CN112630434A - Two-dimensional flow measurement immunochromatographic test strip and preparation method thereof - Google Patents
Two-dimensional flow measurement immunochromatographic test strip and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
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- 239000012528 membrane Substances 0.000 claims abstract description 58
- 238000001514 detection method Methods 0.000 claims abstract description 34
- 238000003908 quality control method Methods 0.000 claims abstract description 27
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- 230000027455 binding Effects 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000010521 absorption reaction Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 46
- 239000002244 precipitate Substances 0.000 claims description 24
- 239000010931 gold Substances 0.000 claims description 20
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- 238000000576 coating method Methods 0.000 claims description 16
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 14
- 241000283707 Capra Species 0.000 claims description 14
- 229940098773 bovine serum albumin Drugs 0.000 claims description 14
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 230000010355 oscillation Effects 0.000 claims description 8
- 239000001509 sodium citrate Substances 0.000 claims description 8
- 238000005507 spraying Methods 0.000 claims description 8
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 8
- 229940038773 trisodium citrate Drugs 0.000 claims description 8
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- 206010028980 Neoplasm Diseases 0.000 abstract description 5
- 238000003317 immunochromatography Methods 0.000 abstract description 5
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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Abstract
The invention relates to a two-dimensional flow-measuring immunochromatographic test strip and a preparation method thereof, and aims to solve the problems of long time consumption and complex operation in enzyme-linked immunosorbent assay (ELISA) for detecting CA153 and CA 125. The two-dimensional flow measurement immunochromatographic test strip is characterized in that a water absorption pad A, a first NC membrane, a gold-labeled binding pad A, a sample pad, a gold-labeled binding pad B, a second NC membrane and a water absorption pad B are sequentially assembled on a bottom plate along the axial direction, a CA125 polyclonal antibody is coated on a detection line A, IgG is coated on a quality control line A, a CA153 polyclonal antibody is coated on a detection line B, IgG is coated on a quality control line B, a colloidal gold solution for marking a CA125 monoclonal antibody is coated on the gold-labeled binding pad A, and a colloidal gold solution for marking the CA153 monoclonal antibody is coated on the gold-labeled binding pad B. The invention develops the immunochromatography test paper for detecting tumor markers CA153 and CA125, and the test paper has the characteristics of rapidness, accuracy and easiness in operation.
Description
Technical Field
The invention belongs to the field of medical detection, and particularly relates to a CA153 and CA125 two-dimensional flow measurement immunochromatographic test strip and a preparation method thereof.
Background
The colloidal gold flow measurement immunochromatography technology is a detection method for detecting a target substance by taking colloidal gold as a labeled probe and based on antigen-antibody specific binding reaction. The structure of the device mainly comprises a sample pad, a combination pad, nitrocellulose (NC membrane), a detection line, a quality control line, a water absorption pad and a bottom plate. Separation was achieved on the NC membrane as macroscopic red bands by the difference in the speed of movement of the capillary forces. LFCA test strips for the detection of human chorionic gonadotropin were first developed since 1980 to diagnose early pregnancy. LFCA is widely used to detect a variety of molecules, including tumor markers, microorganisms, mycotoxins, heavy metals, pesticides, and the like.
CA125 is a serum tumor marker that is secreted by ovarian cancer tumor cells for monitoring the progression of ovarian cancer patients. The change of the expression level is closely related to the disease state of the epithelial ovarian cancer. CA153 is one of the important markers of breast cancer, and has an important relationship with the diagnosis and prognosis of breast cancer. Tumor markers need to be tracked and detected for a long time, so that accurate, rapid and convenient operation is a problem to be solved clinically at present.
Disclosure of Invention
The invention aims to solve the problems of long time consumption and complex operation of enzyme-linked immunosorbent assay for detecting CA153 and CA125, and provides a rapid, simple and convenient two-dimensional flow measurement immunochromatographic test strip and a preparation method thereof.
The two-dimensional flow measurement immunochromatographic test strip is characterized in that a water absorption pad A, a first NC membrane, a gold-labeled binding pad A, a sample pad, a gold-labeled binding pad B, a second NC membrane and a water absorption pad B are sequentially assembled on a bottom plate along the axial direction, a CA125 polyclonal antibody is coated on a detection line A of the first NC membrane, goat anti-mouse IgG is coated on a quality control line A of the first NC membrane, a CA153 polyclonal antibody is coated on a detection line B of the second NC membrane, goat anti-mouse IgG is coated on a quality control line B of the second NC membrane, a colloidal gold solution for marking a CA125 monoclonal antibody is coated on the gold-labeled binding pad A, and a colloidal gold solution for marking a CA153 monoclonal antibody is coated on the gold-labeled binding pad B.
The preparation method of the two-dimensional flow measurement immunochromatographic test strip is realized according to the following steps:
adding a chloroauric acid solution into a round-bottom flask, refluxing and heating to boil, adding a trisodium citrate solution under the stirring condition, reacting until a reaction solution turns red, continuing to heat for 10-15 min, and naturally cooling to room temperature to obtain a colloidal gold solution;
adjusting the pH value of the colloidal gold solution to 8.5-9.5, adding the colloidal gold solution into a centrifugal tube, then adding the CA125 monoclonal antibody, uniformly mixing and reacting for 10-15 min, adding a BSA (bovine serum albumin) aqueous solution, uniformly mixing by vortex oscillation, collecting precipitates after centrifugal treatment, re-suspending the precipitates to the original volume by using a borate buffer solution, performing centrifugal treatment again, and diluting the precipitates to 1/5 of the original volume by using a diluent to obtain the immune gold marked with the CA125 monoclonal antibody;
adjusting the pH value of the colloidal gold solution to 8.5-9.5, adding the colloidal gold solution into a centrifugal tube, then adding a CA153 monoclonal antibody, uniformly mixing and reacting for 10-15 min, adding a BSA aqueous solution, uniformly mixing by vortex oscillation, collecting precipitates after centrifugal treatment, re-suspending the precipitates to the original volume by using a borate buffer solution, performing centrifugal treatment again, and diluting the precipitates to 1/5 of the original volume by using a diluent to obtain the immune gold marked with the CA153 monoclonal antibody;
thirdly, spraying immune gold marked with the CA125 monoclonal antibody on the combination pad A, and performing freeze-drying treatment to obtain a gold-labeled combination pad A;
spraying immune gold marked with the CA153 monoclonal antibody on the binding pad B, and performing freeze-drying treatment to obtain a gold-labeled binding pad B;
coating the CA125 polyclonal antibody on a detection line A of the first NC membrane, coating goat anti-mouse IgG on a quality control line A of the first NC membrane, and drying to obtain the first NC membrane;
coating CA153 polyclonal antibody on a detection line B of a second NC membrane, coating goat anti-mouse IgG on a quality control line B of the second NC membrane, and drying to obtain the second NC membrane;
and fifthly, sequentially assembling the water absorption pad A, the first NC membrane, the gold-labeled combination pad A, the sample pad, the gold-labeled combination pad B, the second NC membrane and the water absorption pad B on the bottom plate along the axial direction to obtain the two-dimensional flow measurement immunochromatographic test strip.
The application method of the two-dimensional flow measurement immunochromatographic test strip for detecting CA153 and CA125 is that serum is dripped on a sample pad, and the sample moves forwards along the test strip due to the capillary action. When moving to the region where the antibody is immobilized, the antigen in the sample undergoes an immunological binding reaction with the antibody, so that the region appears red, thereby achieving detection.
The invention develops the immunochromatography test paper for detecting the tumor markers CA153 and CA125, the test paper has the characteristics of rapidness, accuracy and easy operation, can rapidly and accurately detect the tumor markers, and provides an important detection means for clinical medicine.
Drawings
FIG. 1 is an absorbance spectrum of colloidal gold obtained in example;
fig. 2 is a schematic structural diagram of the two-dimensional flow-measuring immunochromatographic test strip of the present invention, in which 1 represents a water absorbent pad a, 2 represents a first NC membrane, 2-1 represents a quality control line a, 2-2 represents a detection line a, 3 represents a gold-labeled conjugate pad a, 4 represents a sample pad, 5 represents a gold-labeled conjugate pad B, 6 represents a second NC membrane, 6-1 represents a quality control line B, 6-2 represents a detection line B, 7 represents a water absorbent pad B, and 8 represents a bottom plate.
Detailed Description
The first embodiment is as follows: the two-dimensional flow measurement immunochromatography test strip of the embodiment is formed by sequentially assembling a water absorption pad A, a first NC membrane, a gold-labeled binding pad A, a sample pad, a gold-labeled binding pad B, a second NC membrane and a water absorption pad B on a bottom plate along the axial direction, wherein a detection line A of the first NC membrane is coated with a CA125 polyclonal antibody, a quality control line A of the first NC membrane is coated with goat anti-mouse IgG, a detection line B of the second NC membrane is coated with a CA153 polyclonal antibody, a quality control line B of the second NC membrane is coated with goat anti-mouse IgG, a colloidal gold solution for marking a CA125 monoclonal antibody is coated on the gold-labeled binding pad A, and a colloidal gold solution for marking a CA153 monoclonal antibody is coated on the gold-labeled binding pad B.
The water absorption pad and the NC membrane, the NC membrane and the combination pad, and the combination pad and the sample pad in the two-dimensional flow measurement immunochromatography test strip of the embodiment all have a covering and overlapping part, and the length of the covering and overlapping part is 1-2 mm.
The second embodiment is as follows: the preparation method of the two-dimensional flow measurement immunochromatographic test strip of the embodiment is realized according to the following steps:
adding a chloroauric acid solution into a round-bottom flask, refluxing and heating to boil, adding a trisodium citrate solution under the stirring condition, reacting until a reaction solution turns red, continuing to heat for 10-15 min, and naturally cooling to room temperature to obtain a colloidal gold solution;
adjusting the pH value of the colloidal gold solution to 8.5-9.5, adding the colloidal gold solution into a centrifugal tube, then adding the CA125 monoclonal antibody, uniformly mixing and reacting for 10-15 min, adding a BSA (bovine serum albumin) aqueous solution, uniformly mixing by vortex oscillation, collecting precipitates after centrifugal treatment, re-suspending the precipitates to the original volume by using a borate buffer solution, performing centrifugal treatment again, and diluting the precipitates to 1/5 of the original volume by using a diluent to obtain the immune gold marked with the CA125 monoclonal antibody;
adjusting the pH value of the colloidal gold solution to 8.5-9.5, adding the colloidal gold solution into a centrifugal tube, then adding a CA153 monoclonal antibody, uniformly mixing and reacting for 10-15 min, adding a BSA aqueous solution, uniformly mixing by vortex oscillation, collecting precipitates after centrifugal treatment, re-suspending the precipitates to the original volume by using a borate buffer solution, performing centrifugal treatment again, and diluting the precipitates to 1/5 of the original volume by using a diluent to obtain the immune gold marked with the CA153 monoclonal antibody;
thirdly, spraying immune gold marked with the CA125 monoclonal antibody on the combination pad A, and performing freeze-drying treatment to obtain a gold-labeled combination pad A;
spraying immune gold marked with the CA153 monoclonal antibody on the binding pad B, and performing freeze-drying treatment to obtain a gold-labeled binding pad B;
coating the CA125 polyclonal antibody on a detection line A of the first NC membrane, coating goat anti-mouse IgG on a quality control line A of the first NC membrane, and drying to obtain the first NC membrane;
coating CA153 polyclonal antibody on a detection line B of a second NC membrane, coating goat anti-mouse IgG on a quality control line B of the second NC membrane, and drying to obtain the second NC membrane;
and fifthly, sequentially assembling the water absorption pad A, the first NC membrane, the gold-labeled combination pad A, the sample pad, the gold-labeled combination pad B, the second NC membrane and the water absorption pad B on the bottom plate along the axial direction to obtain the two-dimensional flow measurement immunochromatographic test strip.
The third concrete implementation mode: the difference between the present embodiment and the second embodiment is that the mass concentration of the trisodium citrate solution in the first step is 1%.
The fourth concrete implementation mode: the second or third embodiment is different from the first embodiment in that trisodium citrate solution is added in the first step under the condition of stirring to react until the reaction solution turns red, and the heating is continued for 10 min.
The fifth concrete implementation mode: this embodiment differs from one of the second to fourth embodiments in that the pH of the colloidal gold solution is adjusted to 9.0 in the second step.
The sixth specific implementation mode: the difference between this embodiment and one of the second to fifth embodiments is that the centrifugation in the second step is performed at 13000r/min for 15 min.
The seventh embodiment: this embodiment is different from any one of the second to sixth embodiments in that the BSA aqueous solution in step two has a mass concentration of 10%.
The specific implementation mode is eight: this embodiment is different from the second to seventh embodiments in that the temperature of the drying in the fourth step is 37 ℃.
Example (b): the preparation method of the two-dimensional flow measurement immunochromatographic test strip is realized according to the following steps:
adding a chloroauric acid solution into a round-bottom flask, refluxing and heating to boil, adding a trisodium citrate solution with the mass concentration of 1% under the stirring condition, reacting until the reaction solution turns red, continuing to heat for 10min, naturally cooling to room temperature, storing at 4 ℃ for later use, performing full-wavelength scanning within the range of wavelength 400-700nm, and recording the change of an absorption peak (shown in figure 1) to obtain a colloidal gold solution;
adjusting the pH value of the colloidal gold solution to 9.0, adding 200ul of the colloidal gold solution into a centrifugal tube, then adding the CA125 monoclonal antibody, uniformly mixing and reacting for 10min, adding a BSA (bovine serum albumin) aqueous solution with the mass concentration of 10%, uniformly mixing by vortex oscillation, performing centrifugal treatment, collecting a precipitate, re-suspending the precipitate to the original volume by using a borate buffer solution, performing centrifugal treatment again, diluting the precipitate to 1/5 of the original volume by using a diluent, thus obtaining the immune gold marked with the CA125 monoclonal antibody, and storing the immune gold at 4 ℃ in a dark place;
adjusting the pH value of the colloidal gold solution to 9.0, adding the colloidal gold solution into a centrifuge tube, then adding a CA153 monoclonal antibody, uniformly mixing and reacting for 10min, adding a BSA aqueous solution with the mass concentration of 10%, uniformly mixing by vortex oscillation, collecting precipitates after centrifugal treatment, re-suspending the precipitates to the original volume by using a borate buffer solution, performing centrifugal treatment again, diluting the precipitates to 1/5 of the original volume by using a diluent, obtaining the immune gold marked with the CA153 monoclonal antibody, and storing the immune gold at 4 ℃ in a dark place;
thirdly, spraying immune gold marked with the CA125 monoclonal antibody on the combination pad A, and performing freeze-drying treatment to obtain a gold-labeled combination pad A;
spraying immune gold marked with the CA153 monoclonal antibody on the binding pad B, and performing freeze-drying treatment to obtain a gold-labeled binding pad B;
fourthly, coating the CA125 polyclonal antibody on a detection line A of the first NC membrane, coating goat anti-mouse IgG on a quality control line A of the first NC membrane, and drying at 37 ℃ for 1h to obtain the first NC membrane;
coating CA153 polyclonal antibody on a detection line B of a second NC membrane, coating goat anti-mouse IgG on a quality control line B of the second NC membrane, and drying at 37 ℃ for 1h to obtain the second NC membrane;
and fifthly, sequentially assembling the water absorption pad A, the first NC membrane, the gold-labeled combination pad A, the sample pad, the gold-labeled combination pad B, the second NC membrane and the water absorption pad B on the bottom plate along the axial direction to obtain the two-dimensional flow measurement immunochromatographic test strip.
The structure diagram of the two-dimensional flow-measuring immunochromatographic test strip of the embodiment is shown in fig. 1, wherein a quality control line a is located on the left side of a detection line a, and a quality control line B is located on the right side of a detection line B.
The using method comprises the following steps:
the serum is dropped onto the sample pad and due to capillary action, the serum will move to both sides. When moving to the detection line A, the CA125 antigen is specifically combined with the antibody, and the immune gold in the area shows red. And prompting that the content of CA125 in the blood serum is more than 20U/ml, if the color is not developed, prompting that the content of CA125 in the blood is less than 20U/ml, continuously moving the blood serum forwards, and when the blood serum reaches a quality control zone, the area is red, and prompting that the test strip works normally. When the serum moves to the detection line B, the CA153 antigen and the antibody are specifically combined, and the immune gold in the area shows red. And prompting that the content of CA153 in serum is more than 25U/ml, if the color is not developed, prompting that the content of CA125 in blood is less than 25U/ml, continuously moving the serum forwards, and when the serum reaches a quality control band, displaying red color in the area to prompt the test strip to work normally. And the total color reaction time is within 5 min.
And (4) judging a result:
the detection limit of CA125 is 20U/ml, and the detection limit of CA153 is 25U/ml.
When the detection limit A appears red, the quality control line A appears red, and the content of CA125 in the sample is prompted to be more than 20U/ml.
When the detection limit A is not red, the quality control line A is red, which indicates that the content of CA125 in the sample is less than 20U/ml.
When the detection limit B appears red, the quality control line B appears red, and the content of CA153 in the sample is prompted to be more than 25U/ml.
And when the detection limit B is not red, the quality control line B is red, which indicates that the content of CA153 in the sample is less than 25U/ml.
Claims (8)
1. The two-dimensional flow measurement immunochromatographic test strip is characterized in that a water absorption pad A, a first NC membrane, a gold-labeled combination pad A, a sample pad, a gold-labeled combination pad B, a second NC membrane and a water absorption pad B are sequentially assembled on a bottom plate along the axial direction, a CA125 polyclonal antibody is coated on a detection line A of the first NC membrane, goat anti-mouse IgG is coated on a quality control line A of the first NC membrane, a CA153 polyclonal antibody is coated on a detection line B of the second NC membrane, goat anti-mouse IgG is coated on a quality control line B of the second NC membrane, a colloidal gold solution for marking a CA125 monoclonal antibody is coated on the gold-labeled combination pad A, and a colloidal gold solution for marking a CA153 monoclonal antibody is coated on the gold-labeled combination pad B.
2. The preparation method of the two-dimensional flow measurement immunochromatographic test strip is characterized by comprising the following steps:
adding a chloroauric acid solution into a round-bottom flask, refluxing and heating to boil, adding a trisodium citrate solution under the stirring condition, reacting until a reaction solution turns red, continuing to heat for 10-15 min, and naturally cooling to room temperature to obtain a colloidal gold solution;
adjusting the pH value of the colloidal gold solution to 8.5-9.5, adding the colloidal gold solution into a centrifugal tube, then adding the CA125 monoclonal antibody, uniformly mixing and reacting for 10-15 min, adding a BSA (bovine serum albumin) aqueous solution, uniformly mixing by vortex oscillation, collecting precipitates after centrifugal treatment, re-suspending the precipitates to the original volume by using a borate buffer solution, performing centrifugal treatment again, and diluting the precipitates to 1/5 of the original volume by using a diluent to obtain the immune gold marked with the CA125 monoclonal antibody;
adjusting the pH value of the colloidal gold solution to 8.5-9.5, adding the colloidal gold solution into a centrifugal tube, then adding a CA153 monoclonal antibody, uniformly mixing and reacting for 10-15 min, adding a BSA aqueous solution, uniformly mixing by vortex oscillation, collecting precipitates after centrifugal treatment, re-suspending the precipitates to the original volume by using a borate buffer solution, performing centrifugal treatment again, and diluting the precipitates to 1/5 of the original volume by using a diluent to obtain the immune gold marked with the CA153 monoclonal antibody;
thirdly, spraying immune gold marked with the CA125 monoclonal antibody on the combination pad A, and performing freeze-drying treatment to obtain a gold-labeled combination pad A;
spraying immune gold marked with the CA153 monoclonal antibody on the binding pad B, and performing freeze-drying treatment to obtain a gold-labeled binding pad B;
coating the CA125 polyclonal antibody on a detection line A of the first NC membrane, coating goat anti-mouse IgG on a quality control line A of the first NC membrane, and drying to obtain the first NC membrane;
coating CA153 polyclonal antibody on a detection line B of a second NC membrane, coating goat anti-mouse IgG on a quality control line B of the second NC membrane, and drying to obtain the second NC membrane;
and fifthly, sequentially assembling the water absorption pad A, the first NC membrane, the gold-labeled combination pad A, the sample pad, the gold-labeled combination pad B, the second NC membrane and the water absorption pad B on the bottom plate along the axial direction to obtain the two-dimensional flow measurement immunochromatographic test strip.
3. The method for preparing a two-dimensional flow-measuring immunochromatographic test strip according to claim 2, wherein the mass concentration of the trisodium citrate solution in the first step is 1%.
4. The method for preparing a two-dimensional flow-measuring immunochromatographic test strip according to claim 2, wherein in the first step, trisodium citrate solution is added under stirring to react until the reaction solution turns red, and heating is continued for 10 min.
5. The method for preparing a two-dimensional flow-measuring immunochromatographic test strip according to claim 2, wherein the pH of the colloidal gold solution is adjusted to 9.0 in the second step.
6. The method for preparing a two-dimensional flow-measuring immunochromatographic test strip according to claim 2, wherein the centrifugation treatment in the second step is centrifugation for 15min at a speed of 13000 r/min.
7. The method for preparing a two-dimensional flow-measuring immunochromatographic strip according to claim 2, wherein the mass concentration of the BSA aqueous solution in the second step is 10%.
8. The method for preparing a two-dimensional flow-measuring immunochromatographic strip according to claim 2, wherein the temperature for drying in the fourth step is 37 ℃.
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