Colloidal gold immune test paper for detecting thyroglobulin and preparation method of colloidal gold immune test paper

Abstract

The invention provides colloidal gold immune test paper for detecting thyroglobulin and a preparation method of the colloidal gold immune test paper. The colloidal gold immune test paper for detecting thyroglobulin comprises a sample pad, a gold-labeled antibody binding pad, a nitrocellulose membrane and absorbent paper which are sequentially and partially spliced and fixed on a PVC (Polyvinyl Chloride) bottom plate, wherein the gold-labeled antibody combination pad is coated with a mouse anti-human thyroglobulin monoclonal antibody labeled by colloidal gold; a detection line and a quality control line which are separated from each other are arranged on the nitrocellulose membrane, the detection line is located at the 1 / 3 position of the nitrocellulose membrane, and the quality control line is located at the 2 / 3 position of the nitrocellulose membrane; the detection line is coated with an anti-human thyroglobulin specific antibody; and the quality control line is coated with an anti-mouse IgG antibody. The test paper does not need specific equipment and professional technicians, the detection time is short, the result can be interpreted in only about ten minutes, the manpower, material resources and time cost are greatly saved, and the test paper has very high practical value and economic benefit.

Description

Technical Field

[0001] The present invention relates to the technical field of human thyroglobulin immunoassay, in particular to a colloidal gold immunoassay paper for detecting thyroglobulin and a preparation method thereof. Background Art

[0002] Thyroglobulin (Tg) plays a crucial role in the diagnosis and treatment of low-scoring thyroid-related diseases and is a common clinical test. In patients with congenital hypothyroidism, Tg testing can differentiate between complete thyroid deficiency and thyroid dysplasia. Thyroglobulin is a tumor marker for differentiated thyroid cancer (DTC) and serves as an important reference for post-treatment follow-up of patients with DTC. It can also be used to differentiate between subacute thyroiditis and pseudothyrotoxicosis. Tg levels are often significantly elevated in patients with subacute thyroiditis, while those with pseudothyrotoxicosis show no significant changes.

[0003] At present, the detection technology for human thyroglobulin requires professional laboratories and professional technicians to perform the detection. The common methods are radioimmunoassay (RIA) and immunoradiometric analysis (IRMA). Both methods require expensive detection equipment and high manpower and material costs. Labeling requires radionuclide substances, which are divided into two categories: γ-ray and β-ray, and are labeled by direct labeling or indirect labeling. The instruments and reagents have high requirements for operators, and they need to be maintained, calibrated and have indoor quality control before they can be tested. The specimens sent for clinical examination need to be sent to a specific laboratory by a transporter before they can be tested. There are many types of specimens in specific laboratories, and the waiting time for reports is also long, generally taking three hours or more. It can be seen that the detection of thyroglobulin in the existing technology has the following problems: 1. All existing detection methods are highly dependent on laboratory locations and testing personnel, requiring professional technicians to operate in specific laboratories; 2. It requires expensive instruments and reagents for testing and cannot be carried out in smaller hospitals and community service centers.

[0004] 3. The reporting cycle is long. The reporting time of different testing institutions ranges from 3 hours to 24 hours. Therefore, a product suitable for on-site rapid detection of human thyroglobulin is needed. Summary of the Invention

[0005] In order to solve the above problems while meeting clinical needs, the present invention proposes a colloidal gold immunoassay test paper for detecting thyroglobulin, which applies colloidal gold immunochromatography technology to the field of human thyroglobulin detection, realizing the rapid detection of human thyroglobulin. The colloidal gold immunochromatography detection method is simple to operate, rapid (results can be obtained in 10 minutes), and does not require professional equipment. It is suitable for medical institutions at all levels.

[0006] The colloidal gold immunoassay paper for detecting thyroglobulin of the present invention is very easy to use and can be used by anyone according to the instructions, thereby greatly reducing the time for the examinee to go back and forth to the hospital for testing and waiting for the test report.

[0007] Specifically, the present invention provides a method for rapid detection of human thyroglobulin by utilizing the specific binding characteristics between antibodies and antigens and combining them with colloidal gold immunochromatography technology. This method breaks the limitation that human thyroglobulin detection must be limited to specific laboratories and performed by specific personnel, and can achieve rapid detection in the ward without the need to purchase expensive equipment.

[0008] The present invention discloses a colloidal gold immune test paper for detecting thyroglobulin, comprising a sample pad, a gold-labeled antibody binding pad, a nitrocellulose membrane, and absorbent paper, which are partially overlapped on a PVC base plate in sequence. The gold-labeled antibody binding pad is coated with a mouse-derived gold-labeled antibody. The nitrocellulose membrane is provided with a separate detection line and a quality control line, which are located at 1 / 3 and 2 / 3 of the nitrocellulose membrane, respectively. The detection line is coated with an anti-human thyroglobulin-specific antibody, and the quality control line is coated with an anti-mouse IgG antibody. The mouse gold-labeled antibody is a colloidal gold-labeled human thyroglobulin mouse-specific monoclonal antibody.

[0009] In the present invention, the detection line is located at the 1 / 3 position on the nitrocellulose membrane, which makes it easy for the antibody complex formed after the human thyroglobulin contained in the sample binds to the gold-labeled antibody to bind to the anti-human thyroglobulin specific antibody in the detection line area in time to develop color, thereby improving the detection accuracy and shortening the detection time.

[0010] The basic principle of this invention is that after the serum or plasma to be tested is added to the detection well, antibodies bound to tracer gold nanoparticles (pre-coated in a specific area) bind to the thyroglobulin in the sample, forming an antigen-antibody complex. This complex migrates through the detection zone within the detection system via capillary action and then binds to the solid-phase anti-thyroglobulin antibody. When the concentration exceeds a specific concentration, the complex appears as a light red band, the color of which is proportional to the thyroglobulin concentration. This method allows for individual packaging of each test, making it convenient to use and suitable for semi-quantitative testing and rapid testing in hospitals with limited facilities for diagnosis and treatment. It also does not require specialized, expensive testing equipment; interpretation is based solely on the color of the band, greatly facilitating patient and clinician convenience and worthy of nationwide promotion and adoption.

[0011] The specific technical approach of the present invention is as follows: The sample pad is prepared by soaking a glass fiber membrane in a treatment solution comprising 0.01M PBS, 0.1wt% Tween-20, 1wt% bovine serum albumin, and 10wt% sucrose, followed by drying at room temperature and relative humidity of 15% to 30%. A mouse-specific monoclonal antibody to human thyroglobulin is selected and added to a colloidal gold solution (comprising 0.1M Tris-HCl buffer and 20wt% sucrose), stirred evenly, and allowed to stand to modify the antibody surface. After standing for 0.5-24 hours, 1wt% bovine serum albumin is added dropwise, stirred for 0.5-24 hours, and allowed to stand overnight before centrifugation to obtain the colloidal gold-labeled mouse-specific monoclonal antibody to human thyroglobulin. To improve the stability and sensitivity of the colloidal gold antibody, a crosslinker (polyethyleneimine or glutaraldehyde) is added to strengthen the connection between the antibody and the gold nanoparticles. During the preparation of colloidal gold-labeled antibodies, the amount of crosslinker (such as polyethyleneimine or glutaraldehyde) added must be strictly controlled to balance antibody stability and sensitivity. Glutaraldehyde's mechanism of action: Glutaraldehyde forms covalent crosslinks with amino groups (-NH2) on the surface of antibodies and gold nanoparticles through its aldehyde groups, enhancing binding strength. Dosage: Final concentration 0.01%–0.1% (volume ratio), meaning 1–10 μL of 1% glutaraldehyde solution should be added per 1 mL of colloidal gold-antibody mixture. Polyethyleneimine (PEI)'s mechanism of action: PEI electrostatically attracts negatively charged colloidal gold through its positive charge, while its polyamino groups also covalently couple to antibodies. Dosage: Final concentration 0.005%–0.02% (w / v), meaning 0.5–2 μg of PEI (molecular weight 10–25 kDa) should be added per 1 mL of the mixture.

[0012] In the present invention, colloidal gold-labeled antibodies are sprayed onto a gold-labeled antibody conjugate pad and dried at 37°C under ventilation for 5-24 hours to obtain the gold-sprayed conjugate pad, designated as Area A. At the front of Area A is a sample pad for adding the sample, designated as Area S. A nitrocellulose membrane connects the detection area (designated Area T) and the quality control area (designated Area C). Area T is coated with an anti-human thyroglobulin antibody specific for human thyroglobulin, while Area C is coated with a solid-phase anti-mouse IgG antibody.

[0013] During assembly, a polyvinyl chloride (PVC) base, sample pad, gold-labeled antibody conjugate pad, coated nitrocellulose membrane, and absorbent paper are assembled and cut into strips to create a colloidal gold immunochromatographic rapid test strip for detecting human thyroglobulin. When testing, a quantitative sample (20 μl) is added to area S. The sample then permeates and migrates to area A, dissolving the mouse-derived gold-labeled antibody in area A. If the sample contains human thyroglobulin, it binds to the gold-labeled antibody, forming an antigen-antibody complex. This complex, along with the unbound gold-labeled antibody, continues to permeate forward. In area T, the antigen-antibody complex binds to an anti-human thyroglobulin-specific antibody coated there, forming a red reaction line. Unbound gold-labeled antibody continues to migrate to area C, which is coated with anti-mouse IgG. Because the monoclonal antibody in the gold-labeled antibody is mouse IgG, the anti-mouse IgG antibody binds to the unbound gold-labeled antibody, forming a quality control line. The quality control function of area C ensures the performance of the detection system.

[0014] The preparation method of colloidal gold immunoassay paper for detecting thyroglobulin comprises the following steps: (1) Preparation of colloidal gold-labeled antibodies Preparation of colloidal gold solution: 20-40 nm gold nanoparticles (wine red) were generated by sodium citrate reduction using 0.01% HAuCl4 solution.

[0015] Adjust pH to 8.0-8.5 (0.1 M K2CO3 or NaOH).

[0016] Antibody labeling: Add mouse anti-human thyroglobulin monoclonal antibody to colloidal gold solution (containing 0.1 M Tris-HCl, 20% sucrose), stir and let stand for 0.5-24 hours.

[0017] Add 1% BSA to block unbound sites and continue stirring for 0.5 to 24 hours.

[0018] Cross-linker optimization: Glutaraldehyde: final concentration 0.01%–0.1% (1–10 μL 1% glutaraldehyde / mL mixture).

[0019] Polyethyleneimine (PEI): final concentration 0.005%~0.02% (0.5~2μg / mL).

[0020] Purification: Free antibodies were removed by centrifugation and the pellet was resuspended in storage buffer (containing 1% BSA, 0.05% NaN3).

[0021] (2) Sample pad processing The glass fiber membrane was soaked in treatment solution (0.01 M PBS, 0.1% Tween-20, 1% BSA, 10% sucrose).

[0022] Dry at room temperature (15%~30% humidity) and set aside.

[0023] (3) Preparation of gold-labeled antibody binding pad Spray the colloidal gold-labeled antibody onto the glass fiber mat (area A) and dry it at 37°C with exhaust fan for 5 to 24 hours.

[0024] (4) NC membrane coating Test line (T zone): spray anti-human thyroglobulin specific antibody.

[0025] Quality control line (area C): sprayed with anti-mouse IgG antibody.

[0026] Dry at 37℃ and set aside.

[0027] (5) Test strip assembly Paste the following on the PVC base plate: Sample pad (S zone) → Gold-labeled antibody binding pad (A zone) → NC membrane (T / C zone) → Absorbent paper.

[0028] The components overlap by 1-2 mm to ensure chromatographic continuity.

[0029] Cut into 3~5 mm wide test strips and store in a sealed and moisture-proof place.

[0030] The beneficial effects of the above technical solution of the present invention are as follows: To meet the needs of clinical diagnosis and treatment, this paper proposes and designs a colloidal gold immunochromatographic test for human thyroglobulin. This method combines the specific binding of antigen and antibody with gold-labeled chromatography technology. The negatively charged colloidal gold and positively charged protein molecules are firmly bound by electrostatic interactions, acting as a tracer marker for interpretation. This test strip does not require specialized equipment or specialized technicians, and the test time is fast, with results available in approximately ten minutes. This method significantly saves manpower, material, and time costs, and offers high practical value and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1It is a structural diagram of the test paper of the present invention; Figure 2 It is a diagram illustrating the components of the test paper of the present invention. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to specific embodiments.

[0033] Example 1 like Figure 1 and Figure 2 As shown, a colloidal gold immunoassay paper for detecting thyroglobulin in this embodiment includes a sample pad 11, a gold-labeled antibody binding pad 12, a nitrocellulose membrane 13, and an absorbent paper 14 partially overlapped on a PVC base plate 1 in sequence. The gold-labeled antibody binding pad 12 is coated with a mouse-derived gold-labeled antibody. The nitrocellulose membrane 13 is provided with a separate detection line 15 and a quality control line 16. The detection line 15 and the quality control line 16 are respectively located at the 1 / 3 and 2 / 3 positions on the nitrocellulose membrane 13. The test line 15 is coated with anti-human thyroglobulin specific antibody, and the control line 16 is coated with anti-mouse IgG antibody; The mouse gold-labeled antibody is a colloidal gold-labeled human thyroglobulin mouse-specific monoclonal antibody.

[0034] The basic principle is that after the serum or plasma to be tested is added to the detection well of the sample pad 11, antibodies bound to the tracer element gold nanoparticles (pre-coated in a specific area) will bind to the thyroglobulin in the sample, forming an antigen-antibody complex. This complex migrates through the area containing the detection band in the detection system via capillary action and then binds to the solid-phase anti-thyroglobulin antibody. When the concentration is greater than a specific concentration, the complex will appear as a light red stripe, and the color depth of the stripe is proportional to the concentration of thyroglobulin. This method can be packaged individually for each test, making it convenient to use and suitable for semi-quantitative testing and rapid testing in hospitals with poor conditions during diagnosis and treatment. It does not require specific and expensive testing equipment and can be interpreted simply by judging the color depth of the stripe, greatly facilitating the diagnosis of patients and clinicians, and is worthy of nationwide promotion and use.

[0035] Example 2 A method for preparing colloidal gold immunoassay strips for detecting thyroglobulin is described. The sample pad is prepared by soaking a glass fiber membrane in a treatment solution containing 0.01M PBS, 0.1wt% Tween-20, 1wt% bovine serum albumin, and 10wt% sucrose, followed by drying at room temperature and relative humidity of 15% to 30%. A mouse-specific monoclonal antibody to human thyroglobulin is added to a colloidal gold solution (containing 0.1M Tris-HCl buffer and 20wt% sucrose), stirred evenly, and allowed to stand for surface modification. After standing for 0.5-24 hours, 1wt% bovine serum albumin is added dropwise, stirred for 0.5-24 hours, and allowed to stand overnight before centrifugation to obtain the colloidal gold-labeled mouse-specific monoclonal antibody to human thyroglobulin. To improve the stability and sensitivity of the colloidal gold antibody, a crosslinker (polyethyleneimine or glutaraldehyde) is added to strengthen the connection between the antibody and gold nanoparticles. During the preparation of colloidal gold-labeled antibodies, the amount of crosslinker (such as polyethyleneimine or glutaraldehyde) added must be strictly controlled to balance antibody stability and sensitivity. Glutaraldehyde's mechanism of action: Glutaraldehyde forms covalent crosslinks with amino groups (-NH2) on the surface of antibodies and gold nanoparticles through its aldehyde groups, enhancing binding strength. Dosage: Final concentration 0.01%–0.1% (volume ratio), meaning 1–10 μL of 1% glutaraldehyde solution should be added per 1 mL of colloidal gold-antibody mixture. Polyethyleneimine (PEI)'s mechanism of action: PEI electrostatically attracts negatively charged colloidal gold through its positive charge, while its polyamino groups also covalently couple to antibodies. Dosage: Final concentration 0.005%–0.02% (w / v), meaning 0.5–2 μg of PEI (molecular weight 10–25 kDa) should be added per 1 mL of the mixture.

[0036] Colloidal gold-labeled antibodies are sprayed onto the gold-labeled antibody conjugate pad and dried at 37°C under ventilation for 5-24 hours to obtain the gold-sprayed conjugate pad, designated as Area A. At the front of Area A is the sample pad for adding the sample, designated Area S. A nitrocellulose membrane connects the detection area (designated Area T) and the quality control area (designated Area C). Area T is coated with an anti-human thyroglobulin antibody specific for human thyroglobulin, while Area C is coated with a solid-phase anti-mouse IgG antibody.

[0037] During assembly, a polyvinyl chloride (PVC) base, sample pad, gold-labeled antibody conjugate pad, coated nitrocellulose membrane, and absorbent paper are assembled and cut into strips to create a colloidal gold immunochromatographic rapid test strip for detecting human thyroglobulin. When testing, a quantitative sample (20 μl) is added to area S. The sample then permeates and migrates to area A, dissolving the mouse-derived gold-labeled antibody in area A. If the sample contains human thyroglobulin, it binds to the gold-labeled antibody, forming an antigen-antibody complex. This complex, along with the unbound gold-labeled antibody, continues to permeate forward. In area T, the antigen-antibody complex binds to an anti-human thyroglobulin-specific antibody coated there, forming a red reaction line. Unbound gold-labeled antibody continues to migrate to area C, which is coated with anti-mouse IgG. Because the monoclonal antibody in the gold-labeled antibody is mouse IgG, the anti-mouse IgG antibody binds to the unbound gold-labeled antibody, forming a quality control line. The quality control function of area C ensures the performance of the detection system.

[0038] 1. This technical method does not require a professional laboratory and professional technicians. It only requires collecting the patient's blood, separating it into plasma or serum, and dropping it into the detection hole.

[0039] 2. No expensive instruments or equipment are required, and the reagents can be packaged individually. This allows the test to be carried out in small hospitals or hospitals with small sample volumes, alleviating the financial pressure of running the project.

[0040] 3. The detection time is only about 10 minutes, which greatly shortens the waiting time for the report and buys time for timely treatment.

[0041] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A colloidal gold immunoassay for detecting thyroglobulin, characterized in that: It includes a sample pad, a gold-labeled antibody binding pad, a nitrocellulose membrane and absorbent paper which are partially overlapped and fixed on a PVC base plate in sequence; wherein, The gold-labeled antibody binding pad is coated with colloidal gold-labeled mouse anti-human thyroglobulin monoclonal antibody; The nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line is located at 1 / 3 of the nitrocellulose membrane and the quality control line is located at 2 / 3; The test line is coated with anti-human thyroglobulin specific antibody; The quality control line is coated with anti-mouse IgG antibody.

2. The colloidal gold immune test paper according to claim 1, wherein The preparation method of the colloidal gold-labeled mouse anti-human thyroglobulin monoclonal antibody comprises the following steps: The mouse anti-human thyroglobulin monoclonal antibody was mixed with a colloidal gold solution containing 0.1 MT Tris-HCl buffer and 20 wt% sucrose; After standing for 0.5 to 24 hours, 1 wt% bovine serum albumin was gradually added dropwise, stirred for 0.5 to 24 hours, and centrifuged to purify the gold-labeled antibody; Adding a cross-linking agent, wherein the cross-linking agent is glutaraldehyde or polyethyleneimine; the final concentration of glutaraldehyde is 0.01% to 0.1%; The final concentration of polyethyleneimine is 0.005%~0.02%.

3. The colloidal gold immune test paper according to claim 1, wherein The sample pad is prepared by soaking a glass fiber membrane in a treatment solution and then drying the solution. The treatment solution contains 0.01 M PBS buffer, 0.1 wt% Tween-20, 1 wt% bovine serum albumin, and 10 wt% sucrose.

4. The colloidal gold immune test paper according to claim 1, wherein The preparation method of the gold-labeled antibody binding pad is as follows: spraying colloidal gold-labeled mouse anti-human thyroglobulin monoclonal antibody on a glass fiber membrane, and drying the membrane under ventilation at 37° C. for 5 to 24 hours.

5. The colloidal gold immune test paper according to claim 1, wherein The detection method of the test paper comprises the following steps: Add 20 μL of serum or plasma sample to the sample pad; The sample moves along the chromatography direction to the gold-labeled antibody binding pad, dissolving the gold-labeled antibody and forming an antigen-antibody complex; The complex continues to chromatograph until it binds to the coated antibody at the detection line, forming a red band; Unbound gold-labeled antibody binds to anti-mouse IgG antibody at the quality control line to form a quality control band; The thyroglobulin concentration was quantitatively determined by the color intensity of the test line.

6. The colloidal gold immune test paper according to claim 5, characterized in that The detection time is 5 to 15 minutes.

7. The method for preparing the colloidal gold immune test paper according to any one of claims 1 to 6, wherein The steps include: (1) Preparation of colloidal gold-labeled antibodies Preparation of colloidal gold solution: 20-40 nm gold nanoparticles were generated by sodium citrate reduction using 0.01% HAuCl4 solution; Adjust the pH to 8.0-8.5 with 0.1 M K2CO3 or NaOH; Antibody labeling: Add mouse anti-human thyroglobulin monoclonal antibody to the colloidal gold solution, stir and let stand for 0.5-24 hours, add 1% BSA dropwise to block unbound sites, and continue stirring for 0.5-24 hours; Purification: remove free antibodies by centrifugation and resuspend the pellet in storage buffer; (2) Sample pad processing The glass fiber membrane was immersed in a treatment solution containing 0.01 M PBS, 0.1% Tween-20, 1% BSA, and 10% sucrose; dried at room temperature and then used. (3) Preparation of gold-labeled antibody binding pad Spray colloidal gold-labeled antibody onto a glass fiber mat and dry it at 37°C with ventilation for 5 to 24 hours; (4) NC membrane coating Test line: spray point anti-human thyroglobulin specific antibody; Quality control line: spray anti-mouse IgG antibody; Store at 37℃ for drying before use; (5) Test strip assembly Paste the sample pad, gold-labeled antibody binding pad, NC membrane, and absorbent paper on the PVC base plate in sequence; The components overlap by 1-2 mm to ensure chromatographic continuity.

8. The method for preparing colloidal gold immune test paper according to claim 7, wherein Antibody labeling also includes crosslinker optimization steps: Add glutaraldehyde: final concentration 0.01%~0.1%; Polyethyleneimine: final concentration 0.005%~0.02%.

9. Use of the colloidal gold immune test paper according to any one of claims 1 to 6 in preparing a rapid screening kit for abnormal thyroid function, postoperative monitoring of thyroid cancer or congenital thyroid disease.

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