CN111089978A - Colloidal gold immunochromatographic kit for rapidly detecting ovarian cancer tumor marker Legumain and preparation method thereof - Google Patents
Colloidal gold immunochromatographic kit for rapidly detecting ovarian cancer tumor marker Legumain and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain and a preparation method thereof, wherein the kit comprises a PVC (polyvinyl chloride) base plate, a sample pad, a combination pad, a reaction pad and a water absorption pad, the PVC base plate is rectangular, the sample pad and the water absorption pad are symmetrically laid on one group of short edges of the rectangular PVC base plate respectively, the reaction pad is arranged between the sample pad and the water absorption pad, and the combination pad is also arranged between the sample pad and the reaction pad; the reaction pad is provided with a detection line coated with a colloidal gold-labeled mouse anti-LegumainN antibody and a quality control line coated with goat anti-mouse IgG. The colloidal gold is used as a lateral chromatography kit to mark and probe the preparation process to be mature and the performance to be stable; the specificity of the detection result can be enhanced, false positives appearing in the detection process are eliminated, and the detection result is more accurate; the sample size to be detected is small, and the kit is suitable for screening ovarian cancer of wide females, home self-checking and bedside rapid detection.
Description
Technical Field
The invention relates to the field of biological immunochromatographic assay detection methods, in particular to a colloidal gold immunochromatographic assay kit for rapidly detecting an ovarian cancer tumor marker Legumain and a preparation method thereof.
Background
Ovarian cancer is a common malignant tumor in gynecology, the mortality rate of ovarian cancer is the first of all gynecological malignant tumors, and the ovarian cancer is harmful to the health of women. Because ovarian cancer is usually asymptomatic in the early stage and lacks a simple and effective early diagnosis method, about 70% of patients are already in the late stage when the disease is seen, the 5-year survival rate of the late stage patients is 20% -30%, and the 5-year survival rate of the early stage patients is 70-90%. Therefore, early detection of ovarian cancer plays an important role in the selection of treatment regimens and in improving patient prognosis.
At present, the clinical common early detection methods for ovarian cancer mainly comprise gynecological examination, imaging examination, tumor marker detection and the like. When the methods are used in cooperation with each other, although the ovarian cancer can be accurately diagnosed, the requirements on equipment and operators are high, the detection cost is increased, and the popularization and the promotion of early detection of the ovarian cancer are hindered.
The detection of tumor markers is widely used for early screening of tumors, carbohydrate antigen125 (CA 125) is a commonly used tumor marker in diagnosis of ovarian cancer, but the specificity of the detection is low when the detection is used for diagnosis of ovarian cancer, false positive is easy to occur, and the like, so that a new tumor marker needs to be found to replace the detection or realize complementation with the detection.
Legumain is highly expressed in a variety of solid Tumor tissues in mice and humans compared to normal tissues, especially in Tumor cells, neovascular endothelial cells and Tumor-Associated Macrophages (TAMs), but not in corresponding Tumor cell lines cultured in vitro. Liu et al first reported that Legumain was associated with tumorigenesis. They found that Legumain expression was elevated in a variety of murine tumor cells but not in each tumor cell line cultured in vitro by Western blot and immunohistochemical analysis. Liu et al also examined human normal tissues and various solid tumor tissues, and showed that Legumain expression was very low in normal tissues, while Legumain expression was high in various solid tumor tissues (e.g., breast, colon, lung, prostate, ovarian tumors, and central nervous system malignancies).
While Legumain has been clinically and pathologically tested by Western blot and immunohistochemistry methods to confirm whether Legumain expression is associated with tumor differentiation, necrosis, apoptosis and prognosis, Murthy et al performed on 164 primary colon cancers, 34 distal normal mucosal tissues, 89 paracancerous tissues and 33 lymph node metastatic cancer tissue samples by Western blot and immunohistochemistry methods, it was shown that Legumain primary colon cancers, Legumain expression was significantly higher than that in distal normal and paracancerous tissues (P <0.05), but not significantly different from that in metastatic cancer tissue samples (P >0.05), Legumain tumor patients (P ═ 0.01) or stroma (P ═ 0.05), while Legumain was clinically and pathologically tested on Legumain vivo tumor patients, when Legumain vivo, the expression of Legumain tumor patients was significantly higher than that in distal normal and paracancerous tissues (P ═ 0.04), the expression of Legumain metastatic cancer tissue samples was significantly different from that in metastatic cancer tissue samples (P ═ 0.05), while Legumain vivo, the tumor patients, the expression of Legumain vitro, the same was confirmed by Western blot, the presence of antibodies that the Legumain vivo, the tumor patients, the expression of Legumain vivo, the tumor patients was shown to be able to inhibit the proliferation of the tumor metastasis of the tumor, and after the tumor metastasis of the tumor, the tumor metastasis of the tumor, the tumor metastasis of the tumor, the tumor metastasis of the tumor, the tumor was shown.
Disclosure of Invention
The invention aims to provide a colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain and a preparation method thereof, and aims to fill up the blank that Legumain is used as a new biological index and a detection method for Legumain does not appear clinically at present.
In order to achieve the purpose, the invention adopts the following technical scheme: the invention provides a colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain, which comprises a PVC (polyvinyl chloride) base plate, a sample pad, a combination pad, a reaction pad and a water absorption pad, wherein the PVC base plate is rectangular, the sample pad and the water absorption pad are symmetrically laid on one group of short edges of the rectangular PVC base plate respectively, the reaction pad is arranged between the sample pad and the water absorption pad, and the combination pad is also arranged between the sample pad and the reaction pad; the reaction pad is provided with a detection line coated with a colloidal gold-labeled mouse anti-Legumain antibody and a quality control line coated with goat anti-mouse IgG.
Further, the sample pad is treated by the sample pad treatment solution; the sample pad treatment solution comprises phosphate buffer solution, saccharides, bovine serum albumin, neutral salt, surfactant and water.
Further, the water is distilled water, ultrapure water or deionized water.
Further, the concentration of the phosphate buffer solution is 0.01mol/L, and the pH is 6.0-9.0.
Further, the surfactant is selected from Tween or Triton X-100 or PVP or surfactant S7 or surfactant S9.
Further, the concentration of the phosphate buffer was 0.01mol/L, the pH was 7.2, and Na was contained in the phosphate buffer2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%;
the final mass concentration of the sucrose is 3 percent; the final volume concentration of bovine serum albumin is 2%;
the sodium chloride concentration was 300 mM.
Further, the complex of colloidal gold conjugated with the anti-Legumain antibody was suspended and immobilized on the conjugate pad by a heavy suspension consisting of phosphate buffer, inert protein, saccharide and tween.
Further, the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 6.0-9.0; the inert protein is bovine serum albumin or casein or chicken egg white albumin.
The invention also provides a preparation method of the colloidal gold immunochromatographic kit for rapidly detecting the ovarian cancer tumor marker Legumain, which comprises the following specific steps:
step one, preparing colloidal gold: putting ultrapure water into a clean conical flask, putting the clean rotor into the clean conical flask, putting the clean conical flask on a heatable magnetic stirrer, heating the clean conical flask until bubbles uniformly emerge, quickly adding a chloroauric acid solution, immediately adding trisodium citrate after heating to boil, continuing heating, taking down the conical flask, naturally cooling at room temperature, then fixing the volume by using a volumetric flask, and sealing for later use;
step two, antibody labeling: adding potassium carbonate solution into the colloidal gold prepared in the step one, and adjusting the pH value of the solution to 7.5; adding Anti-Legumain-Ab1, mixing at room temperature to make Anti-Legumain-Ab1 fully bind to the colloidal gold particles, and completing the labeling of Anti-Legumain-Ab 1;
step three, sealing: adding a bovine serum albumin solution into the Anti-Legumain-Ab1 labeled by the colloidal gold in the step two, controlling the final mass concentration of the bovine serum albumin to be 1.5%, and uniformly mixing the mixture at room temperature for 30min to seal exposed sites on the colloidal gold particles;
step four, centrifuging the re-suspended and sealed colloidal gold labeled antibody at 10000rpm, discarding the supernatant, and re-suspending the precipitate with 120 mu L of re-suspension;
the resuspension comprises phosphate buffer, bovine serum albumin, sucrose, trehalose and tween;
wherein the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.2; the final mass concentration of bovine serum albumin is 1.5%, the final mass concentration of sucrose is 3%, the final mass concentration of trehalose is 3%, and the final mass concentration of tween is 2%; in phosphate buffer, Na2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%;
step five, preparing a sample pad: soaking the cut glass fiber membrane in the sample pad treatment solution, then drying in a forced air drying oven, taking out to obtain a sample pad, and putting the sample pad into a sealing bag for later use;
step six, preparing a bonding pad: uniformly spreading the Anti-Legumain-Ab1 labeled by the colloidal gold in the step two on a glass fiber membrane, drying to ensure that the colloidal gold particles labeled by the antibody are solidified on the glass fiber membrane to obtain a bonding pad, taking out the bonding pad, putting the bonding pad into a sealed bag, adding a drying agent, and sealing and storing;
step seven, preparing a reaction pad: pasting a nitrocellulose membrane on a PVC plate, and then coating Anti-Legumain-Ab2 with the concentration of 2mg/mL by PBS (phosphate buffer solution) with the concentration of 0.01mol/L and the pH value of 7.2 to be used as a detection line coating antigen; diluting goat anti-mouse IgG to 1mg/mL to be used as a quality control line C coating antigen; scratching a film by a gold spraying film scratching instrument at a speed of 1 mu L/cm, placing the film in a forced air drying machine to obtain a reaction pad, taking out the reaction pad, and placing the reaction pad into a sealing bag for later use;
and step eight, assembling the kit.
The combination pad is attached to the front end of the reaction pad, the reaction pad is pressed for 1mm, the sample pad is attached to the front end of the combination pad, the combination pad is pressed for 2mm, finally the front end of the water absorption paper with the size of 180mm multiplied by 100mm is attached to the rear end of the reaction pad, the reaction pad is pressed for 2mm, the other end of the water absorption paper is attached to the tail end of the PVC plate, the assembly of the test strip is completed, the assembled test strip is placed into a strip cutting machine, the test strip with the width of 4mm is cut into test strips, and the test strips are placed into a card shell, so that the Legumain rapid detection kit is.
Further, in the fifth step, the sample pad treatment solution consists of phosphate buffer solution, sucrose, sodium chloride, bovine serum albumin and tween, wherein the concentration of the phosphate buffer solution is 0.01mol/L, the pH value is 7.2, the concentration of the sodium chloride is 300mM, the final volume concentration of the bovine serum albumin is 2%, the final volume concentration of the tween is 0.05%, and Na in the phosphate buffer solution2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%.
The invention has the beneficial effects that:
the invention provides a colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain and a preparation method thereof, and the colloidal gold is used as a lateral chromatography kit marker to detect the mature preparation process and the stable performance; the specificity of the detection result can be enhanced, false positives appearing in the detection process are eliminated, and the detection result is more accurate; the sample size to be detected is small, and the kit is suitable for screening ovarian cancer of wide females, home self-checking and bedside rapid detection.
2, the invention provides a colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain and a preparation method thereof,
additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The primary objects and other advantages of the invention may be realized and attained by the instrumentalities particularly pointed out in the specification.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings.
FIG. 1 is a schematic view of the structure of the kit of the present invention.
FIG. 2 is a guide chart for determining the detection result of the kit of the present invention.
FIG. 3 is a graph showing the results of detection in the sensitivity test using the kit of example 1.
FIG. 4 is a graph showing the results of the false positive control test using the kit of example 1 and a conventional kit.
Reference numerals: 1-PVC bottom plate, 2-sample pad, 3-combination pad, 4-reaction pad, 41-detection line, 42-quality control line and 5-water absorption pad.
Detailed Description
The technical solutions of the present invention are described in detail below by examples, and the following examples are only exemplary and can be used only for explaining and illustrating the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention. The invention provides a colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain, which comprises a PVC (polyvinyl chloride) base plate 1, a sample pad 2, a combination pad 3, a reaction pad 4 and a water absorption pad 5, wherein the PVC base plate 1 is rectangular, the sample pad 2 and the water absorption pad 5 are symmetrically laid on one group of short edges of the rectangular PVC base plate 1 respectively, the reaction pad 4 is arranged between the sample pad 2 and the water absorption pad 5, and the combination pad 3 is also arranged between the sample pad 2 and the reaction pad 4; the reaction pad 4 is provided with a detection line 41 coated with a colloidal gold-labeled mouse anti-Legumain antibody and a quality control line 42 coated with goat anti-mouse IgG.
Examples
The invention will now be illustrated by means of specific embodiments, without however restricting its scope to the following examples.
Anti-Legumain-Ab1 and Anti-Legumain-Ab2 used in the kit are purchased from Abcom company, and the cargo numbers are Ab183028 and Ab232870 in sequence.
Example one, preparation of a colloidal gold kit for rapid detection of Legumain Anti-Legumain-Ab1 and Anti-Legumain-Ab2 are monoclonal antibodies against different epitopes of Legumain, respectively.
(1) Preparation of colloidal gold
Putting 80mL of ultrapure water into a clean conical flask, putting the clean rotor into the clean conical flask, putting the clean conical flask on a heatable magnetic stirrer, heating the clean conical flask until bubbles emerge uniformly, quickly adding 1mL of 1% chloroauric acid solution, heating the solution to boiling, immediately adding 1mL of 1.5% trisodium citrate, changing the color of the solution in the conical flask from gray black to wine red, continuing to heat the solution for 7min, taking the conical flask down, naturally cooling the solution at room temperature, metering the volume to 100mL by using a volumetric flask, and sealing the volumetric flask for later use.
(2) Antibody labeling
Taking 1mL of the colloidal gold prepared in the step (1), adding a potassium carbonate solution with the concentration of 0.1mol/L, and adjusting the pH value of the solution to 7.5; adding 3 μ L of 2mg/mL Anti-Leguman-Ab 1, mixing at room temperature for 30min to make Anti-Leguman-Ab 1 fully bond to the colloidal gold particles, and completing the labeling of Anti-Leguman-Ab 1;
(3) sealing of
Adding a bovine serum albumin solution into the Anti-Legumain-Ab1 labeled by the colloidal gold in the step (2), controlling the mass final concentration of the bovine serum albumin to be 1.5%, and uniformly mixing for 30min at room temperature to seal exposed sites on the colloidal gold particles;
(4) resuspension
Centrifuging the sealed colloidal gold labeled antibody at 10000rpm for 10min, discarding the supernatant, and resuspending the precipitate with 120 mu L of resuspension liquid; the resuspension is composed of phosphate buffer solution, bovine serum albumin, sucrose, trehalose and tween groupWherein the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.2; the final mass concentration of bovine serum albumin is 1.5%, the final mass concentration of sucrose is 3%, the final mass concentration of trehalose is 3%, and the final mass concentration of tween is 2%; in phosphate buffer, Na2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%;
the preparation method of 1L of resuspension comprises the following steps: taking 600ml of distilled water, wherein the concentration of a phosphate buffer solution is 0.01mol/L, and the pH value is 7.2; the final mass concentration of bovine serum albumin is 1.5%, the final mass concentration of sucrose is 3%, the final mass concentration of trehalose is 3%, and the final mass concentration of tween is 2%; accurately weighing each component, adding into distilled water, stirring, pouring into a 1L volumetric flask, and diluting with distilled water to desired volume for use.
(5) Preparation of sample pad 2
Cutting a GF-06 glass fiber membrane into 220mm multiplied by 100mm, soaking in a sample pad 2 treatment solution for 15min, then drying in a blast drying oven at 37 ℃ for 12h, taking out to obtain a sample pad 2, and putting the sample pad 2 into a sealing bag for later use; the sample pad 2 treatment solution consists of phosphate buffer solution, sucrose, sodium chloride, bovine serum albumin and tween, wherein the concentration of the phosphate buffer solution is 0.01mol/L, the pH is 7.2, the final mass concentration of the sucrose is 3%, the concentration of the sodium chloride is 300mM, the final volume concentration of the bovine serum albumin is 2%, the final volume concentration of the tween is 0.5%, and Na in the phosphate buffer solution2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044 percent, and the balance is water.
Method for preparing 1L of sample pad 2 treatment solution: taking 600ml of distilled water, accurately weighing the components according to the concentration of a phosphate buffer solution of 0.01mol/L, the pH value of 7.2, the final mass concentration of sucrose of 3%, the concentration of sodium chloride of 300mM, the final volume concentration of bovine serum albumin of 2% and the final volume concentration of tween of 0.5%, adding the components into the distilled water, uniformly stirring, pouring the mixture into a 1L volumetric flask, and fixing the volume with the distilled water for later use.
(6) Preparation of the conjugate pad 3
Uniformly spreading the Anti-Legumain-Ab1 marked by the colloidal gold in the step (2) on a GF-06 glass fiber membrane, drying to ensure that colloidal gold particles marked with the antibody are solidified on the glass fiber membrane to obtain a bonding pad 3, taking out the bonding pad and putting the bonding pad into a sealed bag, adding a drying agent, and sealing and storing;
(7) preparation of reaction pad 4
Pasting a nitrocellulose membrane on a PVC plate, and then diluting Anti-Legumain-Ab2 to the concentration of 2mg/mL by using PBS (phosphate buffer solution) with the concentration of 0.01mol/L and the pH value of 7.2 to be used as a coating source of a detection line 41; diluting goat anti-mouse IgG to 1mg/mL to be used as a quality control line 42C coating antigen; scratching a film by a gold spraying film scratching instrument at a speed of 1 mu L/cm, placing at 37 ℃ for forced air drying for 6 hours to obtain a reaction pad 4, taking out and placing in a sealing bag for later use;
(8) kit assembly
The combination pad 3 is attached to the front end of the reaction pad 4, the reaction pad is pressed for 41mm, the sample pad 2 is attached to the front end of the combination pad 3, the combination pad is pressed for 32mm, finally the front end of the water absorption paper with the size of 180mm multiplied by 100mm is attached to the rear end of the reaction pad 4, the reaction pad is pressed for 42mm, the other end of the water absorption paper is attached to the tail end of the PVC plate, the assembly of the test strip is completed, the assembled test strip is placed into a strip cutting machine, the test strip with the width of 4mm is cut into test strips, and the test strip is placed into a card shell, so that the Legumain rapid detection kit can.
Example II method for determining results of colloidal gold-labeled anti-Legumain antibody kit
The result judging method of the Legumain rapid detection kit is shown in fig. 2, when the C quality control band 1 and the T detection band 2 are developed, Legumain with higher sensitivity is contained in the detected sample (corresponding to 3 in fig. 2); when the C control band 1 is developed and the T detection band 2 is not developed, it indicates that Legumain (corresponding to 4 in FIG. 2) with or without sensitivity is not contained in the test sample; if the quality control band 1C does not develop color, the detection effect is invalid no matter whether the T detection band 2 develops color or not (corresponding to 5 in FIG. 2).
Performance detection experiment of Legumain colloidal gold rapid detection kit:
1. the method comprises the following steps of (1) diluting a Legumain standard substance with negative human serum into standard solutions with the concentrations of 400pmol/L, 200pmol/L, 100pmol/L and 50pmol/L respectively, wherein the negative is the negative serum without the Legumain standard substance; then 80. mu.L of standard solutions with different concentrations are respectively dripped into the assembled colloidal gold detection card, and the result is observed and recorded. The double-antibody sandwich method Legumain colloidal gold kit has the negative result that the detection line 41T is not colored, the quality control line 42C is colored, and the positive result that both the detection line 41T and the quality control line 42C are colored; the lowest standard solution concentration for detecting a positive result is the sensitivity of the kit, namely the sensitivity of the kit is 100pmol/L, and the detection result is shown in FIG. 3.
2. False positive control experiment
The difference between the conventional kit and the preparation method of the kit disclosed in example 1 is that the formulation of the treatment solution for the sample pad 2 is different, and the other preparation steps are the same: the sample pad 2 treatment solution disclosed in embodiment 1 of the present invention is composed of a phosphate buffer solution, sucrose, sodium chloride, bovine serum albumin and tween, wherein the concentration of the phosphate buffer solution is 0.01mol/L, the pH is 7.2, the final mass concentration of sucrose is 3%, the concentration of sodium chloride is 300mM, the final volume concentration of bovine serum albumin is 2%, the final volume concentration of tween is 0.05%, and Na in the phosphate buffer solution2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%; the sample pad 2 treatment solution of the common kit consists of phosphate buffer solution, bovine serum albumin, sodium chloride, sucrose and Tween-20, wherein the concentration of the phosphate buffer solution is 0.01mol/L, the pH is 7.2, the concentration of the sodium chloride is 150mM, the volume final concentration of the bovine serum albumin is 10%, the volume final concentration of the Tween-20 is 0.05%, the mass concentration of the sucrose is 7%, and Na in the phosphate buffer solution2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%.
First, 80. mu.L of Legumain negative serum and 80. mu.L of Legumain standard (final concentration is 200pmol/L) serum were added to a common kit, respectively, and the results of the detection were shown in FIGS. 41 and 2; next, two serum samples treated in the same manner were dropped into the kit prepared in example 1 of the present invention, and the results are shown in FIGS. 4, 3 and 4.
FIG. 4 shows the result that 1 is the detection result of the conventional kit for Legumain negative serum, which is shown as Legumain false positive, and illustrates that the detection of Legumain negative blood using the kit treated with the treatment solution of the conventional sample pad 2 may result in false positive, which may cause misdiagnosis of normal population; 2, the detection result of the serum added with Legumain is added into the ordinary kit, and the detection line 41T and the quality control line 42C have good color development, which indicates that the sensitivity and the color development effect of the ordinary kit are not influenced when Legumain with high concentration is added. 3, the detection result of the kit of the embodiment 1 of the invention added with the same Legumain negative serum is displayed as Legumain negative, which shows that in the preparation process of the kit, after the kit sample pad 2 is treated by the sample pad 2 treatment solution disclosed by the invention, the problem of Legumain false positive during the detection of Legumain negative serum (normal human blood is Legumain negative) can be obviously eliminated, and the misdiagnosis rate is greatly reduced; and 4, the detection result of adding high-concentration Legumain into the kit of the embodiment 1 is shown, and the detection line 41T and the quality control line 42C have good color development, which shows that the sensitivity and the color development effect of the kit of the embodiment 1 are not influenced.
And (4) conclusion: the sensitivity of the human Legumain colloidal gold early detection kit prepared by the double-antibody sandwich method is 100pmol/L, the clinical detection requirement of Legumain is met, the Legumain colloidal gold early detection kit prepared by the preparation method disclosed by the invention is low in false positive rate of detection, and the misdiagnosis rate of the kit prepared by a common method is reduced. The kit has the advantages of high sensitivity, high detection speed, simple and convenient operation, easy carrying, no need of special equipment, low cost, no need of professional training, clear and easily identified result, and suitability for bedside detection and family self-detection.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that may be made by those skilled in the art within the technical scope of the present invention will be covered by the scope of the present invention.
Claims (10)
1. A colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain is characterized in that: the kit comprises a PVC base plate (1), a sample pad (2), a combination pad (3), a reaction pad (4) and a water absorption pad (5), wherein the PVC base plate (1) is rectangular, the sample pad (2) and the water absorption pad (5) are symmetrically laid on one group of short edges of the rectangular PVC base plate (1) respectively, the reaction pad (4) is arranged between the sample pad (2) and the water absorption pad (5), and the combination pad (3) is also arranged between the sample pad (2) and the reaction pad (4); the reaction pad (4) is provided with a detection line (41) coated with a colloidal gold-labeled mouse anti-Legumain antibody and a quality control line (42) coated with goat anti-mouse IgG.
2. The colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain as claimed in claim 1, wherein the sample pad (2) is treated with a sample pad (2) treatment solution; the sample pad (2) treatment solution comprises phosphate buffer solution, saccharides, bovine serum albumin, neutral salt, surfactant and water.
3. The colloidal gold immunochromatographic kit for rapid detection of an ovarian cancer tumor marker Legumain as claimed in claim 2, wherein the water is distilled water or ultrapure water or deionized water.
4. The colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain as claimed in claim 2, wherein the concentration of the phosphate buffer is 0.01mol/L and the pH is 6.0 to 9.0.
5. The colloidal gold immunochromatographic kit for rapid detection of an ovarian cancer tumor marker Legumain as claimed in claim 2, wherein the surfactant is selected from tween or Triton X-100 or PVP or surfactant S7 or surfactant S9.
6. The colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain as claimed in claim 2, wherein the concentration of phosphate buffer is 0.01mol/L, the pH is 7.2, and Na is contained in phosphate buffer2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%;
the final mass concentration of the sucrose is 3 percent; the final volume concentration of bovine serum albumin is 2%;
the sodium chloride concentration was 300 mM.
7. The colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain as claimed in claim 2, wherein the complex in which colloidal gold is coupled to the anti-Legumain antibody is suspended and immobilized on the conjugate pad (3) by a resuspension solution consisting of phosphate buffer, inert protein, saccharide and tween.
8. The colloidal gold immunochromatographic kit for rapidly detecting an ovarian cancer tumor marker Legumain as claimed in claim 1, wherein the concentration of the phosphate buffer is 0.01mol/L, and the pH is 6.0 to 9.0; the inert protein is bovine serum albumin or casein or chicken egg white albumin.
9. The preparation method of the colloidal gold immunochromatographic kit for rapidly detecting the ovarian cancer tumor marker Legumain as claimed in any one of claims 1 to 8, which comprises the following specific steps:
step one, preparing colloidal gold: putting ultrapure water into a clean conical flask, putting the clean rotor into the clean conical flask, putting the clean conical flask on a heatable magnetic stirrer, heating the clean conical flask until bubbles uniformly emerge, quickly adding a chloroauric acid solution, immediately adding trisodium citrate after heating to boil, continuing heating, taking down the conical flask, naturally cooling at room temperature, then fixing the volume by using a volumetric flask, and sealing for later use;
step two, antibody labeling: adding potassium carbonate solution into the colloidal gold prepared in the step one, and adjusting the pH value of the solution to 7.5; adding Anti-Legumain-Ab1, mixing at room temperature to make Anti-Legumain-Ab1 fully bind to the colloidal gold particles, and completing the labeling of Anti-Legumain-Ab 1;
step three, sealing: adding a bovine serum albumin solution into the Anti-Legumain-Ab1 labeled by the colloidal gold in the step two, controlling the final mass concentration of the bovine serum albumin to be 1.5%, and uniformly mixing the mixture at room temperature for 30min to seal exposed sites on the colloidal gold particles;
step four, centrifuging the re-suspended and sealed colloidal gold labeled antibody at 10000rpm, discarding the supernatant, and re-suspending the precipitate with 120 mu L of re-suspension;
the resuspension comprises phosphate buffer, bovine serum albumin, sucrose, trehalose and tween;
wherein the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.2; the final mass concentration of bovine serum albumin is 1.5%, the final mass concentration of sucrose is 3%, the final mass concentration of trehalose is 3%, and the final mass concentration of tween is 2%; in phosphate buffer, Na2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%;
step five, preparing a sample pad (2): soaking the cut glass fiber membrane in the sample pad (2) treatment liquid, then drying in a forced air drying oven, taking out to obtain the sample pad (2), and placing in a sealed bag for later use;
step six, preparing the bonding pad (3): uniformly paving the Anti-Legumain-Ab1 marked by the colloidal gold in the step two on a glass fiber membrane, drying to ensure that the colloidal gold particles for marking the antibody are solidified on the glass fiber membrane to obtain a bonding pad (3), taking out the bonding pad, putting the bonding pad into a sealed bag, adding a drying agent, and sealing and storing;
step seven, preparing a reaction pad (4): pasting a nitrocellulose membrane on a PVC plate, and then coating an Anti-Legumain-Ab2 diluted by PBS (phosphate buffer solution) with the concentration of 0.01mol/L and the pH value of 7.2 to be 2mg/mL as a detection line (41); diluting goat anti-mouse IgG to 1mg/mL to be used as a quality control line (42) C coating antigen; scratching a film by a gold spraying film scratching instrument at a speed of 1 mu L/cm, placing the film in a forced air drying machine to obtain a reaction pad (4), taking out the reaction pad and placing the reaction pad into a sealing bag for later use;
step eight, assembling the kit;
the combination pad (3) is attached to the front end of the reaction pad (4), the reaction pad (4) is pressed for 1mm, the sample pad (2) is attached to the front end of the combination pad (3), the combination pad (3) is pressed for 2mm, finally the front end of the water absorption paper with the size of 180mm multiplied by 100mm is attached to the rear end of the reaction pad (4), the reaction pad (4) is pressed for 2mm, the other end of the water absorption paper is attached to the tail end of the PVC plate, the test paper strip is assembled, the assembled test paper strip is placed into a strip cutting machine, the test paper strip is cut into test paper strips with the width of 4mm, the test paper strip is placed into a card shell, and the Legumain rapid detection kit is obtained.
10. The method according to claim 9, wherein in the fifth step, the treatment solution of the sample pad (2) comprises phosphate buffer, sucrose, sodium chloride, bovine serum albumin and tween, wherein the concentration of the phosphate buffer is 0.01mol/L, the pH is 7.2, the concentration of the sodium chloride is 300mM, the final volume concentration of the bovine serum albumin is 2%, the final volume concentration of the tween is 0.05%, and Na in the phosphate buffer is added2HPO4·12H20.26% of O, NaH2PO4·2H2The mass content of O is 0.044%.
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