An antibody against human thyroglobulin and a method for preparing the same and use thereof

Abstract

The application discloses an antibody against human thyroglobulin and a preparation method and application thereof, and relates to the field of human thyroglobulin detection. Specifically, the antibody provided by the application has HCDR1-3 as shown in SEQ ID NO:1, 2 and 3 respectively and LCDR1-3 as shown in SEQ ID NO:4, 5 and 6 respectively; or has HCDR1-3 as shown in SEQ ID NO:7, 8 and 9 respectively and LCDR1-3 as shown in SEQ ID NO:10, 11 and 12 respectively. The antibody provided by the application can specifically recognize human thyroglobulin, the positive coincidence rate, the negative coincidence rate and the total coincidence rate of a detection reagent constructed by using the antibody and Roche control reagent are all 100%; the detection consistency and the interference tolerance threshold are equivalent to those of a reference kit; and the hook effect is significantly better than that of an existing reference kit.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to antibodies against thyroglobulin, their preparation methods, and uses. Background Technology

[0002] Thyroglobulin (Tg) is the most abundant protein in thyroid follicles and is a key backbone and storage form for thyroid hormone synthesis. Tg synthesis is regulated by multiple factors, among which thyroid-stimulating hormone (TSH) is the main positive regulator, while the concentration of iodine in the thyroid gland also affects the degree of iodination and metabolism of Tg through a negative feedback mechanism.

[0003] Catalyzed by thyroid peroxidase (TPO), specific tyrosine residues on the Tg molecule are iodinated to generate monoiodotyrosine (MIT) and diiodotyrosine (DIT), which are further coupled within the Tg molecule to form the biologically active thyroid hormones T3 and T4. When needed by the body, Tg is endocytosed into thyroid follicular cells, where it is hydrolyzed by lysosomal proteases, releasing T3 and T4 into the bloodstream. Therefore, Tg is not only a platform for thyroid hormone synthesis but also an inert carrier for hormone storage; its structural integrity and iodination level directly affect the output of thyroid hormones.

[0004] Under normal physiological conditions, a small amount of thyroid hormone (Tg) can leak into the bloodstream, and serum Tg concentration is relatively stable, mainly affected by thyroid volume and TSH levels, with a normal reference range of approximately 5-40 μg / L. In various pathological states, serum Tg concentration can change significantly. For example, in goiter, thyroiditis, and thyroid cancer, serum Tg levels are often abnormally elevated due to destruction of thyroid follicular structure or increased TSH stimulation. Therefore, serum Tg testing has been widely used for the auxiliary assessment of thyroid function, especially in the postoperative management of patients with differentiated thyroid cancer (DTC), where Tg is considered one of the most sensitive biomarkers for monitoring tumor recurrence or residual tumor.

[0005] However, the clinical value of Tg detection is severely hampered by anti-thyroglobulin antibody (TgAb). In patients with autoimmune thyroid diseases (such as Hashimoto's thyroiditis and Graves' disease), the body's immune system can produce high-affinity autoantibodies targeting different epitopes of Tg. Circulating TgAb can form immune complexes with Tg, competitively binding to detectable antibodies in immunological detection methods (such as chemiluminescence and radioimmunoassay), leading to falsely low or high Tg values ​​and thus misleading clinical decisions. Therefore, accurate detection of TgAb levels is not only crucial for diagnosing autoimmune thyroid diseases but also a prerequisite for correctly interpreting serum Tg data.

[0006] Given the core role of Tg and TgAb detection in the comprehensive management of thyroid diseases, the development of anti-thyroglobulin antibody reagents with both high specificity and high sensitivity is of great significance for improving the accuracy, anti-interference ability, and reliability of Tg immune detection and quantitative detection of autoantibodies. Summary of the Invention

[0007] To improve upon this invention, monoclonal antibodies against thyroglobulin were designed and screened, and their detection advantages were fully demonstrated by comparing them with existing positive reagent kits and other highly efficient detection antibodies. Specifically, the technical solutions described in this invention are as follows.

[0008] On the one hand, this application provides a binding molecule against thyroglobulin.

[0009] In some embodiments, the binding molecule is an antibody against thyroglobulin or an antigen-binding fragment thereof.

[0010] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region.

[0011] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:14; in some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:13. In some specific embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:14; and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:13.

[0012] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:20; in some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:19; and in some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:20; and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:19. In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region as shown in any of the following groups: (1-1) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:14, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:13. (1-2) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:20, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:19.

[0013] In some specific implementations, the CDR is defined according to the numbering system of Kabat, IMGT, Chothia, AbM, or Contact.

[0014] In some specific implementations, the CDR is defined according to Kabat's numbering system.

[0015] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:4, 5, and 6, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:1, 2, and 3, respectively. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:4, 5, and 6, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in SEQ ID NO:1, 2, and 3, respectively.

[0016] In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:1, 2, and 3, respectively.

[0017] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:10, 11, and 12, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:7, 8, and 9, respectively. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:10, 11, and 12, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in SEQ ID NO:7, 8, and 9, respectively.

[0018] In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:10, 11, and 12, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:7, 8, and 9, respectively.

[0019] In some specific embodiments, the antibody or its antigen-binding fragment includes any one of the following groups: (2-1) The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, 2, and 3, respectively; (2-2) The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, 11, and 12, respectively, and the amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, 8, and 9, respectively.

[0020] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:14, and the heavy chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:13; in some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13; in some specific embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13.

[0021] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:20, and the heavy chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:19. In some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:20, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19. In some specific embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:20, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:19.

[0022] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region as shown in any of the following groups: (3-1) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:14, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:13; (3-2) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:20, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:19.

[0023] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a recombinant antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb.

[0024] In some embodiments, the antibody or its antigen-binding fragment further includes an immunoglobulin Fc region. In some specific embodiments, the Fc is selected from IgG1, IgG2, IgG3, or IgG4.

[0025] In some embodiments, the light chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:18, and the heavy chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:17. In some specific embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:18, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:17. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:17.

[0026] In some embodiments, the light chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:24, and the heavy chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:23. In some specific embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:24, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:23. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:24, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:23.

[0027] The present invention also provides a biomaterial.

[0028] In some embodiments, the biomaterial includes any one of the following: (4-1) A nucleic acid molecule, said nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any of the places above; (4-2) Expression vectors / vector sets containing any of the nucleic acid molecules described above; (4-3) Host cells containing any of the nucleic acid molecules or expression vectors / vector groups described above.

[0029] The present invention also provides a method for preparing antibodies or antigen-binding fragments thereof as described in any of the above claims.

[0030] In some embodiments, the method includes culturing host cells as described in any of the preceding embodiments. In some specific embodiments, the cultured host cells express an antibody or an antigen-binding fragment thereof. In some specific embodiments, the method further includes isolating the antibody or the antigen-binding fragment thereof. In some specific embodiments, the method further includes purifying the antibody or the antigen-binding fragment thereof.

[0031] The present invention also provides an anti-thyroglobulin composition.

[0032] In some embodiments, the composition comprises the antibody or antigen-binding fragment thereof described in any of the preceding embodiments.

[0033] The present invention also provides the use of an antibody or antigen-binding fragment thereof as described in any of the preceding claims, or a composition as described in any of the preceding claims.

[0034] In some implementations, the application includes: products for preparing anti-thyroglobulin, or for detecting thyroglobulin not for the purpose of disease diagnosis and treatment; said products are test kits, test strips, or test chips.

[0035] In some specific implementations, the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatographic kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit, or a radioimmunoassay kit; the test strip is a colloidal gold test strip or an ELISA test strip.

[0036] In some specific embodiments, the product includes the antibody as a labeling antibody as described in group (1-1) and the antibody as a coating antibody as described in group (1-2); in some specific embodiments, the product includes the antibody as a labeling antibody as described in group (2-1) and the antibody as a coating antibody as described in group (2-2); in some specific embodiments, the product includes the antibody as a labeling antibody as described in group (3-1) and the antibody as a coating antibody as described in group (3-2).

[0037] In some specific embodiments, the product includes the antibody as described in group (1-1) as a coating antibody and the antibody as described in group (1-2) as a labeled antibody; in some specific embodiments, the product includes the antibody as described in group (2-1) as a coating antibody and the antibody as described in group (2-2) as a labeled antibody; in some specific embodiments, the product includes the antibody as described in group (3-1) as a coating antibody and the antibody as described in group (3-2) as a labeled antibody.

[0038] In some implementations, the thyroglobulin described in any of the above embodiments is human thyroglobulin.

[0039] In some implementations, any of the antibodies described above are monoclonal antibodies. Attached Figure Description

[0040] Figure 1 This represents an SDS-PAGE electrophoresis image. Channel M corresponds to the protein marker; channel 1 corresponds to the 3F11 antibody.

[0041] Figure 2 This represents an SDS-PAGE electrophoresis image. Channel M corresponds to the protein marker; channel 1 corresponds to the 9C8 antibody. Detailed Implementation

[0042] The following description of specific embodiments further illustrates this application, but this is not intended to limit the scope of this disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products; the methods described, unless otherwise specified, are all conventional techniques in the art.

[0043] Example 1: Preparation of anti-human thyroglobulin (Tg) monoclonal antibody 1.1 Animal Immunization Five female Balb / c mice aged 6-8 weeks were selected and immunized with high-purity thyroglobulin (Fitzgerald 30R-AT005) as the antigen. For the first immunization, the antigen was mixed with Freund's complete adjuvant at a dose of 50 μg per mouse, emulsified to a water-in-oil state using a double-syringe method, and then injected subcutaneously at multiple sites (200 μL / mouse) along both sides of the spine, bilateral groins, and bilateral axillae. A second immunization was administered 14 days after the first, with the antigen dose and adjuvant changed to Freund's incomplete adjuvant. The injection method and dose were the same as the first immunization. A third immunization was administered 15 days after the second, with the antigen and Freund's incomplete adjuvant emulsified and injected intraperitoneally at the same dose. Seven days after the third immunization, blood was collected from the tail and serum was separated. Antibody titers were detected using ELISA (Table 1), with a titer of 1:100,000 or higher being the inclusion criterion. Select qualified mice and administer booster immunization by intraperitoneal injection of 100 μg / mouse of the same antigen dissolved in sterile phosphate buffer (without adjuvant) 3 days before cell fusion, in preparation for cell fusion.

[0044] Table 1. Antibody titer detection by ELISA

[0045] 1.2 Construction of B cells (1) Culture and preparation of myeloma cell lines This embodiment uses the SP2 / 0 myeloma cell line, which was adaptively passaged to the logarithmic growth phase before fusion. On the day of fusion, cells were gently blown off the flask wall with a bent dropper and collected in a 50 mL centrifuge tube. The tube was centrifuged at 1000 r / min for 5-10 min. After discarding the supernatant, the pellet was resuspended in 30 mL of culture medium and washed once by centrifugation using the same method. Finally, the supernatant was discarded, and the pellet was resuspended in 20 mL of culture medium for later use.

[0046] (2) Preparation of spleen cells Immunized BALB / c mice were used. Blood was collected by enucleation and serum was separated as a positive control. Mice were euthanized by neck dislocation after blood collection and immersed in 75% alcohol for 5 min. The spleen was aseptically removed in a laminar flow hood and washed in a culture dish containing 10 mL of culture medium, removing surrounding connective tissue. The spleen was punctured with a sterile syringe needle and repeatedly perfused until the spleen tissue turned white. The spleen cell suspension was collected into a 50 mL centrifuge tube and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, and the precipitate was resuspended in 10 mL of culture medium. A small amount was diluted and used for cell counting.

[0047] (3) Cell fusion The spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 10:1 and fused using polyethylene glycol (PEG) as a fusion agent. After fusion, the cells were cultured in HAT selective medium. After 10 days of culture, the hybridoma cell culture supernatant was screened using ELISA. Positive clones were cloned using the limiting dilution method. After four rounds of screening, 18 positive hybridoma cell lines that stably secreted specific antibodies were finally obtained.

[0048] (4) Screening of hybridoma cells (ELISA method) Tg antigen was coated onto 96-well ELISA plates at a concentration of 2 μg / mL and a volume of 50 μL / well, and incubated overnight at 4°C. The next day, the liquid in the wells was discarded, the plates were washed three times and blotted dry. Then, 100 μL of blocking buffer was added to each well, and the plates were blocked at 37°C for 2 h, followed by washing twice and blotting dry. Culture supernatant from 18 hybridoma cell lines was added to each well at 50 μL, with positive, negative, and blank controls included. The plates were incubated at 37°C for 30 min, washed three times, and blotted dry. Next, 50 μL of HRP-labeled goat anti-mouse IgG (diluted 1:20000) was added to each well, and the plates were incubated at 37°C for 30 min, washed three times, and blotted dry. For color development, 100 μL of TMB substrate solution was added to each well, and the plates were incubated at room temperature for 15 min, then stopped with 50 μL / well of 2 mol / L dilute hydrochloric acid. The OD values ​​of each well were read at dual wavelengths of 450 nm and 630 nm.

[0049] The experimental results showed that the OD values ​​of the blank wells were below 0.02, and the OD values ​​of the negative wells were below 0.1. Furthermore, the positive and negative controls showed good differentiation, confirming that the obtained hybridoma cells could secrete antibodies that specifically recognize the Tg antigen (Table 2). In addition, the titers of mouse ocular serum, detected using the same method, were all above 1:100,000, meeting the standards for fusion experiments.

[0050] Table 2. Results of OD value measurement

[0051] 1.3 Production and purification of monoclonal antibodies (1) Induction and collection of ascites From the 18 established positive hybridoma cell lines, two clones with the strongest antibody secretion capacity were selected for mouse ascites preparation. Five Balb / c mice weighing approximately 25 g were pretreated by intraperitoneal injection of 0.5 mL of liquid paraffin; two weeks later, the ascites was further treated with 1×10⁻⁶ liquid paraffin. 6 Hybridoma cells were injected into the peritoneal cavity of mice at a dose of 1 cell per mouse. When the mice's abdomens became noticeably distended and signs of ascites appeared, the ascites fluid was collected, centrifuged at 10,000 rpm for 10 min, cell debris and precipitate were discarded, and the supernatant was collected and stored at -20℃ for later use.

[0052] (2) Purification of antibodies in ascites fluid After thawing the frozen ascites fluid, centrifuge and filter the supernatant through a 0.22 μm filter membrane. Purify the antibody using a Protein A affinity chromatography column, following the product instructions. Collect the eluted target antibody fraction and dialyze it in 20 mM PBS (pH 7.4) for 72 h. After dialysis, sterilely filter through a 0.22 μm filter membrane, aliquot, and store at -20°C for subsequent identification experiments.

[0053] 1.4 Characterization of Monoclonal Antibodies According to conventional sequencing, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the 3F11 antibody are shown in SEQ ID NO:1-3, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:4-6, as detailed in Table 3 below.

[0054] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the 9C8 antibody are shown in SEQ ID NO:7-9, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:10-12. See Table 3 below for details.

[0055] Table 3. Distribution of antibody CDR sequences (Kabat numbers) ; Antibody concentration determination: The absorbance values ​​(A280) and (A260) of the monoclonal antibodies at 280 nm and 260 nm were determined by ultraviolet spectrophotometry. The protein content was 5 mg / mL for 3F11 antibody and 3 mg / mL for 9C8 antibody. The protein content was calculated using the following formula: Protein content (mg / mL) = (A280 × dilution factor) / 1.35.

[0056] Antibody molecular weight determination: Monoclonal antibodies were measured using SDS-PAGE, such as... Figure 1 As shown, the heavy chain of monoclonal antibody 3F11 is approximately 55 kDa, and the light chain is approximately 25 kDa; Figure 2 As shown, the heavy chain of monoclonal antibody 9C8 is approximately 55 kDa, and the light chain is approximately 25 kDa.

[0057] Antibody titer assessment: The titer of the purified ascites monoclonal antibody was determined by indirect ELISA. The results (see Table 4) showed that the titer of the purified antibody was higher than 1:100000.

[0058] Table 4. Results of indirect ELISA assay

[0059] Experimental Example 2: Application of 3F11 and 9C8 antibodies in chemiluminescent reagents for thyroglobulin 2.1 Preparation of monoclonal antibodies coated with magnetic microparticles: Magnetic microparticles with a particle size of 1.5-3 μm, along with EDC and NHS, were added to a 50 mM MES solution at pH 5.0 at a mass ratio of 5:1:2. The magnetic microparticle concentration was 10 mg / mL, the reaction temperature was 37℃, and the reaction time was 30 min. The reacted magnetic microparticles were then conjugated with 3F11 monoclonal antibody at a ratio of 10 μg antibody to 1 mg of magnetic microparticles. The conjugation process was carried out at 25℃ for 5 h. After conjugation, the magnetic microparticles were washed three times with washing buffer, and then added to a phosphate buffer solution containing 2% glycine, 1% BSA, 0.05% Tween 20, 0.05% ProClin 300, and pH 7.4 to achieve a magnetic microparticle concentration of 10 mg / mL. The solution was incubated at 25℃ for 2 h and then stored at 2-8℃ for later use.

[0060] 2.2 Preparation of alkaline phosphatase-labeled monoclonal antibodies (1) Take 1.0 mg AP and dilute it to 10 mg / mL with phosphate buffer containing 0.15 M sodium chloride, and add 0.04 mg TR to it. React at 37 °C for 1 h.

[0061] (2) Take 1 mg of 9C8 antibody, adjust the concentration to 1 mg / mL, add 0.034 mg of SMCC, and react at 37℃ for 1 h.

[0062] (3) Add the alkaline phosphatase that has completed the reaction to the 9C8 antibody after SMCC treatment, mix them evenly at a mass ratio of 1:1, and react at 25℃ for 1h. After the reaction is completed, add 0.1mL of phosphate buffer containing 0.15M sodium chloride, 2% cysteine, and pH=7.4, let stand for 1h, add 1mL of glycerol, and store at -20℃ for later use.

[0063] 2.3 Reagent Performance Testing Magnetic microparticles coated with 3F11 antibody were diluted 20-fold with phosphate buffer containing 0.15M sodium chloride, and alkaline phosphatase-labeled 9C8 antibody was diluted 1000-fold with phosphate buffer containing 0.15M sodium chloride before testing. The test samples were Roche Diagnostics' thyroglobulin assay kit (electrochemiluminescence immunoassay; catalog number: 08906556190, batch number: 92130401). The test included 90 clinical samples.

[0064] Test samples: A total of 90 clinical human serum samples were collected, including 30 low-value serum samples, 30 medium-value serum samples, and 30 high-value serum samples. The concentrations covered the measurement range of the kit. All samples showed no hemolysis, lipemia, or jaundice.

[0065] Interference test method: Sample preparation: Hemoglobin (Hb) was quantitatively added to low, medium, and high concentrations of baseline serum to prepare predetermined gradient hemolytic interference samples. A blank baseline sample without added hemoglobin was set up as a control. Total bilirubin was added to low, medium, and high concentrations of baseline serum to prepare interference samples with different degrees of jaundice. A blank baseline sample was set up as a control group to exclude interference from the matrix itself. Clinical serum confirmed to be highly RF positive was selected and mixed with interference-free baseline serum at a fixed ratio to prepare interference samples with predetermined RF concentrations. A pure baseline serum blank control was set up to ensure accurate and controllable interference concentration. HAMA-positive interference serum was mixed into low, medium, and high concentrations of baseline serum at a fixed ratio to prepare standardized HAMA interference samples. An interference-free blank control group was set up. Three parallel replicates were set up for each interference concentration gradient and each sample concentration. The mean value was used as the final test result to reduce random error. Each sample was tested in parallel according to standard operating procedures. The relative deviation of the detected value after interference was calculated based on the test result of interference-free baseline serum.

[0066] Hook effect test method: For sample preparation, pure antigen standards were used, and negative human serum without antigen interference was used as the dilution matrix. A series of ultra-high concentration antigen samples were prepared through precise gradient dilution, focusing on the critical high and ultra-high concentration ranges. Core characteristic concentrations included 120,000 ng / mL and 150,000 ng / mL. Low, medium, and high conventional concentration samples were also set up as controls. All samples were thoroughly mixed, allowed to stand for stability, and were free from matrix contamination and repeated freeze-thaw cycles. Parallel testing was performed according to standard operating procedures for all gradient high-concentration samples. Three replicates were set for each concentration. The detection luminescence value and measured concentration of the reagents of this invention and the Roche reference kit were recorded. The degree of agreement between the measured value and the theoretical concentration at each concentration point was calculated. Using the linear fitting relationship between concentration and luminescence value within the conventional effective detection concentration range as a benchmark, a hook effect was determined when the measured value of an ultra-high concentration sample was significantly lower than the theoretical concentration, the luminescence signal decreased with increasing antigen concentration, and the absolute value of the relative deviation was >10%.

[0067] 2.4 Experimental Results As can be seen from the results in Table 5, with Roche's reagent kit interpretation criteria as a reference, the interpretation results of this reagent kit show that the positive concordance rate, negative concordance rate, and total concordance rate of the reagent described in this invention and Roche's reagent are all 100%. All indicators show that the results of "test reagent" and "Roche reagent" are completely consistent with each other, with no difference. Kappa=1, which is perfect consistency.

[0068] Table 5. Chemiluminescent microparticle immunoassay results using test reagents and Roche reagents ; Clinical serum samples at low, medium, and high concentration levels were selected, and each sample was measured 10 times using both kits. The coefficient of variation (CV) was calculated. As shown in Table 6 below, the CV values ​​of the kit of this invention at each concentration level are comparable to those of the Roche kit, and even lower.

[0069] Table 6. Precision Analysis ; All 90 clinical samples were tested in parallel using the luminescent antibody pairing system of this invention and the Roche reference kit, respectively, following standard operating procedures. For the Roche kit, the testing procedure was as per its instruction manual. For the reagent of this invention, the procedure was as follows: 35 μl of sample, 50 μl of magnetic microparticle-coated 3F11 antibody working solution, and 50 μl of alkaline phosphatase-labeled 9C8 antibody working solution were mixed, incubated at 37°C for 10 min, followed by magnetic separation and washing. Then, 200 μl of substrate solution was added, and the luminescence value in the reaction vessel was measured using an instrument. The Tg detection concentration for each sample was recorded. Linear regression analysis was performed with the Roche kit result as the x-axis and the result of the system of this invention as the y-axis. The results are shown in Table 7. The linear correlation coefficient was 0.989, indicating consistent detection ranges. The clinical detection results of the two systems were highly consistent and met the requirements for clinical Tg detection.

[0070] Table 7. Correlation and Detection Range Analysis ; The effects of hemolysis, lipemia, jaundice, rheumatoid factor (RF), and heterophile antibody HAMA interference were investigated. The results are shown in Table 8 below. At the given interference concentration, the relative deviation of the detection values ​​of the system of this invention was less than 10%, and the interference tolerance threshold was comparable to that of the reference kit. Hook effect comparison: The antibody pair of this invention did not show a hook effect at a concentration of 150,000 ng / mL, which is superior to the Roche kit at 120,000 ng / mL.

[0071] Table 8. Results of Interference Test and Hook Effect Test

[0072] In summary, the chemiluminescence detection kit constructed using the Tg antibody pair provided by this invention shows highly consistent results with the detection results of the currently mainstream Roche imported kits used in clinical practice. Furthermore, the kit of this invention exhibits superior performance in terms of precision and anti-HOOK capability. The results demonstrate that the antibody pair possesses excellent clinical application performance in a chemiluminescence platform and fully meets the clinical needs for quantitative detection of thyroglobulin.

[0073] The relevant sequences of this invention are as follows: > 3F11 antibody heavy chain variable region QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGNIYPGGDYINYNEKFKGKATLTADTSSSTGYMQLSSLTSEDSAVYFCASTRLTRYFDVWGAGTTVTVSS SEQ ID NO:13 > 3F11 antibody light chain variable region DIQMTQSPSYLAASPGETISINCRASKSISKYLAWFQQKPGKTYKLLIYSGSTLQSGIPSRFSGSGSGEDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK SEQ ID NO:14 > 3F11 antibody heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:15 > 3F11 antibody light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:16 > 3F11 antibody heavy chain QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGNIYPGGDYINYNEKFKGKATLTADTSSSTGYMQLSSLTSEDSAVYFCASTRLTRYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 17 > 3F11 antibody light chain DIQMTQSPSYLAASPGETISINCRASKSISKYLAWFQQKPGKTYKLLIYSGSTLQSGIPSRFSGSGSGEDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 18 > 9C8 antibody heavy chain variable region QVQLQQPGAELVRPGASVNLSCKASGYTFTSYTMNWVRQRPDQGLEWIGRIDPYDTETHYNQKFKDKAILTVDKSSTTAYMQLSSLTSEDSAVYYCARSGNAWFGYWGQGTLVTVSA SEQ ID NO: 19 > 9C8 antibody light chain variable region DIVMTQSPTTMAASPGEKITITCSASSSISSNYLYWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYFCQQGSTLPRTFGGGTKLEIK SEQ ID NO: 20 > 9C8 antibody heavy chain constant region AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 21 > 9C8 antibody light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 22 > 9C8 antibody heavy chain QVQLQQPGAELVRPGASVNLSCKASGYTFTSYTMNWVRQRPDQGLEWIGRIDPYDTETHYNQKFKDKAILTVDKSSSTTAYMQLSSLTSEDSAVYYCARSGNAWFGYWGQGT LVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERT ISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO:23 9C8 Antibody Light Chain DIVMTQSPTTMAASPGEKITITCSASSSISSNYLYWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYFCQQGSTLPRTFGGGTKLEI KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:24 The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. An antibody against thyroglobulin or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment includes any one of the following groups of light chain variable regions and heavy chain variable regions: (1-1) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:14, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:

13. (1-2) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:20, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:

19. The CDR is defined according to the Kabat numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes: (2-1) The amino acid sequences are as shown in SEQ ID NO:4, 5, and 6, respectively, for LCDR1, LCDR2, and LCDR3, and the amino acid sequences are as shown in SEQ ID NO:1, 2, and 3, respectively, for HCDR1, HCDR2, and HCDR3; or (2-2) The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, 11, and 12, respectively, and the amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, 8, and 9, respectively.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment includes any one of the following groups of light chain variable regions and heavy chain variable regions: (3-1) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:14, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:13; (3-2) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:20, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:

19.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv, or dsFv.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, It also includes the immunoglobulin Fc region, wherein the Fc is selected from IgG1, IgG2, IgG3 or IgG4.

6. A biomaterial, characterized in that, The biomaterial includes any one of the following: (1) A nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5; (2) An expression vector / vector group containing the nucleic acid molecules described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the expression vector / vector group described in (2).

7. A method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1-5, characterized in that, The method includes culturing the host cells as described in claim 6 to express an antibody or an antigen-binding fragment thereof; and includes isolating or purifying the antibody or the antigen-binding fragment thereof.

8. A composition for inhibiting thyroglobulin, characterized in that, Includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-5.

9. The use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-5, or the composition as described in claim 8, characterized in that, Test kits, test strips, or test chips for the preparation of anti-thyroglobulin.

10. The application according to claim 9, characterized in that, The detection kit is an ELISA detection kit, immunochromatographic kit, immunoturbidimetric detection kit, magnetic particle detection kit, chemiluminescence detection kit, immunofluorescence detection kit, or radioimmunoassay kit; the test strip is a colloidal gold test strip or an ELISA test strip.

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