CN117665274A - Coating method of valproic acid immunochromatography detection card - Google Patents
Coating method of valproic acid immunochromatography detection card Download PDFInfo
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- CN117665274A CN117665274A CN202211014924.8A CN202211014924A CN117665274A CN 117665274 A CN117665274 A CN 117665274A CN 202211014924 A CN202211014924 A CN 202211014924A CN 117665274 A CN117665274 A CN 117665274A
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- NIJJYAXOARWZEE-UHFFFAOYSA-N Valproic acid Chemical compound CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 title claims abstract description 248
- 238000000576 coating method Methods 0.000 title claims abstract description 233
- 229960000604 valproic acid Drugs 0.000 title claims abstract description 123
- 238000001514 detection method Methods 0.000 title claims abstract description 88
- 238000003317 immunochromatography Methods 0.000 title claims abstract description 38
- 239000011248 coating agent Substances 0.000 claims abstract description 193
- 238000000034 method Methods 0.000 claims abstract description 41
- 239000007853 buffer solution Substances 0.000 claims abstract description 31
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 10
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 67
- 239000000872 buffer Substances 0.000 claims description 57
- 238000001035 drying Methods 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 44
- 239000000427 antigen Substances 0.000 claims description 36
- 102000036639 antigens Human genes 0.000 claims description 36
- 108091007433 antigens Proteins 0.000 claims description 36
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 24
- 229930006000 Sucrose Natural products 0.000 claims description 24
- 239000012528 membrane Substances 0.000 claims description 24
- 239000005720 sucrose Substances 0.000 claims description 24
- 239000007921 spray Substances 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 16
- 239000000020 Nitrocellulose Substances 0.000 claims description 13
- 238000003556 assay Methods 0.000 claims description 13
- 229920001220 nitrocellulos Polymers 0.000 claims description 13
- 239000007790 solid phase Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 8
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 14
- 238000000338 in vitro Methods 0.000 abstract description 2
- 239000003085 diluting agent Substances 0.000 description 11
- 241001494479 Pecora Species 0.000 description 10
- 101000860173 Myxococcus xanthus C-factor Proteins 0.000 description 6
- 230000001133 acceleration Effects 0.000 description 6
- 239000004005 microsphere Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 229940127121 immunoconjugate Drugs 0.000 description 4
- 238000005507 spraying Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 238000013096 assay test Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
-
- 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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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
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Abstract
The invention provides a method for coating a valproic acid immunochromatography detection card, and belongs to the field of in-vitro detection kits. The coating buffer solution used in the coating method of the invention is composed of saccharide substances and phosphate buffer solution as components; the concentration of the phosphate buffer solution is 0.1-0.25M, and the pH of the coating buffer solution is 7.0-9.0. The whole coating process of the coating method is simple to operate, the obtained coating film is suitable for developing a fluorescence immunochromatography valproic acid detection kit, the valproic acid can be quantitatively detected by adopting the detection kit, and the valproic acid detection method is simple and convenient to use, rapid in detection, high in sensitivity, wide in linear range, good in stability, accurate in result and controllable in cost. The method can realize the on-site quick detection of the sample, is little limited by instruments and sites, improves the clinical application of the valproic acid detection reagent of the fluorescence immunochromatography, and has good application prospect.
Description
Technical Field
The invention belongs to the field of in-vitro detection kits, and particularly relates to a method for coating a valproic acid immunochromatography detection card.
Background
Valproic acid is the most commonly used broad spectrum antiepileptic, but valproic acid is a narrow therapeutic window drug, so it is important to closely monitor valproic acid plasma concentrations in patients. The existing detection methods of valproic acid blood concentration mainly comprise mass spectrometry, high performance liquid chromatography, homogeneous enzyme-free method, chemiluminescence method, fluorescence polarization method and the like, some of the methods need expensive instruments and equipment, some of the methods have high requirements on professional operation, the detection steps are complicated, the use time is long, the price is high, and some of the methods also have the problem of poor sensitivity. Therefore, these methods are not beneficial to clinical popularization.
The Chinese patent publication No. CN110954707A discloses a valproic acid derivative and a preparation method thereof, and a kit containing the valproic acid derivative and a preparation method thereof. The valproic acid detection reagent has a linear measurement range of 10-170 mug/mL, and adopts an ELISA method (enzyme-linked immunosorbent assay). The detection reagent has relatively complex operation steps and relatively low sensitivity in the use process, and is not suitable for clinical popularization.
The immunochromatography is a rapid immunological detection method, and utilizes an immune marker to carry out chromatographic separation on a solid phase carrier so as to realize quantitative detection of the content of a detection object. The specific antibody is firstly fixed on a certain zone of a solid phase carrier (such as nitrocellulose membrane), after one end of the dried solid phase carrier is immersed into a sample (urine or serum), the sample moves forward along the membrane due to capillary action, and when the sample moves to the zone fixed with the antibody, the corresponding antigen in the sample is specifically combined with the antibody, so that the specific immunodiagnosis is realized. However, the valproic acid detection kit developed by adopting an immunochromatography method at present has the problems of large variation Coefficient (CV), poor sensitivity and complex process system, can not meet the requirements of users on the accuracy of detection results, and also has the problem of high cost.
Therefore, the valproic acid concentration detection method which is rapid in detection, simple to operate, high in sensitivity, wide in linear range, good in stability, accurate in result and controllable in cost is developed, and the method has important significance in technological improvement and breakthrough on the existing immunochromatography detection test strip.
Disclosure of Invention
The invention aims to provide a method for coating a valproic acid immunochromatography detection card.
The invention provides a coating buffer solution, which consists of saccharide substances and phosphate buffer solution as components; the concentration of the phosphate buffer solution is 0.1-0.25M, and the pH of the coating buffer solution is 7.0-9.0.
Further, the saccharide is one or two of trehalose or sucrose;
preferably, the mass volume concentration of the saccharide is 3-8%; the mass volume concentration of 3-8% means that the sugar substance in 100mL of coating buffer solution is 3-8 g.
More preferably, the mass volume concentration of the carbohydrate substance is 6%.
Further, the coating buffer solution is composed of sucrose and phosphate buffer solution as components;
the mass volume concentration of the sucrose is 6%; the balance of phosphate buffer solution;
the concentration of the phosphate buffer is 0.2M, and the pH of the coating buffer is 8.0.
The invention also provides application of the coating buffer solution in preparing a valproic acid immunochromatography detection card;
preferably, the use of the aforementioned coating buffer for the preparation of a label pad for use in a valproic acid immunochromatographic assay card;
more preferably, the use of the aforementioned coating buffer for coating valproic acid antigens and/or antibodies.
The invention also provides a valproic acid antigen and/or antibody coating method, which uses the coating buffer solution to carry out coating.
Further, the coating method comprises the following steps:
(1) Preparing a coating liquid: diluting valproic acid antigen and/or antibody with the coating buffer solution to prepare valproic acid antigen coating solution and/or valproic acid antibody coating solution;
(2) Marking the prepared coating liquid on a solid phase carrier;
(3) Drying the solid phase carrier.
Further, the method comprises the steps of,
in the step (1), the concentration of the valproic acid antigen coating liquid and/or the valproic acid antibody coating liquid is 0.5-2 mg/mL;
and/or in the step (2), the length of the scribing line is 300mm, and the scribing spray amount is 0.8-1.2 mu L/cm;
and/or in the step (2), the valproic acid antigen coating liquid is marked as a T line on the solid phase carrier, and the valproic acid antibody coating liquid is marked as a C line on the solid phase carrier;
and/or in the step (3), the drying temperature of the drying is 37-42 ℃ and the drying time is 2-18 h.
Further, the method comprises the steps of,
in the step (1), the concentration of the valproic acid antigen coating liquid and/or valproic acid antibody coating liquid is 1mg/mL;
and/or in the step (2), the length of the scribing line is 300mm, and the scribing spray amount is 1 mu L/cm;
and/or, in the step (3), the drying temperature of the drying is 37 ℃ and the drying time is 18 hours;
preferably, the solid support is a nitrocellulose membrane.
The invention also provides a marking pad used in the valproic acid immunochromatography detection card, which is prepared by adopting the coating method.
The invention also provides a valproic acid immunochromatography detection card, which comprises the marking pad.
In the present invention, "coating" refers to a process of binding an antigen or antibody to the surface of a solid support.
In the present invention, normal temperature means 25 ℃.
The immune detection method is expected to become a common method for monitoring the blood concentration of the valproic acid in clinic due to the advantages of simplicity, convenience and rapidness, but the detection kit is dependent on import, and has the defects of high price and short validity period. The valproic acid detection kit developed by adopting the fluorescence immunochromatography in China has the problems of high Coefficient of Variation (CV), CV value up to 15% and poor sensitivity, and can not meet the requirements of users on the accuracy of detection results. Thus, the immunodetection method is difficult to popularize in clinical use.
The invention develops a method for coating the valproic acid immunochromatography detection card, the whole coating process is simple to operate, the coated NC film can be used as one of main components of a time resolution fluorescence immunochromatography kit, and the kit is suitable for developing the valproic acid fluorescence immunochromatography detection kit, can be matched with various detection instruments (manual, semi-automatic and full-automatic instruments) for quantitatively detecting the valproic acid, and meets the various requirements of customers. The valproic acid time-resolved fluorescence immunochromatography detection kit based on the valproic acid coating method has the advantages of no more than 10% of variation coefficient, high detection sensitivity, capability of detecting valproic acid with the concentration of 1.37 mug/mL, even certain distinction between 0.457 mug/mL and 0 mug/mL, sensitivity approaching to that of valproic acid detection reagent of an imported enzyme amplification immunoassay, wide detection linear range, good stability, short detection time of a prepared reagent sample, simple operation and improvement of clinical application of valproic acid detection test strips of the fluorescence immunochromatography.
In summary, the invention provides a method for coating the valproic acid immunochromatography detection card, the whole coating process of the coating method is simple to operate, the obtained coating film is suitable for developing a fluorescence immunochromatography valproic acid detection kit, the valproic acid can be quantitatively detected by adopting the detection kit, and the valproic acid detection kit is simple and convenient to use, rapid to detect, high in sensitivity, wide in linear range, good in stability, accurate in result and controllable in cost. The method can realize the on-site quick detection of the sample, is little limited by instruments and sites, improves the clinical application of the valproic acid detection reagent of the fluorescence immunochromatography, and has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
Example 1 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of trehalose is added into PBS buffer solution to be fixed to 100mL, so as to obtain the coating buffer solution with the mass volume concentration of the trehalose of 6 percent, the concentration of the PBS buffer solution is 0.2M, and the pH value of the coating buffer solution is 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 2 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 3 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 3g of sucrose and 3g of trehalose are added into PBS buffer solution together to reach 100mL, so as to obtain the coating buffer solution with the mass and volume concentration of 3% of sucrose and trehalose, wherein the concentration of the PBS buffer solution is 0.2M, and the pH value of the coating buffer solution is 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 4 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the concentration of the C line coating liquid are both 0.5mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 5 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the concentration of the C line coating liquid are both 2mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 6 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 0.8. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 7 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1.2. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 8 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 37 ℃, and drying overnight for 2 hours to obtain the coated NC film (marking pad).
Example 9 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 42 ℃, and drying overnight for 2 hours to obtain the coated NC film (marking pad).
Example 10 coating method of valproic acid immunochromatographic assay card of the present invention
The coating method of the valproic acid immunochromatography detection card specifically comprises the following steps:
1. preparing a coating buffer solution: 6g of sucrose was added to PBS buffer to a volume of 100mL to give a coating buffer with a sucrose mass volume concentration of 6%, the concentration of PBS buffer was 0.2M, and the pH of the coating buffer was 8.0.
2. Preparing a coating liquid: valproic acid antigen (VPA-Ag) and antibodies (sheep anti-chicken IgY antibodies) were diluted with coating buffer, respectively, to prepare valproic acid antigen coating solution (T-line coating solution) and antibody coating solution (C-line coating solution). The concentration of the T line coating liquid and the C line coating liquid is 1mg/mL.
3. Scribing: the prepared T-line coating liquid and C-line coating liquid were streaked on a nitrocellulose membrane (NC membrane) with a streaking length of 300mm and a streaking spray amount of 1. Mu.L/cm by running a streaking instrument.
4. And (3) drying: and (3) placing the marked NC film in an oven for drying, wherein the drying temperature is 42 ℃, and drying overnight for 18 hours to obtain the coated NC film (marking pad).
Example 11 method for assembling valproic acid immunochromatographic detection card and detection method
1. The assembly method of the valproic acid immunochromatography detection card comprises the following steps:
the dried coated NC film prepared in any one of examples 1 to 10, water absorbing paper and a sample pad are assembled into a detection large plate, cut into test strips of 4.00mm, and assembled into a single-person detection card by using a card shell.
2. The method for detecting the valproic acid by using the valproic acid immunochromatography detection card comprises the following steps:
1) Preparation: 1. Mu.L of fluorescent microsphere antibody conjugate, 98. Mu.L of sample diluent and 1. Mu.L of linear reference are uniformly mixed to obtain a mixed solution for standby. The fluorescent microsphere antibody conjugate is formed by coupling fluorescent microspheres and valproic acid antibodies according to a conventional method in the field.
2) Sample adding: the mixed solution was loaded onto a test reagent card at 90. Mu.L.
3) And (3) detection: after the mixed solution was dropped into the reagent card for 15 minutes, the detection result of the reagent card was read, and luminescence signals (T signal value and C signal value) were detected using a fluorescence immunoassay analyzer, and the signal value of T/C was calculated.
Preparation of sample dilutions: and adding the surfactant S9 into PBS buffer solution, wherein the surfactant S9 accounts for 2g/L (the concentration is 0.2%) of the sample diluent, and mixing and dissolving to obtain the sample diluent, the concentration of the PBS buffer solution is 0.01M, and the pH of the sample diluent is 8.0.
Linear reference: valproic acid standard was dissolved in matrix diluent to prepare 1mg/mL stock solution. The stock solutions were diluted with matrix dilutions to standard solutions of 333.0. Mu.g/mL, 200.0. Mu.g/mL, 111.0. Mu.g/mL, 37.0. Mu.g/mL, 12.333. Mu.g/mL, 4.111. Mu.g/mL, 1.370. Mu.g/mL, 0.457. Mu.g/mL, 0.152. Mu.g/mL, 0. Mu.g/mL, in order. Wherein the matrix diluent is bovine serum.
The linear reference is replaced by a detection sample, and can be used for detecting the valproic acid content in the sample.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 valproic acid immunochromatographic assay test card prepared by different coating methods for detecting valproic acid
1. Different coating buffers
The coated NC films prepared in examples 1 to 3 were assembled into a valproic acid immunochromatographic test card according to the method described in example 11, and valproic acid was detected according to the method described in example 11. And respectively detecting the sample diluent and the linear reference, comparing the detection effects of the sample diluent and the linear reference, and analyzing indexes such as signal values, signal to noise ratios, background reaction values, low-value sensitivity, linear range, thermal acceleration stability and the like. The results of the detection of the different coating buffers are shown in tables 1 and 2.
1) Sensitivity: and judging the signal-to-noise ratio of the T/C signal values measured by the linear reference products with different concentrations and the sample diluent (0 value), wherein the larger the ratio is, the better the sensitivity is. The results are shown in Table 1. The T signal value and the C signal value are the results of fluorescence detection.
TABLE 1 sensitivity test results
2) Thermal stability
The thermal stability detection method comprises the following steps: according to the method, the T/C signal values of different reagent cards at normal temperature (25 ℃) are detected, and then the T/C signal values of the reagent cards after being stored for 9 days at 37 ℃ are detected. To determine its thermal stability.
TABLE 2 Normal temperature control test results
TABLE 3 thermal acceleration 9 days detection results
From the above results, it can be seen that: compared with the embodiment 1 and the embodiment 3, the detection card prepared by using the coated NC film of the embodiment 2 has larger signal-to-noise ratio and better sensitivity; meanwhile, the linear overall deviation is smaller and the thermal stability is better when the detection card prepared by the coated NC film of the embodiment 2 is used for detection. Thus, the coating buffer in example 2 was selected as the optimal coating buffer.
2. Film dividing with different coating liquid concentrations
The coated NC films prepared in examples 2, 4 and 5 were assembled into a valproic acid immunochromatographic test card according to the method described in example 11, and valproic acid was detected according to the method described in example 11. And comparing and verifying by using a linear reference and the fluorescent microsphere antibody conjugate, and confirming the optimal streak concentration according to indexes such as a result analysis signal value, a signal-to-noise ratio, a background reaction value, low-value sensitivity, a linear range and the like. The results are shown in Table 4.
TABLE 4 Linear reference detection results
Samples of equal concentration, with increasing streak concentration, the T/C signal value increases; however, the streak concentration (0.5 mg/mL) used in example 4 was optimal from the signal-to-noise point, and the streak concentration (2 mg/mL) used in example 5 was relatively poor, as compared to the streak concentration of 0.5mg/mL for example 2, which was similar to the streak concentration of 1mg/mL. Considering the linear range of 1-160mg/mL and the signal value of the streak test strip with the signal value of 111mg/mL of calibrator at 0.5mg/mL is only 3644, the streak concentration of 1mg/mL is comprehensively considered as the optimal coating concentration, and the signal-to-noise ratio is high, the detection sensitivity is good, and the linear range is wider.
3. Scribing spray amount selection
The coated NC films prepared in examples 2, 6 and 7 were assembled into a valproic acid immunochromatographic test card according to the method described in example 11, and valproic acid was detected according to the method described in example 11. And comparing and verifying by using a linear reference and the fluorescent microsphere antibody conjugate, and confirming the optimal scribing spray amount according to indexes such as a result analysis signal value, a signal-to-noise ratio, a background reaction value, low value sensitivity, a linear range and the like. The results are shown in Table 5.
TABLE 5 Linear reference detection results
From the signal to noise ratio, the sample with the same concentration has the spraying amount of 1.0 mu L/cm (example 2) more than 0.8 mu L/cm (example 6) more than 1.2 mu L/cm (example 7), which shows that the sensitivity of the detection reagent card prepared with the spraying amount of 1.0 mu L/cm is better than that of the detection reagent card prepared with the spraying amount of 0.8 mu L/cm and the spraying amount of 1.2 mu L/cm. Thus, the spray amount of 1.0. Mu.L/cm was determined as the optimum scribing spray amount.
4. Different temperatures
The coated NC films prepared in examples 2, 8, 9, and 10 were assembled into a valproic acid immunochromatographic test card according to the method described in example 11, and valproic acid was detected according to the method described in example 11. And respectively detecting the sample diluent and the linear reference, comparing the detection effects of the sample diluent and the linear reference, analyzing indexes such as signal value, signal-to-noise ratio, background reaction value, low-value sensitivity, linear range, thermal acceleration stability and the like, and determining the optimal drying temperature and time. The thermal acceleration method is to store the test panel at 37℃for 9 days and then test. The results are shown in tables 6 and 7.
TABLE 6 Linear reference detection results
TABLE 7 thermal acceleration 9 days detection results
From the sensitivity point of view, example 9 > example 2 > example 10 > example 8; from a stable thermal acceleration of 9 days, example 2 > example 10 > example 8 > example 9. To sum up, the drying is carried out at 37℃overnight for 18 hours as the optimal drying temperature time.
In summary, the invention provides a method for coating the valproic acid immunochromatography detection card, the whole coating process of the coating method is simple to operate, the obtained coating film is suitable for developing a fluorescence immunochromatography valproic acid detection kit, the valproic acid can be quantitatively detected by adopting the detection kit, and the valproic acid detection kit is simple and convenient to use, rapid to detect, high in sensitivity, wide in linear range, good in stability, accurate in result and controllable in cost. The method can realize the on-site quick detection of the sample, is little limited by instruments and sites, improves the clinical application of the valproic acid detection reagent of the fluorescence immunochromatography, and has good application prospect.
Claims (10)
1. A coating buffer, characterized in that: the preparation method comprises the following steps of taking saccharide substances and phosphate buffer solution as components; the concentration of the phosphate buffer solution is 0.1-0.25M, and the pH of the coating buffer solution is 7.0-9.0.
2. The coating buffer of claim 1, wherein: the saccharide is one or two of trehalose and sucrose;
preferably, the mass volume concentration of the saccharide is 3-8%;
more preferably, the mass volume concentration of the carbohydrate substance is 6%.
3. The coating buffer according to claim 1 or 2, characterized in that: the compound consists of sucrose and phosphate buffer solution as components;
the mass volume concentration of the sucrose is 6%; the balance of phosphate buffer solution;
the concentration of the phosphate buffer is 0.2M, and the pH of the coating buffer is 8.0.
4. Use of a coating buffer according to any one of claims 1 to 3 for the preparation of a valproic acid immunochromatographic assay card;
preferably, the use of a coating buffer according to any one of claims 1 to 3 for the preparation of a label pad for use in a valproic acid immunochromatographic assay card;
more preferably, the use of a coating buffer according to any one of claims 1 to 3 for coating valproic acid antigens and/or antibodies.
5. A method for coating valproic acid antigen and/or antibody, characterized in that: coating with a coating buffer according to any one of claims 1 to 3.
6. The coating method according to claim 5, wherein: the coating method comprises the following steps:
(1) Preparing a coating liquid: diluting valproic acid antigen and/or antibody with the coating buffer solution according to any one of claims 1-3 to prepare a valproic acid antigen coating solution and/or a valproic acid antibody coating solution;
(2) Marking the prepared coating liquid on a solid phase carrier;
(3) Drying the solid phase carrier.
7. The coating method according to claim 6, wherein:
in the step (1), the concentration of the valproic acid antigen coating liquid and/or the valproic acid antibody coating liquid is 0.5-2 mg/mL;
and/or in the step (2), the length of the scribing line is 300mm, and the scribing spray amount is 0.8-1.2 mu L/cm;
and/or in the step (2), the valproic acid antigen coating liquid is marked as a T line on the solid phase carrier, and the valproic acid antibody coating liquid is marked as a C line on the solid phase carrier;
and/or in the step (3), the drying temperature of the drying is 37-42 ℃ and the drying time is 2-18 h.
8. The coating method according to claim 7, wherein:
in the step (1), the concentration of the valproic acid antigen coating liquid and/or valproic acid antibody coating liquid is 1mg/mL;
and/or in the step (2), the length of the scribing line is 300mm, and the scribing spray amount is 1 mu L/cm;
and/or, in the step (3), the drying temperature of the drying is 37 ℃ and the drying time is 18 hours;
preferably, the solid support is a nitrocellulose membrane.
9. A label pad for use in a valproic acid immunochromatographic test card, characterized in that: it is prepared by the coating method of any one of claims 5 to 8.
10. The utility model provides a valproic acid immunochromatography detection card which characterized in that: comprising the marking pad of claim 9.
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