CN111381039A - Fluorescent test strip for quantitatively detecting thyroglobulin and preparation method thereof - Google Patents
Fluorescent test strip for quantitatively detecting thyroglobulin and preparation method thereof Download PDFInfo
- Publication number
- CN111381039A CN111381039A CN202010191801.6A CN202010191801A CN111381039A CN 111381039 A CN111381039 A CN 111381039A CN 202010191801 A CN202010191801 A CN 202010191801A CN 111381039 A CN111381039 A CN 111381039A
- Authority
- CN
- China
- Prior art keywords
- pad
- thyroglobulin
- test strip
- fluorescent
- buffer solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention relates to a fluorescent test strip for quantitatively detecting thyroglobulin and a preparation method thereof, wherein the test strip sequentially comprises a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad from bottom to top, the combination pad is coated with an antithyroid globulin antibody marked by a nano quantum dot fluorescent microsphere, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with another antithyroid globulin antibody, and the quality control line is coated with goat anti-mouse IgG. The fluorescent test strip prepared by the invention can be used for rapidly detecting the Tg value of the thyroglobulin Tg value of a suspicious lymph node on site and assisting in diagnosing the lymph node metastasis of papillary thyroid carcinoma, and can also be used for detecting the Tg value by virtue of lymph node fine needle puncture aspiration eluent during preoperative, intraoperative and postoperative follow-up visits and assisting in definite diagnosis of the metastasis of the suspicious cervical lymph node papillary thyroid carcinoma. The test strip is simple and convenient in use method, high in detection speed and strong in specificity.
Description
Technical Field
The invention relates to a fluorescent test strip for detecting thyroglobulin and a preparation method thereof, belonging to the technical field of biological reagents.
Background
The operation mode, the subsequent treatment and the follow-up visit of the thyroid cancer patients with cervical lymph node metastasis are completely different, and if the cervical lymph node is cleaned in a preventive manner, the defect is better than the benefit. In the postoperative follow-up visit of thyroid cancer, the recurrence rate of lymph node metastasis is 3% -30%, and the diagnosis rate is 27% -46%. Therefore, it is very important to identify whether or not the cervical lymph node is metastasized at an early stage. Although US-FNA is very specific for the diagnosis of cervical metastatic lymph nodes, it is less sensitive, especially for samples with a lower cell count. Therefore, early detection of cervical lymph node metastasis is one of the major and difficult points of individualized diagnosis and treatment for patients. Thyroglobulin (Tg) is a macromolecular glycoprotein secreted by thyroid follicular epithelial cells, and Tg is not detected in normal lymph nodes, and if Tg is detected at a high concentration in lymph node tissues, the possibility of metastasis of thyroid cancer should be highly warned. The determination of thyroglobulin in fine-needle aspiratory fluid (FNA-Tg) as an eluent by fine needle puncture is a new technology developed in recent years, the operation is simple and accurate, and the subjectivity of pathological diagnosis can be avoided to a certain extent. The FNA-Tg technology is accepted and widely applied to clinic at abroad, but the FNA-Tg technology has not attracted enough attention at home. The existing American and European thyroid society lists FNA-Tg monitoring as an auxiliary diagnosis method for thyroid cancer preoperative and postoperative lymph node evaluation, and provides a new basis for the formulation of a cervical lymph node cleaning strategy. The method adopts an immune labeling technology established on the basis of immunochromatography by adopting a fluorescent microsphere immune chromatography technology, and is characterized in that fluorescent microspheres are taken as a chromogenic marker to be combined with antigen and antibody specific reactions, and a fluorescent quantitative detector is combined to quantitatively detect the concentration of thyroglobulin. The detection method has the advantages of simple result judgment, short detection time, high sensitivity and strong specificity, and is suitable for field detection.
Disclosure of Invention
The invention aims to provide a test strip capable of rapidly and quantitatively analyzing the TG content in a lymph node fine needle puncture eluent, which has high sensitivity and can provide a reference index for making an operation scheme for detecting cervical lymph node metastasis in early clinical stage.
The invention adopts the following technical scheme: the utility model provides a fluorescence test paper strip for quantitative determination thyroglobulin, includes PVC board, sample pad, combination pad, cellulose nitrate membrane and absorbent pad from supreme down in proper order, and the combination pad is wrapped by one kind of anti thyroglobulin antibody that nanometer quantum dot fluorescence microsphere mark, be provided with detection line and quality control line on the cellulose nitrate membrane, another kind of anti thyroglobulin antibody of detection line parcel, sheep anti mouse IgG is wrapped up to the quality control line.
The preparation method of the fluorescent test strip for detecting thyroglobulin comprises the following steps
(1) Sample pad pretreatment: soaking the glass fiber membrane in a sample pad buffer solution for complete soaking, taking out, draining and drying for later use;
(2) pretreatment of the bonding pad: soaking the other glass fiber membrane in the buffer solution of the bonding pad until the other glass fiber membrane is completely soaked, taking out, draining and drying for later use;
(3) preparing a thyroglobulin antibody marked by fluorescent microspheres: adding a reaction buffer solution into the nano quantum dot fluorescent microspheres, uniformly mixing, adding an EDC solution, centrifuging to remove a supernatant, dissolving the reaction buffer solution, and carrying out ultrasonic resuspension; adding an antithyroid globulin antibody into the resuspension, shaking and uniformly mixing, carrying out ultrasonic treatment, and uniformly mixing at room temperature for reaction; centrifuging to remove supernatant, adding BSA solution, centrifuging to remove supernatant, precipitating, dissolving and ultrasonically treating, adding fluorescent microsphere redissolving solution, and storing to form thyroglobulin antibody fluorescent microsphere conjugate solution;
(4) preparing a bonding pad: spraying the thyroglobulin antibody fluorescent microsphere conjugate solution obtained in the step (3) on the treated conjugate pad by using a membrane-scribing metal spraying instrument, wherein the spraying amount is 5-10 mu l/cm, and drying at 37 ℃ overnight to form the thyroglobulin fluorescent microsphere conjugate pad for later use;
(5) preparation of coating film: diluting a thyroglobulin coated antibody to 1-2mg/mL by using a coating buffer solution, diluting goat anti-mouse IgG to 1-2mg/mL, coating the diluted goat anti-mouse IgG on a nitrocellulose membrane, and sticking the nitrocellulose membrane to a PVC (polyvinyl chloride) plate;
(6) assembling the test strip: the water absorption pad, the combination pad and the sample pad are cut to proper sizes respectively, and then the water absorption pad, the combination pad and the sample pad are sequentially pasted on a coated PVC board from top to bottom to form a large board.
Furthermore, the particle size of the nanometer quantum dot fluorescent microsphere is 100-200 nm.
Further, the fluorescent microsphere redissolution in the step (3) contains 0.242% by mass of Tris, 0.1-2% by mass of BSA, 10-20% by mass of sucrose, 1-5% by mass of trehalose, 0.05-0.5% by mass of Proclin300, and the pH value is adjusted to 7.5-8.5.
Further, the sample pad treatment solution in the step (1) comprises 0.6% by mass of Tris, 0.1-2% by mass of sodium caseinate, 0.1-1% by mass of PVP, 0.1-2% by volume of Tween-20, 0.05-0.5% by volume of Proclin300, purified water dissolved to 100mL, and pH adjusted to 8.2.
Further, the buffer solution of the binding pad in the step (2) comprises 0.6% by mass of Tris, 0.1-2% by mass of sodium caseinate, 0.1-1% by mass of PVP and 0.05-0.5% by volume of Proclin300, the purified water is dissolved in 100mL, and the pH value is adjusted to 8.2.
Further, 2-8ug of anti-thyroglobulin antibody is added per mg of fluorescent microspheres in the step (3).
Further, the reaction buffer used in step (3) is MES solution with pH5.5-6 or PBS buffer with pH 6.5-7.5.
Further, adding a reaction buffer solution and 10mg/ml EDC into the fluorescent microsphere solution in the step (3), uniformly mixing at room temperature for 0.5-1h, centrifuging at 10000-15000rpm for 10-30min, dissolving the precipitate with 0.05M/L MES buffer solution or 0.02M/LPBS, and carrying out ultrasonic resuspension; adding 1-20 μ l of antithyroid globulin antibody into the resuspension, shaking and mixing uniformly, performing ultrasonic treatment for 10-12s, and mixing uniformly at room temperature for reaction for 0.5-1 h; centrifuging at 10000-; adding 10% BSA, and uniformly mixing at room temperature for reacting for 1-2 h; centrifuging at 10000-.
The invention has the beneficial effects that: the preparation method is simple, the steps are easy to operate, the quantum dot nano-microsphere one-step method is adopted for marking, the time is shorter, the operation steps are fewer, the combination is stable, the sensitivity is high, and the anti-interference capability is stronger. The prepared fluorescent test strip can rapidly detect the thyroglobulin Tg value of a suspicious lymph node on site and assist in diagnosing the lymph node metastasis of papillary thyroid carcinoma. The test strip is simple and convenient in use method, high in detection speed and strong in specificity. Can quantitatively detect thyroglobulin, fully meets the clinical requirement and has wide application prospect.
Drawings
FIG. 1 is a standard curve of thyroglobulin detected in the present invention.
FIG. 2 is a schematic view of the use state of the fluorescent test strip prepared by the present invention.
Detailed Description
The present invention will be further described with reference to specific examples.
The glass fiber film adopted in the embodiment of the invention is purchased from Shanghai Lianxin; BSA was purchased ex BOVOGEN.
The utility model provides a fluorescence test paper strip for quantitative determination thyroglobulin, includes PVC board, sample pad, combination pad, cellulose nitrate membrane and water absorption pad from supreme in proper order down, and the combination pad is wrapped by one kind of antithyroid globulin antibody that nanometer quantum dot fluorescence microsphere mark, is provided with detection line and quality control line on the cellulose nitrate membrane, and another kind of antithyroid globulin antibody of detection line parcel, quality control line parcel sheep anti mouse IgG.
The specific assembly and cutting method is as follows: it is required to operate under the condition of humidity less than or equal to 30%
(1) Cutting the absorbent pad into 20 x 300mm pieces by a slitter for later use;
(2) cutting the bonding pad into 8 x 300mm by a strip cutting machine for later use;
(3) cutting the sample pad into 17 x 300mm by a slitter for later use;
(4) sequentially adhering the water absorption pad, the combination pad and the sample pad to the coated PVC rubber plate to assemble a large plate;
(5) the large panel was cut to 4mm width with an over speed chopper for further use.
Fluorescent microsphere complex solution: the additive comprises 0.242% of Tris, 0.1-2% of BSA, 10-20% of sucrose, 1-5% of trehalose and 0.05-0.5% of Proclin300 by mass percent, and the pH value is adjusted to 7.5-8.5.
The sample pad treatment solution comprises 0.6% by mass of Tris, 0.1-2% by mass of sodium caseinate, 0.1-1% by mass of PVP, 0.1-2% by volume of Tween-20, 0.05-0.5% by volume of Proclin300, purified water dissolved to 100mL, and pH adjusted to 8.2.
The buffer solution of the combination pad comprises 0.6 percent of Tris, 0.1 to 2 percent of sodium caseinate, 0.1 to 1 percent of PVP and 0.05 to 0.5 percent of Proclin300 in percentage by volume, and the buffer solution is dissolved in purified water to 100mL and the pH value is adjusted to 8.2.
Example 1:
the preparation method of the fluorescent test strip for detecting thyroglobulin comprises the following steps:
(1) sample pad pretreatment: soaking the glass fiber membrane in a sample pad buffer solution for complete soaking, taking out, draining and drying for later use;
(2) pretreatment of the bonding pad: soaking the other glass fiber membrane in the buffer solution of the bonding pad until the other glass fiber membrane is completely soaked, taking out, draining and drying for later use;
(3) preparing a thyroglobulin antibody marked by fluorescent microspheres: adding the nano quantum dot fluorescent microspheres with the particle size of 100nm into a reaction buffer solution and 10mg/ml EDC, uniformly mixing at room temperature for 0.5h, centrifuging at 10000rpm for 10min, dissolving the precipitate with 0.05M/LMES buffer solution or 0.02M/LPBS, and carrying out ultrasonic resuspension; adding 1 μ l of antithyroid globulin antibody into the resuspension, shaking and mixing uniformly, performing ultrasonic treatment for 10s, and mixing uniformly at room temperature for reaction for 0.5 h; centrifuging at 10000rpm for 10min, adding MES or pbs buffer solution for dissolving, and performing ultrasonic resuspension; adding 10% BSA, and uniformly mixing at room temperature for reaction for 1 h; centrifuging at 10000rpm for 10min, dissolving the precipitate with purified water, performing ultrasonic treatment, adding fluorescent microsphere redissolution, and storing to obtain thyroglobulin monoclonal antibody fluorescent microsphere conjugate solution;
(4) preparing a bonding pad: spraying the thyroglobulin antibody fluorescent microsphere conjugate solution obtained in the step (3) on the treated conjugate pad by using a film-scribing metal spraying instrument, wherein the spraying amount is 5 mu l/cm, and drying overnight at 37 ℃ to form the thyroglobulin fluorescent microsphere conjugate pad for later use;
(5) preparation of coating film: diluting a thyroglobulin coated antibody to 1mg/mL by using a coating buffer solution, diluting goat anti-mouse IgG to 1mg/mL, coating the diluted goat anti-mouse IgG on a nitrocellulose membrane, and adhering the nitrocellulose membrane to a PVC (polyvinyl chloride) plate;
(6) assembling the test strip: the water absorption pad, the combination pad and the sample pad are cut to proper sizes respectively, and then the water absorption pad, the combination pad and the sample pad are sequentially pasted on a coated PVC board from top to bottom to form a large board.
Example 2:
the preparation method of the fluorescent test strip for detecting thyroglobulin comprises the following steps:
(1) sample pad pretreatment: soaking the glass fiber membrane in a sample pad buffer solution for complete soaking, taking out, draining and drying for later use;
(2) pretreatment of the bonding pad: soaking the other glass fiber membrane in the buffer solution of the bonding pad until the other glass fiber membrane is completely soaked, taking out, draining and drying for later use;
(3) preparing a thyroglobulin antibody marked by fluorescent microspheres: adding the nano quantum dot fluorescent microspheres with the particle size of 150nm into a reaction buffer solution and 10mg/ml EDC, uniformly mixing at room temperature for 0.8h, centrifuging at 12000rpm for 20min, dissolving the precipitate with 0.05M/LMES buffer solution or 0.02M/LPBS, and carrying out ultrasonic resuspension; adding 10 μ l of antithyroid globulin antibody into the resuspension, shaking and mixing uniformly, performing ultrasonic treatment for 11s, and mixing uniformly at room temperature for reaction for 0.8 h; centrifuging at 12000rpm for 20min, adding MES or pbs buffer solution for dissolving, and performing ultrasonic resuspension; adding 10% BSA, and uniformly mixing at room temperature for reaction for 1.5 h; centrifuging at 12000rpm for 20min, dissolving the precipitate with purified water, ultrasonic treating, adding fluorescent microsphere redissolution, and storing to obtain thyroglobulin monoclonal antibody fluorescent microsphere conjugate solution;
(4) preparing a bonding pad: spraying the thyroglobulin antibody fluorescent microsphere conjugate solution obtained in the step (3) on the treated conjugate pad by using a film-scribing metal spraying instrument, wherein the spraying amount is 8 mu l/cm, and drying overnight at 37 ℃ to form the thyroglobulin fluorescent microsphere conjugate pad for later use;
(5) preparation of coating film: diluting a thyroglobulin coated antibody to 1.5mg/mL by using a coating buffer solution, diluting goat anti-mouse IgG to 1.5mg/mL, coating the diluted goat anti-mouse IgG on a nitrocellulose membrane, and sticking the nitrocellulose membrane to a PVC (polyvinyl chloride) plate;
(6) assembling the test strip: the water absorption pad, the combination pad and the sample pad are cut to proper sizes respectively, and then the water absorption pad, the combination pad and the sample pad are sequentially pasted on a coated PVC board from top to bottom to form a large board.
Example 3:
the preparation method of the fluorescent test strip for detecting thyroglobulin comprises the following steps:
(1) sample pad pretreatment: soaking the glass fiber membrane in a sample pad buffer solution for complete soaking, taking out, draining and drying for later use;
(2) pretreatment of the bonding pad: soaking the other glass fiber membrane in the buffer solution of the bonding pad until the other glass fiber membrane is completely soaked, taking out, draining and drying for later use;
(3) preparing a thyroglobulin antibody marked by fluorescent microspheres: adding the nano quantum dot fluorescent microspheres with the particle size of 200nm into a reaction buffer solution and 10mg/ml EDC, uniformly mixing at room temperature for 1h, centrifuging at 15000rpm for 30min, dissolving the precipitate with 0.05M/LMES buffer solution or 0.02M/LPBS, and carrying out ultrasonic resuspension; adding 20 mul of antithyroid globulin antibody into the resuspension, shaking and uniformly mixing, carrying out ultrasonic treatment for 12s, and uniformly mixing at room temperature for reaction for 1 h; centrifuging at 15000rpm for 30min, adding MES or pbs buffer solution for dissolving, and ultrasonic resuspending; adding 10% BSA, and uniformly mixing at room temperature for reaction for 2 h; centrifuging at 15000rpm for 30min, dissolving the precipitate with purified water, ultrasonic treating, adding fluorescent microsphere redissolution, and preserving to obtain thyroglobulin monoclonal antibody fluorescent microsphere conjugate solution;
(4) preparing a bonding pad: spraying the thyroglobulin antibody fluorescent microsphere conjugate solution obtained in the step (3) on the treated conjugate pad by using a film-scribing metal spraying instrument, wherein the spraying amount is 10 mu l/cm, and drying overnight at 37 ℃ to form the thyroglobulin fluorescent microsphere conjugate pad for later use;
(5) preparation of coating film: diluting a thyroglobulin coated antibody to 2mg/mL by using a coating buffer solution, diluting goat anti-mouse IgG to 2mg/mL, coating the diluted goat anti-mouse IgG on a nitrocellulose membrane, and adhering the nitrocellulose membrane to a PVC (polyvinyl chloride) plate;
(6) assembling the test strip: the water absorption pad, the combination pad and the sample pad are cut to proper sizes respectively, and then the water absorption pad, the combination pad and the sample pad are sequentially pasted on a coated PVC board from top to bottom to form a large board.
1. Diluting the Tg standard substance to 0, 5, 30, 60, 100 and 150ng/ml by using a sample diluent, adding the diluted Tg standard substance to the fluorescent test strip prepared by the invention, reading on a fluorescence immunoassay analyzer after 15-20 minutes, detecting each concentration three times, and taking an average value for fitting. The results are shown in Table 1, and the resulting standard curve is shown in FIG. 1.
TABLE 1
| Signal value | 0.0008 | 0.095 | 0.4106 | 0.6712 | 1.2254 | 1.7328 |
| Concentration (ng/ml) | 0 | 5 | 30 | 60 | 100 | 150 |
The 0 concentration was used for 20 replicates and the signal values are given in Table 2 below.
TABLE 2
Tg standards were tested 10 times at 30ng/ml and 100ng/ml concentrations, respectively, and the mean values were calculated as shown in Table 3.
TABLE 3
| Concentration of | 30 | 100 |
| 1 | 27.51 | 98.68 |
| 2 | 29.58 | 96.67 |
| 3 | 31.25 | 105.47 |
| 4 | 29.06 | 104.25 |
| 5 | 28.3 | 101.79 |
| 6 | 32.15 | 105.86 |
| 7 | 26.68 | 108.26 |
| 8 | 31.52 | 106.15 |
| 9 | 27.5 | 101.06 |
| 10 | 29.43 | 99.52 |
| Mean value | 29.298 | 102.771 |
| SD | 1.87 | 3.79 |
| CV | 6.37％ | 3.68％ |
As can be seen from the results shown in tables 2 and 3, the fluorescent test strip prepared by the method has good test uniformity and the coefficient of variation is less than 10%.
2. Compared with the existing detection test paper adopted clinically, the fluorescence test paper prepared by the invention has high detection sensitivity, the lowest detection limit is less than 0.1ng/ml and the accurate CV detection is less than 10 percent.
Claims (9)
1. A fluorescent test strip for quantitatively detecting thyroglobulin is characterized in that: from supreme PVC board, sample pad, combination pad, nitrocellulose membrane and the pad that absorbs water of including in proper order down, the combination pad parcel has the anti thyroglobulin antibody of nanometer quantum dot fluorescence microsphere mark, be provided with detection line and quality control line on the nitrocellulose membrane, another kind of anti thyroglobulin antibody of detection line parcel, sheep anti mouse IgG is wrapped to the quality control line parcel.
2. The method for preparing a fluorescent test strip for detecting thyroglobulin of claim 1, characterized in that: comprises the following steps
(1) Sample pad pretreatment: soaking the glass fiber membrane in a sample pad buffer solution for complete soaking, taking out, draining and drying for later use;
(2) pretreatment of the bonding pad: soaking the other glass fiber membrane in the buffer solution of the bonding pad until the other glass fiber membrane is completely soaked, taking out, draining and drying for later use;
(3) preparing a thyroglobulin antibody marked by fluorescent microspheres: adding a reaction buffer solution into the nano quantum dot fluorescent microspheres, uniformly mixing, adding an EDC solution, centrifuging to remove a supernatant, dissolving the reaction buffer solution, and carrying out ultrasonic resuspension; adding an antithyroid globulin antibody into the resuspension, shaking and uniformly mixing, carrying out ultrasonic treatment, and uniformly mixing at room temperature for reaction; centrifuging to remove supernatant, adding BSA solution, centrifuging to remove supernatant, precipitating, dissolving and ultrasonically treating, adding fluorescent microsphere redissolving solution, and storing to form thyroglobulin antibody fluorescent microsphere conjugate solution;
(4) preparing a bonding pad: spraying the thyroglobulin antibody fluorescent microsphere conjugate solution obtained in the step (3) on the treated conjugate pad by using a membrane-scribing metal spraying instrument, wherein the spraying amount is 5-10 mu l/cm, and drying at 37 ℃ overnight to form the thyroglobulin fluorescent microsphere conjugate pad for later use;
(5) preparation of coating film: diluting a thyroglobulin coated antibody to 1-2mg/mL by using a coating buffer solution, diluting goat anti-mouse IgG to 1-2mg/mL, coating the diluted goat anti-mouse IgG on a nitrocellulose membrane, and sticking the nitrocellulose membrane to a PVC (polyvinyl chloride) plate;
(6) assembling the test strip: the water absorption pad, the combination pad and the sample pad are cut to proper sizes respectively, and then the water absorption pad, the combination pad and the sample pad are sequentially pasted on a coated PVC board from top to bottom to form a large board.
3. The method of claim 2 for preparing a fluorescent test strip for detecting thyroglobulin, characterized in that: the particle size of the nanometer quantum dot fluorescent microsphere is 100-200 nm.
4. The method of claim 2 for preparing a fluorescent test strip for detecting thyroglobulin, characterized in that: the fluorescent microsphere redissolution in the step (3) comprises 0.242% by mass of Tris, 0.1-2% by mass of BSA, 10-20% by mass of sucrose, 1-5% by mass of trehalose, 0.05-0.5% by mass of Proclin300, and the pH value is adjusted to 7.5-8.5.
5. The method of claim 2 for preparing a fluorescent test strip for detecting thyroglobulin, characterized in that: the sample pad treatment solution in the step (1) comprises 0.6% of Tris by mass, 0.1-2% of sodium caseinate by mass, 0.1-1% of PVP by mass, 0.1-2% of Tween-20 by volume, 0.05-0.5% of Proclin300 by volume, purified water is dissolved to 100mL, and the pH is adjusted to 8.2.
6. The method of claim 2 for preparing a fluorescent test strip for detecting thyroglobulin, characterized in that: the binding pad buffer solution in the step (2) comprises 0.6% of Tris, 0.1-2% of sodium caseinate, 0.1-1% of PVP and 0.05-0.5% of Proclin300 in volume percentage, the purified water is dissolved to 100mL, and the pH value is adjusted to 8.2.
7. The method of claim 2 for preparing a fluorescent test strip for detecting thyroglobulin, characterized in that: and (3) adding 2-8ug of antithyroid globulin antibody into each milligram of the nano quantum dot fluorescent microspheres.
8. The method of claim 2 for preparing a fluorescent test strip for detecting thyroglobulin, characterized in that: the reaction buffer solution adopted in the step (3) is MES solution with pH value of 5.5-6 or PBS buffer solution with pH value of 6.5-7.5.
9. The method of claim 2 for preparing a fluorescent test strip for detecting thyroglobulin, characterized in that: adding a reaction buffer solution and 10mg/ml EDC into the nano quantum dot fluorescent microsphere solution in the step (3), uniformly mixing at room temperature for 0.5-1h, centrifuging at 10000-15000rpm for 10-30min, dissolving the precipitate with 0.05M/LMES buffer solution or 0.02M/LPBS, and carrying out ultrasonic resuspension; adding 1-20 μ l of antithyroid globulin antibody into the resuspension, shaking and mixing uniformly, performing ultrasonic treatment for 10-12s, and mixing uniformly at room temperature for reaction for 0.5-1 h; centrifuging at 10000-; adding 10% BSA, and uniformly mixing at room temperature for reacting for 1-2 h; centrifuging at 10000-.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010191801.6A CN111381039A (en) | 2020-03-18 | 2020-03-18 | Fluorescent test strip for quantitatively detecting thyroglobulin and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010191801.6A CN111381039A (en) | 2020-03-18 | 2020-03-18 | Fluorescent test strip for quantitatively detecting thyroglobulin and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111381039A true CN111381039A (en) | 2020-07-07 |
Family
ID=71221578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010191801.6A Pending CN111381039A (en) | 2020-03-18 | 2020-03-18 | Fluorescent test strip for quantitatively detecting thyroglobulin and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111381039A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113156121A (en) * | 2021-04-16 | 2021-07-23 | 苏州东尼生物技术有限公司 | Colloidal gold test strip for detecting human thyroglobulin |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106932573A (en) * | 2017-04-28 | 2017-07-07 | 天津医科大学总医院 | Detect immunity colloidal gold test paper strip of thyroglobulin and preparation method thereof |
| CN107870234A (en) * | 2017-10-19 | 2018-04-03 | 施康培医疗科技（武汉）有限公司 | A kind of test strips for detecting thyroglobulin concentration and preparation method thereof |
| CN108061727A (en) * | 2017-12-13 | 2018-05-22 | 施康培医疗科技（武汉）有限公司 | A kind of thyroglobulin quantitative testing test paper item and preparation method thereof |
| CN108254577A (en) * | 2018-04-10 | 2018-07-06 | 安徽金标点生物科技有限公司 | A kind of anti-mullerian duct hormone AMH diagnostic kits and preparation method thereof |
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
| CN108398566A (en) * | 2018-03-08 | 2018-08-14 | 江苏瑞安生物技术有限公司 | A kind of fluorescence immune chromatography detection card and preparation method thereof based on quantum dot microsphere |
| CN109187986A (en) * | 2018-08-21 | 2019-01-11 | 施康培医疗科技（武汉）有限公司 | A kind of test strips and preparation method thereof of portable inspectiont people anti-thyroglobulin antibody |
| CN109212208A (en) * | 2018-10-25 | 2019-01-15 | 郑州大学 | Test strips of detection tetanus antibody and preparation method thereof and application method are chromatographed based on quantum dot fluorescence microballoon |
| CN109799334A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | Fluorescent microsphere joint-detection device of cardiac marker and preparation method thereof |
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
-
2020
- 2020-03-18 CN CN202010191801.6A patent/CN111381039A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106932573A (en) * | 2017-04-28 | 2017-07-07 | 天津医科大学总医院 | Detect immunity colloidal gold test paper strip of thyroglobulin and preparation method thereof |
| CN107870234A (en) * | 2017-10-19 | 2018-04-03 | 施康培医疗科技（武汉）有限公司 | A kind of test strips for detecting thyroglobulin concentration and preparation method thereof |
| CN108061727A (en) * | 2017-12-13 | 2018-05-22 | 施康培医疗科技（武汉）有限公司 | A kind of thyroglobulin quantitative testing test paper item and preparation method thereof |
| CN108398566A (en) * | 2018-03-08 | 2018-08-14 | 江苏瑞安生物技术有限公司 | A kind of fluorescence immune chromatography detection card and preparation method thereof based on quantum dot microsphere |
| CN108254577A (en) * | 2018-04-10 | 2018-07-06 | 安徽金标点生物科技有限公司 | A kind of anti-mullerian duct hormone AMH diagnostic kits and preparation method thereof |
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
| CN109187986A (en) * | 2018-08-21 | 2019-01-11 | 施康培医疗科技（武汉）有限公司 | A kind of test strips and preparation method thereof of portable inspectiont people anti-thyroglobulin antibody |
| CN109212208A (en) * | 2018-10-25 | 2019-01-15 | 郑州大学 | Test strips of detection tetanus antibody and preparation method thereof and application method are chromatographed based on quantum dot fluorescence microballoon |
| CN109799334A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | Fluorescent microsphere joint-detection device of cardiac marker and preparation method thereof |
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113156121A (en) * | 2021-04-16 | 2021-07-23 | 苏州东尼生物技术有限公司 | Colloidal gold test strip for detecting human thyroglobulin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2015020210A1 (en) | Hemolytic streptococcus diagnostic immunochromato reagent, kit, and detection method | |
| EP3324185B1 (en) | Immunoassay and immunochromatographic kit | |
| US8999730B2 (en) | Immunochromatography method | |
| KR101919330B1 (en) | High sensitivity immunochromatographic method and kit for immunochromatograph | |
| US20140322724A1 (en) | Homogeneous competitive lateral flow assay | |
| EP3076177B1 (en) | Immunochromatography-assisted detection method | |
| JP2012211849A (en) | Highly sensitive immunochromatography method | |
| CN108398565A (en) | Immunofluorescence for detecting dog C reactive protein chromatographs detection card and preparation method | |
| TWI639002B (en) | Chromatographic medium | |
| CN106546750A (en) | A kind of preparation of microalbumin Quantitative detection test strips and detection method | |
| CN111381039A (en) | Fluorescent test strip for quantitatively detecting thyroglobulin and preparation method thereof | |
| CN117310164B (en) | Hepatitis C virus core antigen detection test strip and kit | |
| EP2770324B1 (en) | Chromatography method, chromatography kit, and method of producing an insoluble carrier for chromatography | |
| JP5238190B2 (en) | Immunoassay in the presence of hyaluronic acid and products used therefor | |
| CN114460287A (en) | Detection method and kit for neutralizing antibody | |
| CN114324879A (en) | Novel qualitative detection kit for coronavirus neutralizing antibody based on colloidal gold double-antibody sandwich method | |
| CN111208308A (en) | Preparation method of fluorescence immunochromatographic test paper for assisting ovarian cancer diagnosis based on FSH rapid detection | |
| US20180196042A1 (en) | Homogeneous competitive lateral flow assay | |
| CN109507420A (en) | The rapid detection method of buffalo Fasciola gigantica antibody | |
| JP6595215B2 (en) | Immunochromatographic analyzer, manufacturing method thereof, and immunochromatographic analysis method | |
| CN115248308A (en) | Application of poly-levodopa nanoparticles in preparation of immunochromatography detection test strip | |
| CN115951046A (en) | Human serum amyloid A time-resolved fluorescence immunochromatographic test paper and application | |
| CN115420893A (en) | PSEP colloidal gold test paper, kit, preparation method and application thereof | |
| JP5265423B2 (en) | Chromatographic method | |
| CN117074670A (en) | Kit for detecting typhoid antigens in faeces/serum samples and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |