Anti-T3 antibody mAb32 or antigen binding fragment, preparation method and application

Abstract

The invention belongs to the technical field of immunodetection, and discloses an anti-T3 antibody mAb32 or an antigen binding fragment, the anti-T3 antibody mAb32 or the antigen binding fragment comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the LCDR2 is IAS, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO.3; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown as SEQ ID NO. 4-6. When the anti-T3 antibody mAb32 is used for detecting a T3 standard antigen, the detection sensitivity is lower than 0.2 ng / ml, and when a clinical sample is detected through a magnetochemiluminescence method, the correlation with R clinical comparison is good within the sample range of 1-80 pmol / L.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and specifically to anti-T3 antibody mAb32 or antigen-binding fragments thereof and applications thereof. Background Art

[0002] T3, triiodothyronine, is an important component of thyroid hormone and an indicator of thyroid hormone. In the human body, about 99.7% of T3 specifically binds to thyroid hormone binding protein (TBG) and exists in a bound state; while the truly metabolically active free T3 (FT3) accounts for only about 0.3%. T3 can accelerate the cell metabolism of carbohydrates, fats, proteins and other substances, thereby accelerating the body's metabolism. It works together with thyroxine (T4) to regulate the body's energy consumption and material conversion. The production of T3 is activated by thyroid-stimulating hormone (TSH). When T3 is released into the blood, its increased concentration will inhibit the production of TSH in the anterior pituitary, forming a negative feedback regulation mechanism to stabilize the level of thyroid hormone in the blood. At the same time, T3 is a specific indicator for diagnosing hyperthyroidism and is also of great reference value for estimating whether hyperthyroidism recurs. Increased levels are seen in hyperthyroidism, early stage of triiodothyronine-type hyperthyroid crisis, iodine-deficiency goiter, hyperthyroidism, chronic active hepatitis, primary biliary cirrhosis, etc., while decreased levels are seen in hypothyroidism, thyroid adenoma, chronic thyroiditis, etc.

[0003] At present, mass spectrometry is commonly used for T3 detection, but mass spectrometry detection requires complex and time-consuming sample pre-treatment processes and high clinical automation requirements, making it difficult to apply on a large scale. Therefore, immunoassays are usually used in clinical testing. Immunoassay technology is simple to operate and fast to detect, and is a practical alternative to small molecule mass spectrometry detection. Since small molecules lack antigenic epitopes, competitive methods are currently commonly used in clinical immunoassays. Competitive methods have low sensitivity, are prone to false positives, and have poor specificity.

[0004] Therefore, there is an urgent need to develop highly specific and sensitive monoclonal antibodies for the T3 sandwich method to realize the double antibody sandwich method for detecting T3. Summary of the invention

[0005] In order to overcome the above technical problems, the present application provides an anti-T3 antibody mAb32 or antigen-binding fragment with good specificity and high affinity, which can successfully realize the rapid detection of T3 in an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and is of great significance in the field of diagnosis and treatment of thyroid diseases.

[0006] On the one hand, the present application provides an anti-T3 antibody mAb32 or an antigen-binding fragment, comprising a light chain variable region and a heavy chain variable region. The light chain variable region comprises complementarity-determining regions LCDR1, LCDR2 and LCDR3. The amino acid sequence of LCDR1 is ESIFGY (SEQ ID NO.1), the amino acid sequence of LCDR2 is IAS (SEQ ID NO.2), and the amino acid sequence of LCDR3 is QQGYISSNVDNA (SEQ ID NO.3).

[0007] In certain embodiments, the heavy variable region comprises complementarity-determining regions HCDR1, HCDR2 and HCDR3. The amino acid sequence of HCDR1 is GFDLSTYA (SEQ ID NO.4), the amino acid sequence of HCDR2 is ISNSGTT (SEQ ID NO.5), and the amino acid sequence of HCDR3 is shown as ARGHSTSSGSYFAAYGMDP (SEQ ID NO.6).

[0008] In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO.7. Specifically, the full length of VL is 110 amino acids, and the number of amino acids in the 4 domains of its FR are 26, 17, 36 and 10 respectively, and the number of amino acids in the 3 domains of LCDR are 6, 3 and 12 respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-100aa respectively, and their amino acid sequences are ESIFGY (SEQ ID NO.1), IAS (SEQ ID NO.2), QQGYISSNVDNA (SEQ ID NO.3) respectively.

[0009] In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO.8. Specifically, the full length of VH is 125 amino acids, and the number of amino acids in the 4 domains of its FR are 25, 17, 38 and 11 respectively, and the number of amino acids in the 3 domains of HCDR are 8, 7 and 19 respectively. HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-114aa respectively, and their amino acid sequences are GFDLSTYA (SEQ ID NO.4), ISNSGTT (SEQ ID NO.5), ARGHSTSSGSYFAAYGMDP (SEQ ID NO.6) respectively.

[0010] In some embodiments, the antigen-binding fragment is one of F(ab’)2, Fab’, Fab, Fv, scFv, dsFv, bispecific antibody, and the minimal antibody recognition unit; preferably, the species from which the remaining sequences of the antibody are derived include one or more of rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

[0011] On the other hand, the present application also provides a biological material, which includes a polynucleotide, a vector, or a cell. The polynucleotide encodes the anti-T3 antibody mAb32 or the antigen-binding fragment; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector, or is capable of expressing the anti-T3 antibody mAb32 or the antigen-binding fragment.

[0012] On the other hand, the present application also provides a method for preparing the anti-T3 antibody mAb32 or the antigen-binding fragment, including culturing the cell; optionally, the cell is prepared by transforming a polynucleotide encoding the anti-T3 antibody mAb32 or the antigen-binding fragment into the cell. The polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0013] In some embodiments, the cell is a eukaryotic cell, preferably a mammalian cell, and more preferably a 293 cell or a CHO cell.

[0014] On the other hand, the present application also provides the use of the anti-T3 antibody mAb32 or the antigen-binding fragment or the biological material in any of the following: 1) Detecting T3 for non-diagnostic and non-therapeutic purposes; 2) Preparing a product for detecting T3; 3) Purifying T3; 4) Preparing a product for purifying T3.

[0015] On the other hand, the present application also provides a detection reagent or a detection kit, which contains the anti-T3 antibody mAb32 or the antigen-binding fragment, or the biological material.

[0016] In some embodiments, the detection kit is an immunodetection kit prepared by a double antibody sandwich ELISA or chemiluminescence method.

[0017] Compared with the prior art, the anti-T3 antibody mAb32 or the antigen-binding fragment of the present application can bind to T3 with high specificity and has high affinity. Its association constant Ka can reach 7.5×10 8L / mol. The anti-T3 monoclonal antibody mAb32 of the present application can also be prepared into various immunoassay kits for detecting T3. In particular, it is applied to immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence method. The double-antibody sandwich chemiluminescence platform detects the T3 standard antigen, and the detection sensitivity is lower than 0.2 ng / ml. The magnetic chemiluminescence method detects clinical samples, and within the range of 1-80 pmol / L samples, it has a good correlation with the clinical comparison R. Brief Description of the Drawings

[0018] Figure 1 It is the electrophoresis pattern of the full-length amplification products of the mAb32 heavy chain and light chain. M is the DNA molecular weight Marker.

[0019] Figure 2 It is the mAb32 sandwich ELISA method for detecting cross proteins and target antigen samples, and the ordinate is the detected OD value.

[0020] Figure 3 It is the mAb32 magnetic particle chemiluminescence method for detecting the T3 standard antigen. The abscissa is the concentration of the T3 standard antigen (ng / ml), and the ordinate is the detected luminescence value. The R of the standard curve 2 = 0.9911, the linear detection range is 0.2-10 ng / mL, and the sample concentration calculation formula is deduced: y = 340165x - 125265.

[0021] Figure 4 It is the mAb32 magnetic particle chemiluminescence method for detecting clinical samples. The abscissa is the free T3 concentration (pmol / L) of the clinical sample, and the ordinate is the detected luminescence value. The R of the standard curve 2 = 0.931, the linear detection range is 1-80 pmol / L, and the sample concentration calculation formula is deduced: y = 34523x - 134239. Detailed Embodiments

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to the conventional conditions, the conditions described in the laboratory manual, or the conditions recommended by the manufacturer.

[0023] Example 1 Preparation of Rabbit Monoclonal Antibody for T3 Sandwich Method 1) Preparation of Immunogen Prepare the T3 complex, and the purity of the immunogen is above 90%, meeting the purity requirements for preparing monoclonal antibodies.

[0024] 2) Animal Immunization The prepared T3 complex was emulsified with complete Freund's adjuvant at a volume ratio of 1:1 and used to immunize New Zealand white rabbits weighing about 2 kg by subcutaneous injection. The immunization dose was 800 μg per rabbit. The second immunization was carried out two weeks later, emulsified with incomplete Freund's adjuvant at a volume ratio of 1:1, and the immunization dose was 400 μg per rabbit. After two immunizations, tail blood was taken and the serum titer was measured by gradient dilution using the ELISA method; based on the standard that the OD450 at an ELISA titer of 128000 was greater than 1.0, it was determined whether to collect PBMCs or continue immunization according to the results, and the rabbit with the highest antibody titer was selected for PBMCs collection.

[0025] 3) Isolation of PBMCs, sorting of specific B cells, cloning and recombination The rabbit was fixed supine on the operating table, the hair at the heart area was removed, the skin was disinfected with alcohol, and the most obvious heart beat was selected for puncture with a 50 ml syringe. After the needle penetrated the heart, blood rushed into the syringe. After obtaining the required blood volume, the needle was quickly withdrawn, and the whole blood in the syringe was transferred into a sterile 50 ml tube, mixed with an equal volume of PBS, and slowly added drop by drop above the lymphocyte separation solution. Centrifugation was carried out at 400×g at room temperature for 30 minutes. After centrifugation, the liquid surface was divided into four layers from top to bottom: yellow plasma layer, white film layer (i.e., mononuclear cell layer), separation solution layer, and red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation solution, and then rabbit PBMCs could be obtained.

[0026] Antigen-specific B cells were further sorted from rabbit PBMCs for culture. The supernatant of the cultured B cells was screened for positive clones using an ELISA plate coated with the antigen. After the cells of the positive clones were collected and lysed, RNA was extracted and reverse transcribed into cDNA. The full-length sequences of the light and heavy chains of the naturally paired rabbit monoclonal antibody were amplified from the corresponding positive clone cDNA, and a rabbit monoclonal antibody expression vector was constructed by cloning and recombination methods, and the sequence was determined by sequencing. The results of the amplified full-length PCR products were as Figure 1 .

[0027] 4) Preparation and purification of monoclonal antibodies In order to obtain multiple strains of rabbit monoclonal antibodies that recognize the T3 small molecule, the rabbit monoclonal antibody heavy chain and light chain genes were loaded on the expression vector, and the plasmid was transfected into KEK293 cells. After transfection for 120 - 144 hours, the culture supernatant contained recombinant rabbit monoclonal antibodies that recognize the T3 small molecule. The cell suspension was collected, centrifuged, and the supernatant was taken for antibody purification by affinity chromatography. The concentration of the purified monoclonal antibody was measured by the BCA method, and then it was aliquoted and freeze-dried. The purified antibody was named rabbit monoclonal antibody mAb32.

[0028] Example 2 Identification of rabbit monoclonal antibody in T3 sandwich method 1) Specificity identification of rabbit monoclonal antibody Detected by indirect ELISA method. The enzyme-labeled plate was coated with cross protein and T3 complex antigen at a concentration of 1 μg / ml and incubated overnight at 4°C. The enzyme-labeled plate was blocked with PBST containing 1% BSA, and 10 4 times diluted purified rabbit monoclonal antibody was added, and the reaction was carried out at 37°C for 50 min. The plate was washed 3 times with PBST, HRP-goat anti-rabbit IgG secondary antibody was added, and the reaction was carried out at 37°C for 50 min. The plate was washed 5 times with PBST, TMB was added for color development for 10 min, the termination solution was added, and the absorbance at A450 was measured with an enzyme-labeled instrument.

[0029] Figure 2 The results showed that the reaction between cross protein and rabbit monoclonal antibody mAb32 was negative, and the OD450 values were all less than 0.1; the reaction between T3 complex and mAb32 antibody was positive, and the OD value was much higher than that of cross protein, indicating that the sandwich ELISA rabbit monoclonal antibody of this application specifically recognized the T3 complex.

[0030] 2) Determination of the affinity constant of rabbit monoclonal antibody The affinity constant (Ka) was determined by non-competitive ELISA method.

[0031] Coating: The antigen was diluted with carbonate buffer to concentrations of 1, 0.5, 0.1, 0.05 μg / mL, and added to a 96-well enzyme-labeled plate at 100 μL / well for coating respectively, and incubated at 4°C for 24 h; Blocking: The plate was washed 4 times with PBST, and BSA solution was added at 200 μL / well and incubated at 37°C for 2 h; Adding monoclonal antibody: The plate was washed 4 times with PBST, and the rabbit monoclonal antibody was serially diluted starting from 100 μg / mL with carbonate buffer, 100 μL was added to each well, and incubated at 37°C for 2 h; Adding enzyme-labeled secondary antibody: The plate was washed 4 times with PBST, and 100 μL of HRP-labeled goat anti-rabbit Ig secondary antibody diluted 1:10000 was added to each well and placed at 37°C for 30 min; Color development and termination: The plate was washed 4 times with PBST, 100 μL of substrate chromogenic solution was added to each well, and the reaction was carried out in the dark at 37°C for 15 min; 50 μL of 1.0 mol / L H2SO4 termination solution was added to each well to terminate the reaction; Detection: The absorbance value (A450nm) at a wavelength of 450 nm was measured.

[0032] With the logarithm of antibody concentration as the abscissa and the OD value as the ordinate, an S-shaped curve was plotted. After calculation, the affinity constant Ka of the sandwich ELISA monoclonal antibody mAb32 was 7.5×10 8 L / mol.

[0033] 3) Antibody pairing for sandwich ELISA To select the best combination of the coated antibody and the detection antibody, the T3 complex antibody was coated on an ELISA plate and incubated overnight at 4°C. The next day, the ELISA plate was taken out, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and then washed 3 times with PBST; 100 μl of the T3 complex with a concentration of 20 ng / ml was added to each well and incubated at 37°C for 1 hour; after incubation, the ELISA plate was taken out, washed 3 times with PBST, and HRP-labeled rabbit monoclonal antibody mAb32 was added as the detection antibody and incubated at 37°C for 1 hour. The plate was washed 5 times with PBST, TMB substrate was added, and color development was carried out at 37°C for 10 min. After taking it out, the stop solution was added, and the OD450 reading was measured on an ELISA reader. According to the OD value of the sample and the background value of the negative control, the most ideal antibody pair was selected, and the pairing screening results are shown in Table 1.

[0034] Table 1 Screening Results of Antibody Pairing Experiments It can be seen that the antibody mAb32 involved in the present application is the best for sandwich detection.

[0035] Example 3 Analysis of Monoclonal Antibody Variable Region Genes and Amino Acid Sequences Using the recombinant plasmid of the antibody as the DNA template, sequencing primers for the light chain variable region and the heavy chain variable region were designed according to the 5' end vector sequences of the light chain and the heavy chain on the template, and sequencing was carried out using the sequencer ABI 3730. The nucleotide sequences of the light chain and the heavy chain variable regions of the rabbit monoclonal antibody were obtained by sequencing.

[0036] Using the Internet, on http: / / www.imgt.org, the IMGT / V-QUEST analysis software was used to analyze the sequencing data of the nucleotide sequences of the light chain variable region and the heavy chain variable region respectively. The amino acid sequence of the light chain variable region of the rabbit monoclonal antibody mAb32 is shown as SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.8.

[0037] The full length of VL is 110 amino acids. The amino acid numbers of the 4 domains of FR are 26, 17, 36, and 10 respectively. The amino acid numbers of the 3 domains of LCDR are 6, 3, and 12 respectively. The regions of LCDR1, LCDR2, and LCDR3 are 27aa-32aa, 50aa-52aa, and 89aa-100aa respectively, and their amino acid sequences are: ESIFGY (SEQ ID NO.1), IAS (SEQ ID NO.2), QQGYISSNVDNA (SEQ ID NO.3).

[0038] The full length of VH is 125 amino acids. The number of amino acids in the 4 domains of its FR is 25, 17, 38, and 11 respectively. The number of amino acids in the 3 domains of HCDR is 8, 7, and 19 respectively. HCDR1, HCDR2, and HCDR3 are 26aa - 33aa, 51aa - 57aa, and 96aa - 114aa respectively, and their amino acid sequences are GFDLSTYA (SEQ ID NO.4), ISNSGTT (SEQ ID NO.5), and ARGHSTSSGSYFAAYGMDP (SEQ ID NO.6) respectively.

[0039] Example 4 Sandwich Rabbit Monoclonal Antibody for Magnetic Particle Chemiluminescence Immunoassay 1. Detection Principle and Method Magnetic particle chemiluminescence immunoassay technology based on the principle of sandwich method is adopted. Biotin-labeled T3 antibody is used to immobilize the biotinylated antibody on SA magnetic beads. ALP-conjugated T3 complex rabbit monoclonal antibody is used. At the same time, T3, ALP substrate, and corresponding buffer components are placed on the Cosmai fully automatic magnetic particle chemiluminescence analyzer, and the instrument program is set for detection. The result is judged as positive when the signal-to-noise ratio of the luminescence value is greater than 2.0. The magnitude of the luminescence value reflects the amount of enzyme-labeled antibody bound, which is proportional to the concentration of T3 in the specimen. The standard curve is plotted according to the luminescence values of the measured standards. Figure 3 As such, the concentration value of T3 in the test specimen can be obtained from the standard curve.

[0040] 2. Composition of the Magnetic Particle Chemiluminescence Detection Kit for Detecting T3 1) SA Magnetic Beads Bound with Biotin-T3 Antibody: Take 50 μl of magnetic beads into a 0.5 ml centrifuge tube, place it on the magnetic rack, remove the supernatant after 1 min; wash the magnetic beads 3 times with 0.5 ml of antibody diluent; add a certain amount of biotin-labeled T3 antibody and rotate and mix at room temperature for 60 min; after magnetic separation, resuspend in magnetic storage buffer, and the working concentration is 0.5 mg / ml.

[0041] 2) mAb32 Conjugated with ALP: First, reduce the antibody mAb32 with 2-IT; second, form the ALP-SMCC intermediate, and finally couple the ALP-SMCC with the reduced antibody. After the coupling is completed, dilute it to the working concentration with ALP storage buffer.

[0042] 3) Washing Buffer: It is a conventional PBST with pH 7.4, containing 0.05% Proclin300, and is prepared as a 20-fold concentrated solution.

[0043] 4) Chemiluminescence Chromogenic Solution: Purchased from Aivide Biotech.

[0044] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered and sterilized by filtration.

[0045] 6) Standard: Small molecule T3, diluted to 5 μg / ml with PBS containing 1% BSA, 5% sucrose, 10% glycerol, and 0.05% Proclin 300 as the diluent, filtered and sterilized by filtration, and aseptically aliquoted.

[0046] 3. Detection of T3 clinical samples Treat clinical samples with different T3 concentrations. Using the T3 antibody as the coating antibody and the mAb32 antibody as the detection antibody, detect clinical samples with different concentrations using the above detection method. The detection results are shown in Figure 4 .

[0047] According to the result data, the rabbit monoclonal antibody described in this application for chemiluminescent immunoassay reagent with magnetic microparticles has a good correlation with clinical comparison within the range of 1 - 80 pmol / L samples.

[0048] In summary, applying the sandwich method rabbit monoclonal antibody mAb32 of T3 in this application to an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, detecting the T3 standard antigen by the double antibody sandwich chemiluminescence platform, the detection sensitivity is lower than 0.2 ng / ml. Detecting clinical samples by the magnetic chemiluminescence method, within the range of 1 - 80 pmol / L samples, its clinical coincidence rate > 0.93, significantly higher than the traditional competitive detection method, and the process is simple, breaking through the limitations of the traditional competitive method.

[0049] This specific embodiment is only an explanation of this application and does not limit this application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as it is within the scope of the claims of this application, it is protected by the patent law.

Claims

1. An anti-T3 antibody mAb32 or antigen-binding fragment thereof, characterized in that: It comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.1, the LCDR2 comprises the amino acid sequence IAS, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.

3.

2. The anti-T3 antibody mAb32 or antigen-binding fragment according to claim 1, characterized in that: The heavy variable region includes complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR1 includes the amino acid sequence shown in SEQ ID NO.4, the HCDR2 includes the amino acid sequence shown in SEQ ID NO.5, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO.

6.

3. The anti-T3 antibody mAb32 or antigen-binding fragment according to claim 1 or 2, characterized in that: The light chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:7; the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

8.

4. The anti-T3 antibody mAb32 or antigen-binding fragment according to claim 1 or 2, characterized in that: The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody and antibody minimum recognition unit; preferably, the remaining sequence of the antibody is derived from species including rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, One or more of goat, sheep, cow, pig, horse, monkey and human.

5. Biomaterial, characterized in that The biological material includes a polynucleotide, a vector or a cell, wherein the polynucleotide encodes the anti-T3 antibody mAb32 or the antigen-binding fragment according to any one of claims 1 to 4; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector, or is capable of expressing the anti-T3 antibody mAb32 or the antigen-binding fragment according to any one of claims 1 to 4.

6. The method for preparing the anti-T3 antibody mAb32 or antigen-binding fragment according to any one of claims 1 to 4, characterized in that: Comprising culturing the cell as claimed in claim 5; Optionally, the cell is obtained by transforming a polynucleotide encoding the anti-T3 antibody mAb32 or antigen-binding fragment into the cell, the polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprises co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.

7. The preparation method according to claim 6, characterized in that: The cell is a eukaryotic cell, preferably a mammalian cell, more preferably a 293 cell or a CHO cell.

8. Use of the anti-T3 antibody mAb32 or antigen-binding fragment according to any one of claims 1 to 4 or the biomaterial according to claim 5 in any of the following: 1) Testing T3 for non-diagnostic and treatment purposes; 2) preparing products for detecting T3; 3) Used to purify T3; 4) Preparation of products for purification of T3.

9. A detection reagent or a detection kit, characterized in that: The detection reagent or detection kit comprises the anti-T3 antibody mAb32 or antigen-binding fragment according to any one of claims 1 to 4, or the biological material according to claim 5.

10. The detection reagent or detection kit according to claim 9, characterized in that: The detection kit is an immunodetection kit prepared by double antibody sandwich ELISA or chemiluminescence method.

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