a detection line;
coating a second quality control antibody on the nitrocellulose membrane to form a quality control line spaced from the detection line, wherein the second quality control antibody can be specifically combined with the first quality control antibody;
and arranging a sample pad, a water absorption pad, the combination pad coated with an antibody microsphere coupling compound and the nitrocellulose membrane provided with the detection line and the quality control line on a backing to obtain the cortisol detection immune test paper, wherein two ends of the combination pad are respectively lapped with the sample pad and the nitrocellulose membrane, one end of the nitrocellulose membrane, which is far away from the combination pad, is lapped with the water absorption pad, and the detection line is closer to the combination pad than the quality control line.
In one embodiment, the step of coating the antibody microsphere conjugate complex on the conjugate pad comprises:
mixing a cortisol monoclonal antibody, a first quality control antibody and a labeled microsphere to prepare a labeled solution containing the antibody microsphere coupling compound;
the labeling solution was sprayed on the conjugate pad and dried.
In one embodiment, the preparation of the labeling solution containing the antibody microsphere conjugate complex comprises the following steps:
preparing a labeled microsphere solution from the labeled microsphere, mixing the labeled microsphere solution with the cortisol monoclonal antibody and the first quality control antibody, separating and taking a precipitate, and dissolving the precipitate by adopting PBS-TBN (phosphate buffer solution-tert-butyl ether) to prepare the labeled solution containing the antibody microsphere coupling compound.
In one embodiment, in the labeled microsphere solution, the concentration of the labeled microspheres is (800-1200) μ g/100 μ L; the dosage ratio of the total amount of the cortisol monoclonal antibody and the first quality control antibody to the labeled microsphere solution is (5-20) mu g/100 mu L.
Drawings
FIG. 1 is a schematic diagram of an embodiment of a cortisol test strip;
FIG. 2 is a graph of the linear relationship between the theoretical concentration and the detected concentration of a sample;
FIG. 3 is a graph of the linear dependence of cortisol calibrator concentration on sensitivity corresponding to test using the dipsticks prepared in preparative example and comparative example 1.
Detailed Description
In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The cortisol is a steroid micromolecule substance, has antigenicity and no immunogenicity, so that the difficulty in obtaining a high-specificity cortisol antibody is high. In blood samples, a plurality of steroid small molecular substances with similar structures to the cortisol generally exist, the substances can interfere with the detection of the cortisol, and meanwhile, the small molecular substances are easily affected by steric hindrance and matrix effects in the detection process. The problems of low sensitivity, narrow linear range and insufficient accuracy generally exist in the immunochromatography detection of small molecular substances.
In view of the above problems, an embodiment of the present invention provides a cortisol test strip and a method for manufacturing the same. The structure of the cortisol-testing strip will be described in detail below with reference to the method for manufacturing the cortisol-testing strip.
Referring to fig. 1, the cortisol test strip includes a
The
When the cortisol detection immune test paper works, a sample to be detected is contacted with the
If the sample to be detected contains cortisol, cortisol in the sample and the cortisol antigen coated by the
If the sample to be detected does not contain cortisol, the cortisol monoclonal antibody on the antibody microsphere coupled complex on the
If the
The inventor finds that a cortisol monoclonal antibody and a first quality control antibody are coupled on a marked microsphere together to form an antibody microsphere coupled complex, and then the antibody microsphere coupled complex is coated on a
In a specific example, the cortisol test strip is a canine/feline cortisol test strip. Correspondingly, the cortisol monoclonal antibody is a canine/feline cortisol monoclonal antibody, and the cortisol antigen is a canine/feline cortisol antigen.
In one example, the
In some of these embodiments, the
Wherein, the
In some of these embodiments, the step of coating the antibody microsphere conjugate complex on the
Further, step S11 includes the following steps: and preparing the labeled microspheres into a labeled microsphere solution, mixing the labeled microsphere solution with the cortisol monoclonal antibody and the first quality control antibody, separating and taking precipitates, and dissolving the precipitates by adopting PBS-TBN (phosphate buffer solution-tert-butyl ether) to prepare the labeled solution containing the antibody microsphere coupling compound. Wherein the PBS-TBN mainly comprises PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, and pH 7.4).
Furthermore, in step S11, the concentration of the labeled microspheres in the labeled microsphere solution is (800-1200) μ g/100 μ L; the dosage ratio of the total amount of the cortisol monoclonal antibody and the first quality control antibody to the labeled microsphere solution is (5-20) mu g/100 mu L. Wherein the marked microsphere further comprises a step of activating the marked microsphere before use. Further, in step S11, the mixing is carried out under the condition of room temperature (25 ℃) for 1.5 to 3 hours. Further, the amount of PBS-TBN is adjusted so that the volume of the labeling solution containing the antibody microsphere coupling complex is the same as the volume of the labeling microsphere solution.
Further, in the step S12, the spraying amount of the marking solution sprayed on the
Further, in the preparation step of the antibody microsphere coupling compound or the labeling solution containing the antibody microsphere coupling compound, the mass ratio of the cortisol monoclonal antibody to the first quality control antibody is (1-2): 1. Preferably, in the step of preparing the antibody microsphere conjugate complex or the labeling solution containing the antibody microsphere conjugate complex, the mass ratio of the cortisol monoclonal antibody to the first quality control antibody is 1: 1.
In some of these embodiments, the labeled microspheres are selected from fluorescent latex microspheres, colloidal gold microspheres, or magnetic bead microspheres. Further, the marked microspheres are selected from fluorescent latex microspheres, and the diameter of the fluorescent latex microspheres is 0.1-1 μm; the emission wavelength of the fluorescent latex microspheres is 180 nm-800 nm.
The
In some embodiments, one of the first and second quality control antibodies is selected from a goat anti-rabbit IgG antibody and the other is selected from a rabbit anti-mouse IgG antibody.
Further, the first quality control antibody is selected from a goat anti-rabbit IgG antibody, and the second quality control antibody is selected from a rabbit anti-mouse IgG antibody.
In a specific example, the
In one specific example, the
In some of these examples, the
In some embodiments, the
Specific application examples are as follows.
Test paper preparation example
The cortisol test strip shown in FIG. 1 is prepared as follows.
The sample pad can be prepared by the following method: dissolving protein with buffer solution, adding a small amount of surfactant, adjusting pH to 7, adding blocking agent to obtain mixed solution, spraying the mixed solution onto sample pad at a spraying amount of 6 μ L/cm, and air drying at 50 deg.C for 24 hr in drying oven.
Coating the antibody microsphere coupling compound on the binding pad: the activated fluorescent latex microspheres are prepared into an activated fluorescent latex microsphere solution with the concentration of 1000 mug/100 mug. Adding a canine-feline cortisol monoclonal antibody and a goat-anti-rabbit IgG antibody into the activated fluorescent latex microsphere solution, wherein the addition amount of the canine-feline cortisol monoclonal antibody and the goat-anti-rabbit IgG antibody is as follows: adding 5 mu g of the activated fluorescent latex microspheres into each 100 mu L of the solution, mixing for 2 hours at room temperature, centrifuging, dissolving the obtained precipitate by adopting PBS-TBN, and adjusting the volume of the obtained solution to be equal to that of the added activated fluorescent latex microspheres to obtain a labeling solution. Uniformly spraying a labeling solution containing a cortisol monoclonal antibody labeled by fluorescent latex microspheres and a goat anti-rabbit IgG antibody on a bonding pad, wherein the spraying amount is 6 mu L/cm, and placing the bonding pad in a drying oven for 24 hours at 50 ℃ for forced air drying to obtain the fluorescent latex microsphere.
Preparation of a detection line: diluting the canine/feline cortisol antigen to 1mg/mL by using Tris buffer solution with the pH value of 6-8 according to the spraying amount of 20 mu L/30cm2And (3) scribing on the nitrocellulose membrane, and placing the nitrocellulose membrane in a drying oven for forced air drying at 50 ℃ for 72h to obtain the detection line.
Preparing a quality control line: the concentration of rabbit anti-mouse IgG antibody is diluted to 1.0mg/mL according to the spraying amount of 20 mu L/30cm2And (3) scribing on the nitrocellulose membrane, wherein the line is parallel to the detection line and is separated by 5-10 mm, and the drying condition is the same as the detection line, so that a quality control line is obtained.
Linear range of one
The prepared cortisol detection immune test paper is adopted to test 1-5 samples to be tested respectively. The sample to be tested 1-5 is a cortisol calibrator with theoretical concentrations of 20, 80, 200, 500 and 1000nmol/L respectively. Then, cortisol detection immune test paper is adopted to respectively test 1-5 samples to be tested, and then a fluorometer is used to read the corresponding cortisol reading on the cortisol detection immune test paper, so that the obtained detection results are shown in table 1.
TABLE 1
The theoretical concentration and the mean value of the detection values in table 1 were plotted on the abscissa and ordinate (in nmol/L), and the linear correlation coefficient R2 was 0.9996, indicating that the linear range of the detection concentration of the test paper was 20nmol/L to 1000 nmol/.
Second, precision
The prepared cortisol detection immune test paper is used for testing low-value quality control products and high-value quality control products, each quality control product is tested for 20 times, the average value and the variation coefficient of the 20 times of tests are calculated, and the results are shown in table 2.
TABLE 2
Three, relative deviation
The prepared cortisol test immunotest paper is used for testing cortisol reference substances with the concentrations of 80nmol/L and 500nmol/L, the mean value and the relative deviation of the test results of the samples are calculated for 3 times in each concentration test, and the results are shown in the following table 3.
TABLE 3
Sample concentration (nmol/L)
80
500
1
81.652
510.297
2
79.687
523.459
3
83.266
521.083
Mean value
81.535
518.27967
Bias％
-2％
-4％
Fourth, minimum detection limit
The cortisol reference substance with zero concentration is determined by using the prepared cortisol detection immune test paper, the determination is repeated for 20 times, the average value and the standard deviation of the results of the concentration values measured for 20 times are calculated, and the lowest detection limit is calculated, wherein the results are shown in the following table 4.
TABLE 4
The test strip adopted in the comparative example 1 has basically the same structure as the test strip prepared in the preparation example, except that: the combination pad is coated with not the antibody microsphere coupling compound, but the labeled microsphere is respectively labeled with the cortisol monoclonal antibody and the first quality control antibody. Specifically, the bonding pad of comparative example 1 was prepared as follows: adding a canine-feline cortisol monoclonal antibody and a goat-anti-rabbit IgG antibody into the two activated fluorescent latex microsphere solutions respectively, wherein the addition amounts of the canine-feline cortisol monoclonal antibody and the goat-anti-rabbit IgG antibody are as follows: adding 5 mu g of each of the activated fluorescent latex microsphere solutions into 100 mu L of the activated fluorescent latex microsphere solutions, mixing for 2 hours at room temperature, centrifuging, dissolving the obtained precipitate by adopting PBS-TBN, and adjusting the volume of the obtained solution to be equal to the volume of the added activated fluorescent latex microsphere solution to obtain two parts of labeling solutions. And uniformly spraying the two parts of the marking solution on the bonding pad in sequence, wherein the spraying amount is 6 mu L/cm, and placing the bonding pad in a drying oven for 24 hours at 50 ℃ for forced air drying.
The prepared cortisol detection immune test paper and the test paper prepared in the comparative example 1 are adopted to respectively test cortisol calibrators with theoretical concentrations of 20, 80 and 200 nmol/L. The corresponding cortisol concentration reading on the cortisol-detecting strip was then read by a fluorometer and the calculated sensitivity was calculated as shown in table 5. Accordingly, a linear correlation graph of the concentration of the cortisol calibrator and the sensitivity corresponding to the test using the test strips prepared in preparation example and comparative example 1 was obtained, as shown in FIG. 3, in which the abscissa is the concentration of cortisol calibrator nmol/L and the ordinate is C/T (signal intensity). As is apparent from Table 5 and FIG. 3, the sensitivity of the cortisol-detecting immunological test strip prepared as described above is superior to that of the test strip prepared in comparative example 1.
TABLE 5
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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