Thyroglobulin detection kit and detection method, sample puncture detection needle and application

Abstract

The application relates to a thyroglobulin detection kit and a detection method, a sample puncture detection needle and application. The kit comprises a sample diluent and a sample puncture detection needle; a first coated carrier and a second coated carrier are sequentially arranged in the needle barrel along the axial direction of the needle barrel; the first coated carrier and the second coated carrier are matched with the gap of the needle barrel; the first coated carrier is close to the end of the needle head, the outer side of the first coated carrier is coated with a material capable of specifically combining with an antibody or an antigen of a human species; the second coated carrier is arranged at the end away from the needle head, and the outer side of the second coated carrier is coated with a material capable of specifically combining with an immune complex; a colorimetric card is arranged on the outer wall of the needle barrel. The technical problem solved is how to realize on-site rapid detection of the antibody or antigen concentration in the thyroglobulin, and overcome the defects that the prior art cannot meet the instantaneity and on-site nature of the operation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology, and specifically relates to a thyroglobulin detection kit, a semi-quantitative thyroglobulin detection method, a sample puncture needle, and the application of the sample puncture needle in the semi-quantitative detection of thyroglobulin. Background Technology

[0002] Thyroglobulin (Tg) is a large glycoprotein secreted by the follicular epithelium of the thyroid gland. It is commonly found in normal thyroid tissue, well-differentiated thyroid cancer tissue, and metastatic lymph nodes. Moreover, its concentration in tissue fluid is much higher than that in serum. Therefore, in the follow-up of differentiated thyroid cancer after surgery, if a high concentration of Tg is detected in extrathyroidal tissues, the possibility of thyroid cancer metastasis should be highly suspected.

[0003] The existing technologies for detecting thyroglobulin mainly include the following methods:

[0004] One method is FNA-Tg detection, which requires fine-needle aspiration, followed by smear preparation, fixation with anhydrous ethanol, and hematoxylin and eosin (HE) staining. All aspirated lesion locations must be clearly marked with their corresponding specimens, ensuring a one-to-one correspondence before being sent to pathology for cytological examination. The examination also involves repeated centrifugation and testing. This method is complex and time-consuming, making it unsuitable for clinical situations, especially during surgery, where immediate results are crucial, and thus failing to meet the immediate needs of on-site testing.

[0005] Another method is the "intraoperative rapid pathological section" method, which is the gold standard for determining whether thyroid cancer has metastasized to the cervical lymph nodes. The routine examination of this method takes about 40 minutes and cannot obtain test results in time. At the same time, the implementation of this method requires relevant pathology professionals, experienced pathologists, and very expensive and sophisticated equipment. The high cost of equipment and the shortage of professional personnel make it difficult to popularize and promote this method. This method also cannot meet the requirements of immediacy and on-site operation. Summary of the Invention

[0006] The main objective of this invention is to provide a thyroglobulin detection kit, a semi-quantitative thyroglobulin detection method, a sample puncture needle, and the application of the sample puncture needle in the semi-quantitative detection of thyroglobulin. The technical problem to be solved is how to achieve rapid on-site testing of antibody or antigen concentrations in thyroglobulin, overcoming the shortcomings of existing technologies that cannot meet the immediacy and on-site requirements of surgery, thus making it more suitable for practical use.

[0007] The objective of this invention and the technical problem it solves are achieved by the following technical solution. A thyroglobulin detection kit according to this invention comprises:

[0008] The sample diluent contains an antibody or antigen, and an immunoreaction buffer solution for diluting the antibody or antigen; the antibody or antigen is at least labeled with colored microspheres.

[0009] A sample puncture test needle includes a needle tip, a syringe, a first coating carrier, a second coating carrier, and a colorimetric card; the first and second coating carriers are sequentially disposed within the syringe along its axial direction; the first and second coating carriers are fitted with the syringe with a clearance fit; the first coating carrier is disposed at the end of the syringe near the needle tip; the outer coating material of the first coating carrier is capable of specifically binding antibodies or antigens of a human species; the second coating carrier is disposed at the end of the syringe opposite to the needle tip; the outer coating material of the second coating carrier is capable of specifically binding immune complexes; and

[0010] Colorimetric card.

[0011] The objectives of this invention and the technical problems it addresses can be further achieved by the following technical measures.

[0012] Preferably, in the aforementioned thyroglobulin detection kit, the antibody or antigen includes a detection antibody or antigen; the detection antibody or antigen is labeled with colored microspheres; the colored microspheres are selected from any one of fluorescent microspheres, quantum dots, colloidal gold, and latex microspheres.

[0013] Preferably, in the aforementioned thyroglobulin detection kit, when the antibody or antigen includes only the detection antibody, the outer coating material of the second coating carrier is a capture antibody capable of specifically binding to the antigen to be detected; when the antibody or antigen includes only the detection antigen, the outer coating material of the second coating carrier is a secondary antibody capable of specifically binding to the antibody to be detected.

[0014] Preferably, in the aforementioned thyroglobulin detection kit, the antibody or antigen further includes a capture antibody; the capture antibody is labeled with biotin or fluorescein.

[0015] Preferably, in the aforementioned thyroglobulin detection kit, the outer coating material of the second coating carrier is as follows:

[0016] When the capture antibody is labeled with biotin, the outer coating material of the second coating carrier is streptavidin;

[0017] When the capture antibody is labeled with fluorescein, the outer coating material of the second coating carrier is a fluorescein antibody.

[0018] The objective of this invention and the technical problem it solves are achieved through the following technical solution. A semi-quantitative detection method for thyroglobulin according to this invention includes the following steps:

[0019] 1) Aspiration to extract thyroglobulin test samples;

[0020] 2) Inject a specified amount of test sample directly into the sample diluent, mix well, react, and obtain a solution containing immune complexes; the sample diluent contains antibodies or antigens, and an immunoreaction buffer solution for diluting the antibodies or antigens; the antibodies or antigens are at least labeled with colored microspheres;

[0021] 3) The solution containing the immune complex is sequentially passed through a first coating carrier and a second coating carrier; the outer side of the first coating carrier is coated with a component that can specifically bind to antibodies or antigens of human species; the outer side of the second coating carrier is coated with a component that can specifically bind to the immune complex.

[0022] 4) Read the test results of thyroglobulin using a colorimetric card.

[0023] Preferably, the aforementioned semi-quantitative detection method for thyroglobulin is implemented using the aforementioned thyroglobulin detection kit.

[0024] The objective of this invention and the technical problem it solves are achieved by the following technical solution. A sample puncture detection needle according to this invention includes a needle tip, a syringe barrel, and a piston rod arranged sequentially. The needle tip is disposed at one end of the syringe barrel; the piston rod is disposed at the other end of the syringe barrel and is axially movable along the inner wall of the syringe barrel. It further includes:

[0025] A first coating carrier is disposed inside the syringe near the needle tip; the outer coating material of the first coating carrier is capable of specifically binding to antibodies or antigens of a human species; the first coating carrier is spaced and fitted with the syringe.

[0026] The second coating carrier is disposed inside the syringe at one end away from the needle; the outer coating material of the second coating carrier can specifically bind immune complexes; the second coating carrier is in close fit with the syringe; both the first coating carrier and the second coating carrier are disposed between the needle and the piston rod.

[0027] Preferably, in the aforementioned sample puncture detection needle, both the first coating carrier and the second coating carrier are movably engaged with the inner wall of the syringe; the first coating carrier and the second coating carrier are coated at least on the sidewalls parallel to the inner wall of the syringe; a filter screen is provided at the connection between the syringe and the needle tip; the pore size of the filter screen is 1 μm; the sample puncture detection needle also includes a scale line; the scale line is located on the outer wall of the syringe near the needle tip and is used to measure the volume of the sample to be detected.

[0028] The objective of this invention and the technical problem it solves are achieved by the following technical solution. According to this invention, an application of the aforementioned sample puncture detection needle in the semi-quantitative detection of thyroglobulin is provided, wherein the outer coating material of the second coating carrier is matched with the type of the sample to be tested and the sample diluent; specifically, the following steps are included:

[0029] 1) The test sample is drawn through the needle and placed in the sample diluent to react, thus obtaining an immune complex solution;

[0030] 2) The immune complex solution is drawn into the syringe through the needle by the movement of the piston rod; the immune complex solution first flows through the gap between the first coated carrier and the syringe to trap the body's own antibodies or antigens, and then flows through the gap between the second coated carrier and the syringe to capture the immune complex.

[0031] 3) Read the test results of thyroglobulin using a colorimetric card.

[0032] By employing the above technical solutions, the thyroglobulin detection kit, the semi-quantitative thyroglobulin detection method, the sample puncture needle, and the application of the sample puncture needle in the semi-quantitative detection of thyroglobulin proposed in this invention have at least the following advantages:

[0033] The thyroglobulin detection kit, semi-quantitative thyroglobulin detection method, sample puncture needle, and application of the sample puncture needle in semi-quantitative thyroglobulin detection proposed in this application allow for sample collection via a needle. The sample is then added to a sample diluent. When detecting the antigen concentration in thyroglobulin, the sample diluent includes antibodies and an immune reaction buffer. When detecting the antibody concentration in thyroglobulin, the sample diluent includes antigens and an immune reaction buffer. The antibodies or antigens in the sample diluent are at least labeled with colored microspheres, and may also be labeled with biotin or fluorescein. The sample reacts in the sample diluent, potentially generating a substance containing biotin or fluorescein. An immune complex solution containing "photosensitive antibody-capture antibody-detection antigen-detection antibody-color microspheres", "detection antigen-detection antibody-color microspheres", or "detection antibody-detection antigen-color microspheres" is used. The immune complex solution first flows through a first coated carrier, where the coating material can trap the body's own antibodies or antigens to avoid or reduce their impact on the subsequent color development, thereby reducing or avoiding false negative or false positive detection errors. The immune complex solution then flows through a second coated carrier, where the coating material specifically binds to the immune complex, causing it to develop color. The detection result for thyroglobulin is then read using a colorimetric card. The technical solution of this invention does not require extensive pretreatment of the test sample or repeated centrifugation. It only requires injecting the extracted test sample directly into the sample diluent for reaction, and then passing the resulting immune complex solution sequentially through the first and second coated carriers to obtain the concentration of antibodies or antigens in thyroglobulins. This enables real-time and effective inference or even judgment of related conditions, effectively solving the problem that existing technologies cannot meet the requirements of immediacy and on-site operation.

[0034] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the sample puncture detection needle of the present invention;

[0036] Figure 2 This is a schematic diagram of the structure of the first or second coated carrier of the sample puncture detection needle of the present invention in conjunction with the syringe.

[0037] Figure 3 It shows Figure 2 A schematic diagram of the cross section along AA. Detailed Implementation

[0038] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following, in conjunction with the accompanying drawings and preferred embodiments, details the specific implementation, structure, features and effects of a thyroglobulin detection kit, a semi-quantitative thyroglobulin detection method, a sample puncture needle and its application in the semi-quantitative detection of thyroglobulin according to the present invention.

[0039] This invention proposes a thyroglobulin detection kit, which includes a sample diluent and a sample puncture needle. The sample diluent is used to induce a complex reaction in the test sample to generate an immune complex; the sample puncture needle can be used to extract the test sample and inject it into the sample diluent, and can also be used to draw the immune complex solution generated by the complex reaction into a syringe for capture and color development to obtain the test result.

[0040] The sample diluent contains antibodies or antigens, and an immunoreaction buffer solution for diluting the antibodies or antigens. The immunoreaction buffer solution can be any form of buffer solution commonly used in the art, such as any one or more of phosphate buffer, MES / HEPES, carbonate buffer, or citrate buffer, etc., without specific limitations in this invention.

[0041] When the indicator to be detected is the concentration of antigen in thyroglobulin, the sample diluent contains an antibody and an immunoreaction buffer solution to dilute the antibody; when the indicator to be detected is the concentration of antibody in thyroglobulin, the sample diluent contains an antigen and an immunoreaction buffer solution to dilute the antigen.

[0042] The sample diluent includes antibodies or antigens, which may contain only detection antibodies or antigens for binding to the colored microspheres, or may also contain capture antibodies for binding biotin or fluorescein. The detection antibodies or antigens are labeled with colored microspheres; the colored microspheres are selected from any one of fluorescent microspheres, quantum dots, colloidal gold, and latex microspheres.

[0043] When the antibody or antigen includes only a detection antibody, the outer coating material of the second coating carrier is a capture antibody capable of specifically binding to the antigen to be detected; when the antibody or antigen includes only a detection antigen, the outer coating material of the second coating carrier is a secondary antibody capable of specifically binding to the antibody to be detected; the secondary antibody is an antibody extracted from other species that is anti-human antibody.

[0044] The molar ratio of biotin to the capture antibody is 1:10 to 100; preferably, the molar ratio is 1:20; the molar ratio of fluorescein to the capture antibody is 1:1 to 100; preferably, the molar ratio is 1:5.

[0045] The outer coating material of the second coating carrier is as follows:

[0046] When the capture antibody is labeled with biotin, the outer coating material of the second coating carrier is streptavidin. When the outer coating material of the second coating carrier is streptavidin, the coating process is as follows: Prepare a 0.06 mol / L citrate-sodium citrate buffer solution with a pH of 4.8; then dilute streptavidin to a concentration of 1 μg / ml to 50 μg / ml, preferably 10 μg / ml; then add 0.02 mg of CH-((CH2)n-NHS)3 to a 1 mg streptavidin solution, where n is selected as 5-15, preferably 10; stir and incubate at room temperature for 20 min. This solution serves as the second coating solution. The N-hydroxysuccinimide ester (-NHS) on this molecule can specifically bind to the amino group on streptavidin, thereby modifying streptavidin into polystreptavidin and enhancing the adsorption capacity of streptavidin on polystyrene materials. The coated region of the second coated carrier was then immersed in the second coating solution, followed by immersion in a blocking solution for blocking. The blocking solution was a 0.02 mol / L phosphate buffer, which also contained 1% BSA and 0.1% biological preservative. The carrier was then removed and dried. Finally, the second coated carrier, coated with the components of the second coating solution, was installed in a syringe near the piston rod for later use.

[0047] When the capture antibody is labeled with fluorescein, the outer coating material of the second coating carrier is a corresponding fluorescein antibody. The coating process for the outer coating of the second coating carrier with fluorescein antibody is as follows: Prepare a 0.06 mol / L citrate-sodium citrate buffer solution with a pH of 4.8; then dilute the fluorescein antibody to 1 μg / ml to 50 μg / ml, preferably 5 μg / ml, and use this solution as the second coating solution. Then immerse the coating region of the second coating carrier in the second coating solution, and then immerse it in a blocking solution for blocking; the blocking solution is a 0.02 mol / L phosphate buffer solution, which also contains 1% BSA and 0.1% biological preservative; remove and dry. Finally, install the second coating carrier coated with the components of the second coating solution into the syringe near the piston rod for later use.

[0048] In the sample diluent, the volume ratio of the antibody or antigen to the immune reaction buffer is 1:300 to 3000; generally, 1:300 is preferred. When both capture antibody and detection antibody are included, their molar ratio is preferably 1:1 to ensure effective formation of immune complexes without wasting material costs.

[0049] In the thyroglobulin detection kit, each vial typically contains 0.05 ml to 0.5 ml of sample diluent; preferably 0.2 ml.

[0050] The sample puncture needle included in the thyroglobulin detection kit, as shown in the attached... Figure 1 As shown, it includes a needle 31, a syringe 32, a first coating carrier 41, and a second coating carrier 42; the first and second coating carriers are sequentially disposed within the syringe along the axial direction of the syringe; the first and second coating carriers are fitted with the syringe with a gap; the first coating carrier is disposed at one end of the syringe near the needle; the outer coating material of the first coating carrier can specifically bind to antibodies or antigens of human species; generally, it can be coated with secondary antibodies, such as donkey anti-human secondary antibody, mouse anti-human secondary antibody, or rabbit anti-human secondary antibody. The first coating carrier has a coating solution concentration of 1 μg / ml to 50 μg / ml, preferably 10 μg / ml. When the captured antibody is a labeled fluorescein, the outer coating material of the first coating carrier can also be coated with streptavidin. The specific process of coating with streptavidin is as described above. When the immune complex solution flows through the first coating carrier, the first coating carrier can retain the body's own antibodies or antigens to avoid or reduce their influence on the colorimetric results, thereby avoiding or reducing false positive or false negative results. The second coating carrier is disposed at the end of the syringe opposite to the needle tip. The outer coating material of the second coating carrier can specifically bind to the immune complex. When the immune complex solution flows through the second coating carrier, the second coating carrier can capture the immune complex, causing the linked colored microspheres to accumulate there, thereby identifying the number of antibodies or antigens captured by the second coating material based on the intensity of the color development.

[0051] The thyroglobulin detection kit also includes a colorimetric card (not shown in the figure); the colorimetric card can exist independently; or, the colorimetric card can also be placed on the outer wall of the syringe for easy storage and colorimetric comparison; by visually comparing the color of the colored microspheres accumulated at the second coating material with the standard color of the colorimetric card, the concentration result of the detected antibody or antigen can be obtained.

[0052] The above-mentioned technical solution for detecting thyroglobulin does not require sample pretreatment. It can be directly injected into the sample diluent for reaction, and the reaction product can be aspirated into a syringe for colorimetric reading. It is not only fast, but can also be used for detection in any situation.

[0053] This invention also proposes a semi-quantitative detection method for thyroglobulin, which includes the following steps:

[0054] 1) Aspiration to extract thyroglobulin test samples;

[0055] 2) Inject a specified amount of test sample directly into the sample diluent, mix well, react, and obtain a solution containing immune complexes; the sample diluent contains antibodies or antigens, and an immunoreaction buffer solution for diluting the antibodies or antigens; the antibodies or antigens are at least labeled with colored microspheres;

[0056] 3) The solution containing the immune complex is sequentially passed through a first coating carrier and a second coating carrier; the outer side of the first coating carrier is coated with a component that can specifically bind to antibodies or antigens of human species; the outer side of the second coating carrier is coated with a component that can specifically bind to the immune complex.

[0057] 4) Read the test results of thyroglobulin using a colorimetric card.

[0058] The above detection method can be achieved using the aforementioned thyroglobulin detection kit.

[0059] This invention also proposes a sample puncture detection needle, as shown in the attached document. Figure 1 As shown, it includes a needle 31, a syringe 32, and a piston rod 34 arranged sequentially. The needle is located at one end of the syringe. The needle can be selected and designed according to the puncture needs, and must ensure a vacuum cleanliness standard for the surgical environment. The syringe is transparent to facilitate observation of the color emitted by the second coated carrier for colorimetric reading. The piston rod is located at the other end of the syringe and can move axially along the inner wall of the syringe. It also includes:

[0060] The first coating carrier 41 is disposed inside the syringe near the needle tip; the outer coating material of the first coating carrier can specifically bind to antibodies or antigens of human species; the first coating carrier is in inter-species fit with the syringe.

[0061] The second coating carrier 42 is disposed inside the syringe at one end away from the needle; the outer coating material of the second coating carrier can specifically bind immune complexes; the second coating carrier is in close fit with the syringe; both the first coating carrier and the second coating carrier are disposed between the needle and the piston rod.

[0062] In order to accurately measure the number of test samples taken, the sample puncture test needle may also be provided with a scale line 33; the scale line is set on the outer wall of the syringe near the end of the needle tip, and is used to measure the volume of the test sample, so as to realize the quantitative extraction of the test sample and improve the detection accuracy.

[0063] To facilitate intuitive colorimetric comparison, the sample puncture detection needle also includes a colorimetric card (not shown in the figure); the colorimetric card is disposed on the outer wall of the syringe at the end opposite to the needle tip, and serves as a colorimetric standard for reading the test results. The colorimetric card can be any standard component well-known to those skilled in the art, and this invention does not impose specific limitations on it.

[0064] To prevent tissues, red blood cells, macromolecules, impurities, etc. from entering the syringe and affecting subsequent testing, the present invention can set a filter screen at the connection between the syringe and the needle to achieve effective filtration; generally, the pore size of the filter screen is preferably 1μm.

[0065] The graduation line interval is 50 μl; when using a sample puncture needle to extract thyroid fluid for testing, the preferred sample volume is 50–300 μl, and the graduation lines are set to 50 μl, 100 μl, 150 μl, 200 μl, and 300 μl; when detecting thyroglobulin, the preferred sample volume is 100 μl to effectively ensure that the extracted concentration of the specified antigen is close to the true concentration.

[0066] The first coating carrier is spaced apart from the scale line along the axis of the syringe, that is, there is a certain gap between it and the maximum volume scale line, so that when quantitatively extracting the test sample, if no shaking is performed, the test sample will not come into contact with the first coating carrier, thereby avoiding the reaction between the test sample and the sample diluent.

[0067] To facilitate the installation of the first and second coating carriers, the present invention allows the first and second coating carriers to be movably snapped onto the inner wall of the syringe, as shown in the attached figure. Figure 2 and attached Figure 3 As shown, protrusions can be provided on the sidewalls of either radial end of the first coating carrier and the second coating carrier, and grooves can be provided on the inner wall of the syringe corresponding to the protrusions, so as to achieve snap-fit ​​by using the protrusions and grooves; of course, the number of protrusions can be designed and adjusted according to actual needs, and can be distributed at intervals around the axial direction of the first coating carrier and the second coating carrier.

[0068] To facilitate observation of color development and colorimetric comparison, the outer walls of the first and second coating carriers (excluding the aforementioned protrusions) are configured with a gap fit to the inner wall of the syringe. This allows the immune complex solution to flow through the gap into the first and second coating carriers, contacting the coating components on them. The accumulated color of the colored microspheres is primarily displayed in the gap between the outer walls of the first and second coating carriers and the inner wall of the syringe, thereby effectively improving the clarity of color development and the convenience of colorimetric comparison.

[0069] The first coating carrier and the second coating carrier are coated at least on the sidewalls parallel to the inner wall of the syringe.

[0070] The first and second coating carriers are rigid structures attached by immersion in a specific coating solution. The shapes of the first and second coating carriers can be columnar or disc-shaped. For example, the material can be polystyrene. At least the outer surfaces of the first and second coating carriers are irradiated with cobalt source gamma rays to enable them to adsorb antibodies or antigens. Thus, when the immune complex solution flows through the first and second coating carriers, human antibodies or antigens can be immediately adsorbed on the outer surface of the first coating carrier to form a trapping effect on human autoantibodies or antigens, thus avoiding their influence on the detection results. At the same time, exogenous (non-human) antibodies or antigens can be immediately adsorbed on the outer surface of the second coating carrier to form the capture and accumulation of colored microspheres, producing different depths of color, which can then be read by colorimetric reading using a colorimetric card.

[0071] This invention also proposes an application of the aforementioned sample puncture detection needle in the semi-quantitative detection of thyroglobulin, wherein the outer coating material of the second coating carrier is matched with the type of the sample to be tested and the sample diluent, as detailed above; specifically including the following steps:

[0072] 1) The test sample is drawn through the needle and placed in the sample diluent to react, thus obtaining an immune complex solution;

[0073] 2) The immune complex solution is drawn into the syringe through the needle by the movement of the piston rod; the immune complex solution first flows through the gap between the first coated carrier and the syringe to trap the body's own antibodies or antigens, and then flows through the gap between the second coated carrier and the syringe to capture the immune complex.

[0074] 3) Read the test results of thyroglobulin using a colorimetric card.

[0075] The present invention will be further described below with reference to specific embodiments, but this should not be construed as a limitation on the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention still fall within the scope of protection of the present invention.

[0076] Unless otherwise specified, all materials and reagents mentioned below are commercially available products well known to those skilled in the art; unless otherwise specified, all methods described are methods known in the art. Unless otherwise defined, the technical or scientific terms used should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0077] Example 1

[0078] Thyroid biopsy combined with detection of antigen concentration in thyroglobulin (TG)

[0079] 1. Preparation of sample diluent

[0080] 1.1 Biotin-labeled thyroglobulin capture antibody:

[0081] 1.1.1 Antibody pretreatment:

[0082] Select an ultrafiltration column with appropriate cutoff, add 200 μl of phosphate buffered saline solution (PBS, concentration 0.1 mol / L, pH 7.2, the same below), add 1 mg of capture antibody with a concentration of 5 mg / mL, mix well; centrifuge at 4000 rpm for 5 min at 4℃, and discard the filtrate.

[0083] Add 200 μl of phosphate buffer solution to the ultrafiltration column and mix well; centrifuge at 4000 rpm for 5 min at 4°C and discard the filtrate. Repeat steps 1 to 3 times.

[0084] Mix the remaining liquid in the ultrafiltration column and let it stand at room temperature for 1 minute. Invert the ultrafiltration column and place it in a new ultrafiltration tube. Centrifuge at 4000 rpm for 5 minutes at 4°C and collect the liquid.

[0085] Add 50 μl of phosphate buffer solution to the ultrafiltration column, mix well, and let stand for 1 min. Invert the ultrafiltration column and centrifuge at 6000 rpm for 2 min at 4 °C, then collect the liquid.

[0086] The collected filtrates were combined, and the concentration of the capture antibody was adjusted to 2 mg / ml with phosphate buffer solution and stored at 4°C for later use.

[0087] 1.1.2 Biotin labeling:

[0088] Biotin was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg / ml. It was then added to the capture antibody solution prepared in the previous step at a molar ratio of 1:20 between biotin and the capture antibody molecules. The mixture was then reacted at room temperature in the dark with stirring for 1 hour.

[0089] 1.1.3 Remove free biotin and other reagents by dextran gel separation / dialysis bag or ultrafiltration tube.

[0090] 1.1.4 Store the capture antibody in 0.02M phosphate buffer.

[0091] 1.2 Red latex microspheres labeled with thyroglobulin to detect antibodies:

[0092] 1.2.1 Cleaning:

[0093] Take 12.5 μL of red latex microspheres (4% solid content) and add them to 487.5 μL of 2-(N-morpholino)ethanesulfonic acid buffer (MES, concentration 50 mmol / L, pH 6.0, the same below). After sonicating and mixing, freeze at high speed and centrifuge 1 to 3 times. During the process, pay attention to avoid agglomeration and clumping.

[0094] 1.2.2 Primary activation:

[0095] Take the cleaned red latex microspheres and add them to 500 μl of 2-(N-morpholino)ethanesulfonic acid buffer. After sonicating and mixing, add 10 μl of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in 2-(N-morpholino)ethanesulfonic acid buffer solution. The solution is obtained by dissolving 10 mg of EDC solute in 1 ml of 2-(N-morpholino)ethanesulfonic acid buffer. Shake well and react at room temperature for 30 min. After the first activation, centrifuge the microspheres at high speed, carefully observing during the process to avoid agglomeration.

[0096] 1.2.3 Connecting the spacer arm:

[0097] Take one activated red latex microsphere, add 100 μl of 2-(N-morpholino)ethanesulfonic acid buffer, sonicate to mix, and then add 10 μg of carboxyl extension molecule (COOH)2-CH-(CH2). 12 -NH2, shake well and react at room temperature for 30 min, then couple and centrifuge the microspheres at high speed under refrigeration.

[0098] 1.2.4 Secondary activation:

[0099] Take the centrifuged red latex microspheres with spacers, add 500 μl of 2-(N-morpholino)ethanesulfonic acid buffer, sonicate to mix, then add 20 μl of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in 2-(N-morpholino)ethanesulfonic acid buffer solution (solution as in 1.2.2); shake well and react at room temperature for 30 min. After secondary activation, centrifuge the microspheres at high speed.

[0100] 1.2.5 Antibody conjugation:

[0101] Take the red latex microspheres after secondary activation, add 100 μl of 2-(N-morpholino)ethanesulfonic acid buffer, mix by sonication, add 20 μg of detection antibody with a concentration of 5 mg / mL, stir at room temperature in the dark for 2 h, and after coupling, centrifuge the microspheres at high speed. During the process, pay attention to avoid agglomeration and clumping.

[0102] 1.2.6 Closure:

[0103] Take the coupled red microspheres and add 500 μl of blocking buffer. The blocking buffer (hereinafter the same) is a solution of 50 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid, which also includes 1% bovine serum albumin (BSA) and 0.1% preservative (P-300) at a pH of 8.0. After sonication and blocking in the dark for 30 min, the microspheres are centrifuged at high speed. During the process, pay attention to avoid agglomeration and clumping.

[0104] 1.2.7 Save:

[0105] Add the labeled red microspheres to 50 μl of blocking buffer, sonicate to mix, and store for later use.

[0106] 1.3 Preparation of the buffer system:

[0107] Prepare the buffer system according to the proportions in Table 1 below:

[0108] Table 1

[0109]

[0110] Weigh the above reagents and place them in a clean container. Add purified water to make up to 1000 mL, dissolve and mix well, and measure the pH value using a pH meter to determine that it is 7.10–7.60.

[0111] 1.4 Preparation of sample diluent:

[0112] The biotin-labeled capture antibody prepared in this embodiment was diluted with the buffer system diluent at a volume ratio of 1:300; the color microsphere-labeled detection antibody prepared in this embodiment was diluted with the buffer system diluent at a volume ratio of 1:300; the biotin-labeled capture antibody diluent and the color microsphere-labeled detection antibody diluent were mixed so that the molar ratio of capture antibody to detection antibody in the mixture was 1:1.

[0113] The above mixture was dispensed into 0.2 ml bottles.

[0114] 2. Wrapping and installation of the coating carrier:

[0115] 2.1 First coating carrier coating:

[0116] Secondary antibody coating was performed on the outer surface of the first coating vector. The coating process was as follows: a 0.06 mol / L citrate-sodium citrate buffer solution with a pH of 4.8 was prepared; then, donkey anti-human secondary antibody was dissolved in the citrate-sodium citrate buffer solution and its concentration was controlled to be 10 μg / ml, which was used as the first coating solution for the first coating vector.

[0117] The coated region of the first coated carrier was immersed in the first coating solution overnight at 4°C. The next day, it was removed and then immersed in the blocking solution overnight at 4°C. The blocking solution was a 0.02 mol / L phosphate buffer, which also contained 1% BSA and 0.1% biological preservative by mass. The carrier was then removed and dried.

[0118] The first coating carrier, coated with the first coating liquid component, is installed in the syringe near the needle tip for later use.

[0119] 2.2 Second coating carrier coating:

[0120] Streptavidin was coated on the outer surface of the second coating carrier. The coating process was as follows: a 0.06 mol / L citrate-sodium citrate buffer solution with a pH of 4.8 was prepared; then streptavidin was diluted to a concentration of 10 μg / ml, and 0.02 mg of CH-((CH2)n-NHS)3 (n = 10) was added to a 1 mg streptavidin solution. The solution was stirred and incubated at room temperature for 20 min. This solution was used as the second coating solution.

[0121] The coated region of the second coated carrier was immersed in the second coating solution overnight at 4°C. The next day, it was removed and then immersed in the blocking solution overnight at 4°C. The blocking solution was a 0.02 mol / L phosphate buffer solution, which also contained 1% BSA and 0.1% biological preservative by mass. The carrier was then removed and dried.

[0122] The second coating carrier, coated with the second coating liquid component, is installed in the syringe near the piston rod for later use.

[0123] 3. Testing process:

[0124] The specific steps are as follows:

[0125] 1) Perform a puncture on the lymph node tissue in the thyroid region;

[0126] 2) Use a sample aspiration needle to extract 100 μl of thyroid fluid;

[0127] 3) Inject the aspirated thyroid fluid into a disposable diluent and mix well. Then, use the sample needle again to draw all the immune complex solution into the syringe. The liquid flows through the internal pores to the first and second coated carrier regions. As the extraction force increases, the immune complex is immobilized in the coated region by biotin-streptavidin, and the color deepens with increasing TG concentration. Compare with the colorimetric card to achieve semi-quantitative detection of thyroglobulin (TG).

[0128] 4. Evaluation of test data:

[0129] The above detection steps were repeated 5 times, and the detection results are shown in Table 2 below. The results were consistent each time, all being L5, indicating that the repeatability of the method described in this embodiment meets the requirements.

[0130] The test samples were examined using existing, accurate quantitative detection methods, and the results are shown in Table 2 below:

[0131] Table 2

[0132] Specimen testing Method of this embodiment Existing technical methods ng / mL 1 L5 240.68 2 L5 220.37 3 L5 235.37 4 L5 242.33 5 L5 244.35

[0133] The colorimetric card for red latex microspheres is shown in Table 3 below; the corresponding quantitative results standards are shown in Table 4 below.

[0134] Table 3

[0135]

[0136] Table 4

[0137] Tg (ng / mL) L1 ＜30 L2 30～80 L3 80～140 L4 140～200 L5 200～250 L6 250～300 L7 300～350 L8 350～400 L9 ＞400

[0138] As can be seen from the detection results in Tables 2 and 4 above, the concentration range of the semi-quantitative detection method of the present invention is consistent with the accurate quantitative detection results in the prior art, indicating that the detection results obtained by the semi-quantitative detection method of the present invention are accurate and reliable.

[0139] Example 2:

[0140] Same as Example 1. The only difference is:

[0141] 1) The thyroglobulin capture antibody is labeled with fluorescein; during fluorescein labeling, the molar ratio of fluorescein to capture antibody molecules is 1:5; the detection antibody is labeled with red latex microspheres as in Example 1.

[0142] 2) The first coating carrier is coated with streptavidin, and the coating steps of streptavidin are the same as in Example 1;

[0143] 3) Second coating carrier coating:

[0144] The outer surface of the second coating vector was coated with fluorescein antibody. The coating process was as follows: a 0.06 mol / L citrate-sodium citrate buffer solution with a pH of 4.8 was prepared; then the fluorescein antibody was dissolved in the citrate-sodium citrate buffer solution and its concentration was controlled to be 5 μg / ml, which was used as the second coating solution for the second coating vector.

[0145] The coated region of the second coated carrier was immersed in the second coating solution overnight at 4°C. The next day, it was removed and then immersed in the blocking solution overnight at 4°C. The blocking solution was a 0.02 mol / L phosphate buffer solution, which also contained 1% BSA and 0.1% biological preservative by mass. The carrier was then removed and dried.

[0146] The second coating carrier, coated with the second coating liquid component, is installed in the syringe near the piston rod for later use.

[0147] 4) Mix the fluorescently labeled capture antibody diluent with the colored microsphere labeled detection antibody diluent to make the molar ratio of capture antibody to detection antibody in the mixture 1:1.

[0148] Evaluation of test data:

[0149] The above detection steps were repeated 5 times, and the detection results are shown in Table 5 below. The results were consistent each time, all being L5, indicating that the repeatability of the method described in this embodiment meets the requirements.

[0150] The test samples were examined using existing, accurate quantitative detection methods, and the results are shown in Table 5 below:

[0151] Table 5

[0152]

[0153]

[0154] Similar to Example 1, the colorimetric card for the red latex microspheres is shown in Table 3; the corresponding quantitative result standards are shown in Table 4. Based on the detection results in Table 5, it can be seen that the concentration range of the semi-quantitative detection method of the present invention is consistent with the accurately quantified detection results in the prior art, indicating that the detection results obtained by the semi-quantitative detection method of the present invention are accurate and reliable.

[0155] Example 3

[0156] Detection of thyroglobulin (Tg) antibody concentration by thyroid biopsy

[0157] Same as Example 1. The only difference is:

[0158] 1) The sample diluent does not contain capture antigens, therefore the step of capturing antigen labeling is not included;

[0159] 2) The thyroglobulin detection antigen was labeled using fluorescent microspheres, and the labeling steps were the same as those for the red latex microspheres in Example 1.

[0160] 3) Preparation of sample dilution solution: The fluorescent microsphere-labeled detection antigen prepared in this embodiment is diluted with the buffer system dilution solution at a volume ratio of 1:300, and dispensed into 0.2 ml bottles.

[0161] 4) The first coating carrier is coated with streptavidin, and the coating steps of streptavidin are the same as in Example 1;

[0162] 5) The second coating vector is coated with mouse anti-human secondary antibody, and the coating steps for the secondary antibody are the same as in Example 1;

[0163] Evaluation of test data:

[0164] The above detection steps were repeated 5 times, and the detection results are shown in Table 6 below. The results were consistent each time, all being L2, indicating that the repeatability of the method described in this embodiment meets the requirements.

[0165] The test samples were examined using existing, accurate quantitative detection methods, and the results are shown in Table 6 below:

[0166] Table 6

[0167] Specimen testing Method of this embodiment Existing technical methods (IU / mL) 1 L2 180.33 2 L2 167.54 3 L2 163.49 4 L2 164.58 5 L2 166.91

[0168] The fluorescent microsphere colorimetric card is shown in Table 7 below; the corresponding quantitative result standards are shown in Table 8 below.

[0169] Table 7

[0170]

[0171] Table 8

[0172] Tg antibody (IU / mL) L1 ＜120 L2 120～200 L3 200～300 L4 300～400 L5 400～500 L6 500～600 L7 600～700 L8 700～800 L9 ＞800

[0173] As can be seen from the detection results in Tables 6 and 8 above, the concentration range of the semi-quantitative detection method of the present invention is consistent with the accurate quantitative detection results in the prior art, indicating that the detection results obtained by the semi-quantitative detection method of the present invention are accurate and reliable.

[0174] Example 4

[0175] Same as Example 1. The only difference is:

[0176] 1) The sample diluent does not contain capture antibodies, therefore the step of labeling with capture antibodies is not included;

[0177] 2) Preparation of sample dilution solution: The red latex microsphere-labeled detection antibody prepared in this embodiment is diluted with the buffer system dilution solution at a volume ratio of 1:300, and dispensed into 0.2 ml vials.

[0178] 3) The first coating vector is coated with rabbit anti-human secondary antibody, and the coating steps for the secondary antibody are the same as in Example 1;

[0179] 4) Second coating carrier coating:

[0180] The capture antibody is coated onto the second coating vector. The specific steps are as follows: Prepare a 0.06 mol / L citrate-sodium citrate buffer with a pH of 4.8; then dissolve the capture antibody in the citrate-sodium citrate buffer and control its concentration to 2 μg / ml as the second coating solution for the second coating vector.

[0181] The coated region of the second coated carrier was immersed in the second coating solution overnight at 4°C. The next day, it was removed and then immersed in the blocking solution overnight at 4°C. The blocking solution was a 0.02 mol / L phosphate buffer solution, which also contained 1% BSA and 0.1% biological preservative by mass. The carrier was then removed and dried.

[0182] The second coating carrier, coated with the second coating liquid component, is installed in the syringe near the piston rod for later use.

[0183] Evaluation of test data:

[0184] The above detection steps were repeated 5 times, and the detection results are shown in Table 9 below. The results were consistent each time, all being L5, indicating that the repeatability of the method described in this embodiment meets the requirements.

[0185] The test samples were examined using existing, accurate quantitative detection methods, and the results are shown in Table 9 below:

[0186] Table 9

[0187] Specimen testing Method of this embodiment Existing technical methods (ng / mL) 1 L5 240.68 2 L5 220.25 3 L5 235.37 4 L5 242.33 5 L5 244.35

[0188] Similar to Example 1, the colorimetric card for the red latex microspheres is shown in Table 3; the corresponding quantitative result standards are shown in Table 4. Based on the detection results in Table 9 above, it can be seen that the concentration range of the semi-quantitative detection method of the present invention is consistent with the accurately quantified detection results in the prior art, indicating that the detection results obtained by the semi-quantitative detection method of the present invention are accurate and reliable.

[0189] The technical features in the claims and / or specification of this invention can be combined, and the combination is not limited to the combinations obtained through reference in the claims. Technical solutions obtained by combining the technical features in the claims and / or specification are also within the scope of protection of this invention.

[0190] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A thyroglobulin detection kit, characterized in that, It includes: A sample diluent containing an antibody or antigen, and an immunoreaction buffer solution for diluting the antibody or antigen; The antibody or antigen is at least labeled with colored microspheres; A sample puncture test needle includes a needle tip, a syringe, a first coating carrier, and a second coating carrier. The first and second coating carriers are sequentially disposed within the syringe along its axial direction. The first and second coating carriers are in a clearance fit with the syringe. The first coating carrier is disposed at the end of the syringe near the needle tip. The outer coating material of the first coating carrier is capable of specifically binding antibodies or antigens of a human species. The second coating carrier is disposed at the end of the syringe opposite to the needle tip. The outer coating material of the second coating carrier is capable of specifically binding immune complexes. and Colorimetric card; The sample diluent contains a capture antibody and a detection antibody, wherein the capture antibody is labeled with biotin or fluorescein, and the detection antibody is labeled with the colored microspheres; When the capture antibody is labeled with biotin, the outer coating material of the second coating carrier is streptavidin; when the capture antibody is labeled with fluorescein, the outer coating material of the second coating carrier is fluorescein antibody. The immune complex is a biotin or fluorescein-capture antibody-antigen to be tested-detection antibody-colored microspheres.

2. The thyroglobulin detection kit according to claim 1, characterized in that, The antibody or antigen includes a detection antibody or antigen; the detection antibody or antigen is labeled with colored microspheres; the colored microspheres are selected from any one of fluorescent microspheres, quantum dots, colloidal gold and latex microspheres.

3. A semi-quantitative detection method for thyroglobulin, characterized in that, It includes the following steps: 1) Aspiration to extract thyroglobulin test samples; 2) Inject a specified amount of test sample directly into the sample diluent, mix well, react, and obtain a solution containing immune complexes; the sample diluent contains antibodies or antigens, and an immunoreaction buffer solution for diluting the antibodies or antigens; the antibodies or antigens are at least labeled with colored microspheres; 3) The solution containing the immune complex is sequentially passed through a first coating carrier and a second coating carrier; the outer side of the first coating carrier is coated with a component that can specifically bind to antibodies or antigens of human species; the outer side of the second coating carrier is coated with a component that can specifically bind to the immune complex. 4) Read the test results of thyroglobulin using a colorimetric card; The sample diluent contains a capture antibody and a detection antibody, wherein the capture antibody is labeled with biotin or fluorescein, and the detection antibody is labeled with the colored microspheres; When the capture antibody is labeled with biotin, the outer coating material of the second coating carrier is streptavidin; when the capture antibody is labeled with fluorescein, the outer coating material of the second coating carrier is fluorescein antibody. The immune complex is a biotin or fluorescein-capture antibody-antigen to be tested-detection antibody-colored microspheres.

4. The semi-quantitative detection method for thyroglobulin according to claim 3, characterized in that, The method is implemented using the thyroglobulin detection kit according to any one of claims 1 to 2.

5. An application of a sample puncture detection needle in the semi-quantitative detection of thyroglobulin, the sample puncture detection needle comprising a needle tip, a syringe barrel, and a piston rod arranged sequentially, the needle tip being disposed at one end of the syringe barrel; the piston rod being disposed at the other end of the syringe barrel and capable of axial movement along the inner wall of the syringe barrel, characterized in that, It also includes: A first coating carrier is disposed inside the syringe near the needle tip; the outer coating material of the first coating carrier is capable of specifically binding to antibodies or antigens of a human species; the first coating carrier is spaced and fitted with the syringe. The second coating carrier is disposed inside the syringe at one end away from the needle; the outer coating material of the second coating carrier can specifically bind immune complexes; the second coating carrier is in close fit with the syringe; both the first coating carrier and the second coating carrier are disposed between the needle and the piston rod. The outer coating material of the second coating carrier is matched with the type of the sample to be tested and the sample diluent; specifically, it includes the following steps: 1) The test sample is drawn through the needle and placed in the sample diluent to react, thus obtaining an immune complex solution; 2) The immune complex solution is drawn into the syringe through the needle by the movement of the piston rod; the immune complex solution first flows through the gap between the first coated carrier and the syringe to trap the body's own antibodies or antigens, and then flows through the gap between the second coated carrier and the syringe to capture the immune complex. 3) Read the thyroglobulin test results using a colorimetric card; The sample diluent contains a capture antibody and a detection antibody, wherein the capture antibody is labeled with biotin or fluorescein and the detection antibody is labeled with colored microspheres; When the capture antibody is labeled with biotin, the outer coating material of the second coating carrier is streptavidin; when the capture antibody is labeled with fluorescein, the outer coating material of the second coating carrier is fluorescein antibody. The immune complex is a biotin or fluorescein-capture antibody-antigen to be tested-detection antibody-colored microspheres.

6. The application of the sample puncture detection needle according to claim 5 in the semi-quantitative detection of thyroglobulin, characterized in that, Both the first and second coating carriers are movably engaged with the inner wall of the syringe; the first and second coating carriers are coated at least on the sidewalls parallel to the inner wall of the syringe; a filter screen is provided at the connection between the syringe and the needle; the filter screen has a pore size of 1 μm; the sample puncture detection needle also includes a scale line; the scale line is located on the outer wall of the syringe near the needle, and is used to measure the volume of the sample.

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