Anti-T3 antibody mAb68 or antigen binding fragment, preparation method and application

Abstract

The invention belongs to the technical field of immunodetection, and discloses an anti-T3 antibody mAb68 or an antigen binding fragment, the anti-T3 antibody mAb68 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 TAS, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO.3; the heavy 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 mAb68 is used for detecting a T3 standard antigen, the detection sensitivity is lower than 0.2 ng / ml, a clinical sample is detected through a magnetochemiluminescence method, and 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, specifically to anti-T 3 Antibody mAb68 or antigen-binding fragment and its use. Background Art

[0002] Triiodothyronine (T 3 ) is an important component of thyroid hormone and an indicator of thyroid hormone. 3 About 99.7% of it is specifically bound to thyroid hormone binding protein (TBG) and exists in a bound state; the free T 3 (FT 3 ) accounts for only about 0.3%. 3 It is involved in almost every physiological process of the body, including growth and development, metabolism, regulating body temperature and heart rate, etc. It is one of the key factors in maintaining normal metabolism of the human body. 3 It is a specific indicator for diagnosing hyperthyroidism and is also of great reference value for estimating whether hyperthyroidism has recurred. Increased levels are seen in hyperthyroidism, early stage of triiodothyronine-type hyperthyroid crisis, iodine-deficient goiter, hyperthyroidism, chronic active hepatitis, primary biliary cirrhosis, etc., while decreased levels are seen in hypothyroidism, thyroid adenoma, chronic thyroiditis, etc.

[0003] Currently, T 3 The "gold standard" for detection is mass spectrometry, 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 practice. 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, poor specificity, are prone to false positives, and have complex processes.

[0004] Therefore, it is urgent to develop 3 Sandwich method with high specificity and high sensitivity monoclonal antibodies, realizing double antibody sandwich method to detect T 3 . Summary of the invention

[0005] In order to overcome the above technical problems, the present application provides an anti-T 3 The mAb68 antibody or antigen-binding fragment can be successfully used in immunoassay kits prepared by double antibody sandwich ELISA or chemiluminescence method to detect T 3 Rapid detection is of great significance in the diagnosis and treatment of thyroid diseases.

[0006] On the one hand, the present application provides an anti-T 3 antibody mAb68 or antigen-binding fragment, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises complementarity-determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 is QNINSW (SEQ ID NO.1), the amino acid sequence of LCDR2 is TAS, and the amino acid sequence of LCDR3 is QQGYIGSNVENA (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 GFSLSSYA (SEQ ID NO.4), the amino acid sequence of HCDR2 is INTGGFP (SEQ ID NO.5), and the amino acid sequence of HCDR3 is shown as ARGHTINNNYYFAAYGMDP (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 109 amino acids, the number of amino acids in the 4 domains of its FR are 26, 16, 36 and 10 respectively, the number of amino acids in the 3 domains of LCDR are 6, 3 and 12 respectively, and the regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 49aa-51aa and 88aa-99aa respectively, and their amino acid sequences are QNINSW (SEQ ID NO.1), TAS (SEQ ID NO.2), QQGYIGSNVENA (SEQ ID NO.3).

[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, the number of amino acids in the 4 domains of its FR are 25, 17, 38 and 11 respectively, the number of amino acids in the 3 domains of HCDR are 8, 7 and 19 respectively, and HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-114aa respectively, and their amino acid sequences are GFSLSSYA (SEQ ID NO.4), INTGGFP (SEQ ID NO.5), ARGHTINNNYYFAAYGMDP (SEQ ID NO.6).

[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 sequence of the antibody is derived includes 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 biomaterial, which includes a polynucleotide, a vector, or a cell, and the polynucleotide encodes the anti-T 3 antibody mAb68 or an antigen-binding fragment; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector, or is capable of expressing the anti-T 3 antibody mAb68 or an antigen-binding fragment.

[0012] On the other hand, the present application also provides a method for preparing the anti-T 3 antibody mAb68 or an antigen-binding fragment, which includes culturing the cell; optionally, the cell is prepared by transforming a polynucleotide encoding the anti-T 3 antibody mAb68 or an 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-T 3 antibody mAb68 or an antigen-binding fragment or the biomaterial in any of the following: 1) Detecting T for non-diagnostic and non-therapeutic purposes 3 ; 2) Preparing a product for detecting T 3 ; 3) Purifying T 3 ; 4) Preparing a product for purifying T 3 ;

[0015] On the other hand, the present application also provides a detection reagent or a detection kit, which contains the anti-T 3 antibody mAb68 or an antigen-binding fragment, or the biomaterial.

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

[0017] Compared with the prior art, the anti-T 3 antibody mAb68 or antigen-binding fragment thereof can bind to T 3 with high specificity and high affinity, and its affinity constant Ka can reach 8×10 8 L / mol. The anti-T 3 monoclonal antibody mAb68 of the present application can also be prepared into various immunoassay kits for detecting T 3 , especially, it can be applied to immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence method. The double-antibody sandwich chemiluminescence platform detects the T 3 standard antigen, and the detection sensitivity is lower than 0.2 ng / ml. When detecting clinical samples by the magnetic chemiluminescence method, within the range of 1-80 pmol / L samples, the correlation with the R clinical comparison is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is an electrophoresis diagram of the full-length amplification products of the heavy chain and light chain of mAb68, and M is the DNA molecular weight Marker.

[0019] Figure 2 It is the detection of cross proteins and target antigen samples by the mAb68 sandwich ELISA method, and the ordinate is the detected OD value.

[0020] Figure 3 It is the detection of the T 3 standard antigen by the mAb68 magnetic particle chemiluminescence method. The abscissa is the concentration (ng / ml) of the T 3 standard antigen, and the ordinate is the detected luminescence value. The R 2 of the standard curve = 0.9951, the linear detection range is 0.2-10 ng / mL, and the sample concentration calculation formula is deduced: y = 415501x - 238223.

[0021] Figure 4 It is the detection of clinical samples by the mAb68 magnetic particle chemiluminescence method. The abscissa is the free T 3 concentration (pmol / L) of the clinical sample, and the ordinate is the detected luminescence value. The R 2 of the standard curve = 0.9556, the linear detection range is 1-80 pmol / L, and the sample concentration calculation formula is deduced: y = 35177x - 142086. DETAILED DESCRIPTION OF THE 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 generally carried out under conventional conditions, the conditions described in laboratory manuals, or the conditions recommended by the manufacturer.

[0023] Example 1 T 3 Preparation of sandwich rabbit monoclonal antibody 1) Preparation of immunogen Prepare T 3 complex, and the purity of the immunogen is above 90%, meeting the purity requirements for preparing monoclonal antibodies.

[0024] 2) Animal immunization The above-prepared T 3 complex was emulsified with complete Freund's adjuvant at a volume ratio of 1:1, and New Zealand white rabbits weighing about 2 kg were immunized by subcutaneous injection. The immunization dose was 800 μg / 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 / 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 128,000 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 on 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 pulled out, 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. Centrifuge at 400×g for 30 minutes at room temperature. 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. Carefully aspirate the mononuclear cell layer and wash it with PBS to remove platelets and lymphocyte separation solution to obtain rabbit PBMCs.

[0026] Continue to sort antigen-specific B cells 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. RNA was extracted from the collected and lysed positive clone cells 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. 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 are as Figure 1 .

[0027] 4) Preparation and purification of monoclonal antibodies To obtain multiple strains of rabbit monoclonal antibodies that recognize T 3 small molecules, the heavy and light chain genes of rabbit monoclonal antibodies were loaded onto an expression vector, and the plasmid was transfected into KEK293 cells. After transfection for 120 - 144 hours, the culture supernatant containing recombinant rabbit monoclonal antibodies that recognize T 3 small molecules was obtained. The cell suspension was collected, centrifuged, and the supernatant was taken. The antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then it was aliquoted and lyophilized. The purified antibody was named rabbit monoclonal antibody mAb68.

[0028] Example 2 T 3 Identification of sandwich rabbit monoclonal antibody 1) Specificity identification of rabbit monoclonal antibody The indirect ELISA method was used for detection. The enzyme - linked immunosorbent assay (ELISA) plate was coated with cross - linked protein and T 3 complex antigen at a concentration of 1 μg / ml and incubated overnight at 4°C. The ELISA plate was blocked with PBST containing 1% BSA. 10 - fold 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, then 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, and the stop solution was added. The absorbance at 450 nm (A450) was measured with an enzyme - linked immunosorbent assay reader. 4 The results showed that the reaction between cross - linked protein and rabbit monoclonal antibody mAb68 was negative, and the OD450 values were all less than 0.1; the reaction between T

[0029] Figure 2 complex and mAb68 antibody was positive, and the OD value was much higher than that of cross - linked protein, indicating that the sandwich rabbit monoclonal antibody of the present application specifically recognizes T 3 complex. 3 The sandwich rabbit monoclonal antibody specifically recognizes T 3 complex.

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

[0031] Coating: The antigen was diluted with carbonate buffer to concentrations of 1, 0.5, 0.1, 0.05 μg / mL, and 100 μL / well was added to a 96 - well ELISA plate 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; Add enzyme-labeled secondary antibody: Wash the plate 4 times with PBST. Add 100 μL of HRP enzyme-labeled goat anti-rabbit Ig secondary antibody diluted 1:10,000 to each well and incubate at 37 °C for 30 min; Color development and termination: Wash the plate 4 times with PBST. Add 100 μL of substrate chromogenic solution to each well and react at 37 °C in the dark for 15 min; Add 50 μL of 1.0 mol / L H 2 SO 4 termination solution to terminate the reaction; Detection: Measure the absorbance value (A450nm) at a wavelength of 450 nm.

[0032] Using the logarithm of the antibody concentration as the abscissa and the OD value as the ordinate, plot an S-shaped curve. After calculation, T 3 The affinity constant Ka of the sandwich monoclonal antibody mAb68 = 8×10 8 L / mol.

[0033] 3) Antibody pairing for sandwich method To select the best combination of coating antibody and detection antibody, coat the enzyme-labeled plate with the T 3 complex antibody and incubate overnight at 4 °C. Take out the enzyme-labeled plate the next day, wash it once with PBST, block it with 1% BSA solution at 37 °C for 2 hours, and wash it 3 times with PBST; Add 100 μl of the T 3 complex with a concentration of 20 ng / ml to each well and incubate at 37 °C for 1 hour; After incubation, take out the enzyme-labeled plate, wash it 3 times with PBST, add the HRP-labeled rabbit monoclonal antibody mAb68 as the detection antibody respectively, and incubate at 37 °C for 1 hour. Wash it 5 times with PBST, add the TMB substrate, and develop color at 37 °C for 10 min. After taking it out, add the termination solution and measure the OD450 reading on the microplate reader. According to the OD value of the sample and the background value of the negative control, select the most ideal antibody pair. The pairing screening results are shown in Table 1.

[0034] Table 1 Screening results of antibody pairing experiment It can be seen that the antibody mAb68 involved in this 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, design sequencing primers for the light chain variable region and heavy chain variable region according to the light chain and heavy chain 5'-end vector sequences on the template, and perform sequencing using the sequencer ABI 3730. The nucleotide sequences of the light chain and heavy chain variable regions of the rabbit monoclonal antibody are obtained by sequencing.

[0036] Using the Internet, IMGT / V-QUEST analysis software was used at http: / / www.imgt.org to analyze the sequencing results of the nucleotide sequences of the light chain variable region and the heavy chain variable region. The amino acid sequence of the light chain variable region of the rabbit monoclonal antibody mAb68 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0037] The total length of VL is 109 amino acids, the number of amino acids in the four domains of FR are 26, 16, 36 and 10 respectively, the number of amino acids in the three domains of LCDR are 6, 3 and 12 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 49aa-51aa and 88aa-99aa respectively, and their amino acid sequences are: QNINSW (SEQ ID NO.1), TAS (SEQ ID NO.2), QQGYIGSNVENA (SEQ ID NO.3).

[0038] The total length of VH is 125 amino acids, the amino acid numbers of the four FR domains are 25, 17, 38 and 11 respectively, the amino acid numbers of the three HCDR domains 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 GFSLSSYA (SEQ ID NO.4), INTGGFP (SEQ ID NO.5), ARGHTINNNYYFAAYGMDP (SEQ ID NO.6), respectively.

[0039] Example 4T 3 Sandwich rabbit monoclonal antibody for magnetic microparticle chemiluminescent immunoassay 1. Detection principles and methods The method uses magnetic particle chemiluminescent immunoassay technology based on the principle of double antibody sandwich method. 3 Antibodies, biotinylated antibodies were fixed to SA magnetic beads, ALP was coupled to T 3 Complex rabbit monoclonal antibody, and T 3 Place the ALP substrate and the corresponding buffer components on the Cosma fully automatic magnetic microparticle chemiluminescence instrument and set the instrument program for detection. The result is judged to be positive based on the luminescence value signal-to-noise ratio greater than 2.0. The size of the luminescence value reflects the amount of bound enzyme-labeled antibody, which is related to the T in the sample. 3 The standard curve is drawn according to the luminescence value of the measured standard, such as Figure 3 , T in the sample to be tested 3 The concentration value can be obtained from the standard curve.

[0040] 2. Detection of T 3 Composition of the magnetic particle chemiluminescence detection kit 1) SA magnetic beads conjugated with biotin-T 3 antibody: Take 50 μl of magnetic beads into a 0.5 ml centrifuge tube, place it on a magnetic stand, 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 T 3 antibody, rotate and mix at room temperature for 60 min; after magnetic separation, resuspend in magnetic preservation buffer, with a working concentration of 0.5 mg / ml.

[0041] 2) mAb68 conjugated with ALP: First, reduce the antibody mAb68 using 2-IT; secondly, form the ALP-SMCC intermediate, and finally couple the ALP-SMCC with the reduced antibody. After the coupling is completed, dilute to the working concentration with ALP preservation buffer.

[0042] 3) Washing buffer: It is a conventional PBST with pH 7.4, containing 0.05% Proclin 300, 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.

[0045] 6) Standard: T 3 Small molecule, using PBS containing 1% BSA, 5% sucrose, 10% glycerol, and 0.05% Proclin 300 as the diluent, dilute the sample to 5 μg / ml, filter and sterilize, and dispense aseptically.

[0046] 3. Detection of clinical samples for T 3 Process clinical samples with different T concentrations, using the T 3 antibody as the coating antibody and the mAb68 antibody as the detection antibody, detect clinical samples with different concentrations using the above detection method, and the detection results are shown in 3 Figure 4 .

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

[0048] In summary, applying the sandwich rabbit monoclonal antibody mAb68 of T 3 in this application to the immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, detecting T on the double antibody sandwich chemiluminescence platform​3 Standard antigen, with a detection sensitivity lower than 0.2 ng / ml. When detecting clinical samples by the magnetic chemiluminescence method, within the sample range of 1 - 80 pmol / L, its clinical coincidence rate > 0.95, which is significantly higher than that of the traditional competitive detection method. Moreover, the process is simple, breaking through the limitations of the traditional competitive method.

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

Claims

1. An anti-T3 antibody mAb68 or an 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, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO.1, the amino acid sequence of the LCDR2 is TAS, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO.

3.

2. The anti-T3 antibody mAb68 or antigen-binding fragment according to claim 1, characterized in that: The heavy variable region includes complementary determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO.4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.

6.

3. The anti-T3 antibody mAb68 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 has at least 95% identity with 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 has at least 95% identity with the amino acid sequence shown in SEQ ID NO:

8.

4. The anti-T3 antibody mAb68 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 mAb68 or the antigen-binding fragment described in any one of claims 1-4; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector, or is capable of expressing the anti-T3 antibody mAb68 or the antigen-binding fragment described in any one of claims 1-4.

6. The method for preparing the anti-T3 antibody mAb68 or antigen-binding fragment thereof 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 mAb68 or 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.

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 mAb68 or the 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 mAb68 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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