Monoclonal antibodies against human st2 and uses thereof

Abstract

The present application relates to the technical field of biology, and in particular to a kind of anti-human ST2 monoclonal antibody and its related application.The affinity high, specific pair antibody of mouse hybridoma cell technology is obtained, is used for detection and capture respectively, and can be used for the detection of clinical sample.A method-time resolution microsphere immunochromatography detection for clinical sample detection is provided, which can be used for quantitative detection of ST2 protein in clinical sample, the correlation coefficient is greater than 0.99, the test range is 1-250ng / ml, compared with commercial pair antibody, the detection lower limit is better than commercial antibody, and the sensitivity is higher.Using the pair antibody for clinical sample detection 10 times, CV is less than 15%, and stability is good.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against human ST2 and its related applications. Background Technology

[0002] Interleukin-33 (IL-33) is a member of the IL-1 cytokine family and has a dual function: it can induce the activation of NF-κB and MAPK, and as a transcription factor, it can participate in the regulation of gene expression. ST2 is a member of the IL-1R superfamily, mainly expressed in cardiomyocytes, and also expressed on various immune cells. ST2 precursor mRNA, after selective splicing, can be divided into three different isoforms: transmembrane ST2L, secretory ST2 (soluble ST2, sST2), and the variant form ST2V. ST2L is expressed on Th2 lymphocytes, NK cells, NKT cells, mast cells, and myeloid cells such as monocytes, dendritic cells, and granulocytes. The IL-33 / ST2 cellular pathway can promote Th1 and Th2 immune responses and has various cardiovascular protective effects, including anti-atherosclerosis, anti-myocardial hypertrophy, and anti-myocardial fibrosis. Under mechanical stress, cardiomyocytes produce large amounts of soluble ST2, which acts as a "decoy receptor," competitively binding to IL-33, reducing cardioprotective effects, and leading to myocardial remodeling and dysfunction. Clinically, the level of ST2 in the blood is measured for the diagnosis and prognostic assessment of heart failure. In normal individuals, ST2 levels are ≤35 ng / ml, while in patients with poor prognosis, levels are >35 ng / ml. Furthermore, ST2 testing helps assess the risk of acute coronary syndrome (ACS), and high ST2 levels are associated with a higher incidence of cardiovascular events.

[0003] Currently, the main methods for detecting ST2 in clinical practice include chemiluminescence, immunochromatography, and latex turbidimetry. These methodologies are all based on the principle of double-antibody sandwich method for antigen detection. Therefore, it is crucial to obtain monoclonal paired antibodies for ST2 with high affinity and strong specificity, which is also a key factor limiting the clinical detection of ST2. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention has developed ST2 monoclonal paired antibodies that can be used for clinical detection, for detection and capture, respectively; in addition, this invention also provides a method for clinical detection of ST2 antigen, which can quantitatively detect the content of ST2 in blood.

[0005] To address the above problems, the technical solution provided by this invention is as follows:

[0006] A monoclonal antibody against human ST2, wherein the monoclonal antibody is 1D2, the amino acid sequence of the heavy chain variable region of the 1D2 antibody is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO:3.

[0007] A monoclonal antibody against human ST2, wherein the monoclonal antibody is 5C6, the amino acid sequence of the heavy chain variable region of the 5C6 antibody is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO:5.

[0008] A monoclonal antibody against human ST2, wherein the monoclonal antibody is 1C3, the amino acid sequence of the heavy chain variable region of the 1C3 antibody is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO:7.

[0009] A monoclonal antibody against human ST2, wherein the monoclonal antibody is 120, the amino acid sequence of the heavy chain variable region of antibody 120 is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO:9.

[0010] A monoclonal antibody against human ST2, wherein the monoclonal antibody is 120-1, the amino acid sequence of the heavy chain variable region of antibody 120-1 is SEQ ID NO:10, and the amino acid sequence of the light chain variable region is SEQ ID NO:11.

[0011] This invention develops ST2 monoclonal paired antibodies that can be used for clinical detection, for detection and capture, respectively. These paired antibodies are obtained through mouse hybridoma cell screening technology, addressing one or more of the aforementioned needs or limitations, and providing a methodology for clinical sample detection—time-resolved fluorescent microsphere immunochromatography—for the quantitative detection of ST2 levels in blood.

[0012] The method includes the following steps:

[0013] 1) Recombinant expression of ST2: The eukaryotic expression plasmid of ST2 was obtained by molecular cloning technology. Recombinant expression was performed in 293 cells and high-purity recombinant ST2 protein was obtained by affinity purification.

[0014] 2) Hybridoma cell screening: Recombinant ST2 protein was used to perform four rounds of immunization. After immunization, the spleen of the mice was removed to isolate B cells. The isolated B cells were fused with myeloma cells. Positive clones were screened by methods such as pressure screening, limiting dilution, and ELISA detection. Finally, the heavy and light chain sequences of the antibody were obtained by molecular biology.

[0015] 3) Expression of ST2 antibody: The ST2 heavy and light chain sequences were synthesized and codons were optimized. The sequences were then constructed into eukaryotic expression vectors, such as pCDNA3.1, pCDNA, PTT5 and other transient transfection vectors. Stable transfection vectors containing the resistance gene can also be used. In this example, the expression was carried out in 293 cells by transient transfection in a vector containing the constant region of mouse IgG.

[0016] 4) ST2 antibody purification: ST2 antibody was purified by affinity using protein A packing material. The elution buffer was 0.1-0.2M glycine (pH=2.5-3.0). Finally, it was neutralized with 1-2M Tris-HCl (pH=8.0-8.5). The dialysate could be Tris-HCl buffer or PBS buffer, with a suitable pH of 6.0-8.0 and an optimal pH of 7.0.

[0017] 5) ELISA method for detecting the affinity of ST2 antibody: Coat each well with 500 ng / ml-2000 ng / ml recombinant ST2 protein, 100 μl per well. In the example of this invention, the concentration is 500 ng / ml. Coat at 4°C for 20 hours. Wash the plate 5 times with PBST the next day. Add 100 μg / ml of ST2 antibody and serially dilute 15 wells in a 3-fold gradient. Set up negative wells, i.e., replace the antibody with the diluent. Incubate at 37°C for 2 hours.

[0018] 6) SPR assay for affinity and pairing: SPR assay for affinity and pairing:

[0019] Affinity and pairing detection was performed based on the principle of surface plasmon resonance (SPR). The ST2 antigen was diluted to 30 μg / ml, and 50 μl was pipetted into the corresponding well of the Ni NTA chip. The cap was closed, and the start button was clicked to perform ligand immobilization for 5 min. After the immobilization program was completed, the ST2 sample was discarded, the sample was patted dry, and the diluted antibody was simultaneously added to the target chip wells according to the concentration gradient using a multi-channel pipette, 50 μL / well. The cap was closed, and the start button was quickly clicked on the software to perform the analyte-ligand binding kinetics detection process for 5 min. After the process was completed, the antibody sample was discarded, the sample was patted dry, 150 μl of dilution buffer was added to the experimental wells, the cap was closed, and the start button was clicked to perform the analyte dissociation process for 10 min. Finally, the data were statistically analyzed.

[0020] The same method can be used for ST2 antibody pairing detection. Dilute the ST2 antigen to 20ug / ml, aspirate 50ul into the corresponding well of the Ni NTA chip, close the cap, and click the start button to perform ligand fixation for 5 minutes. After the fixation program is completed, discard the ST2 sample, pat dry, add 50ul of the diluted first ST2 antibody, close the cap, and then quickly click the start button on the software to perform the analyte-ligand binding detection process for 5 minutes. After the process is completed, discard the antibody sample, pat dry, add 50ul of the diluted second ST2 antibody, close the cap, and then quickly click the start button on the software to perform the analyte-ligand binding detection process for 5 minutes. After the process is completed, discard the antibody sample, and finally perform pairing data analysis.

[0021] 7) Time-Resolved Microsphere Immunochromatography: Capture antibody and rabbit anti-chicken IgY were coated onto the NC membrane, with the capture antibody as the T line and the rabbit anti-chicken IgY as the C line. The membrane was dried overnight at 45°C. The detection antibody was coupled onto the time-resolved microspheres, with 1 mg of time-resolved microspheres coupled to 0.1 mg of antibody. After coupling, the microspheres were sprayed onto the conjugate pad and dried overnight at 45°C. The NC membrane, gold pad, sample pad, and absorbent paper were assembled, cut into strips, and loaded into the immunochromatographic cartridge for blank and antigen testing. The sample volume for both blank and antigen was 80 μL, and the detection time was 10 min. Finally, the data were analyzed and statistically analyzed.

[0022] The beneficial effects of this invention are as follows: This invention develops ST2 monoclonal paired antibodies that can be used for clinical detection, specifically for detection and capture. These paired antibodies are obtained through mouse hybridoma cell screening technology, and a methodological approach for clinical sample detection—time-resolved fluorescence microsphere immunochromatography—is provided for the quantitative detection of ST2 levels in blood. Recombinant ST2 protein obtained through eukaryotic cells can be used as a clinical testing standard. Paired antibodies with high affinity and specificity were obtained through mouse hybridoma cell technology, used for detection and capture, and applicable to clinical sample detection. A method for clinical sample detection—time-resolved microsphere immunochromatography—is provided, which can be used for the quantitative detection of ST2 protein in clinical samples, with a correlation coefficient >0.99 and a test range of 1-250 ng / ml. Using this paired antibody to test clinical samples 10 times, the CV is less than 15%, indicating good stability. Attached Figure Description

[0023] Figure 1 This is a graph of serum titer ELISA assay data;

[0024] Figure 2 These are SDS-PAGE electrophoresis images of five hybridoma cell lines after antibody recombinant expression.

[0025] Figure 3This is a graph showing the ELISA activity assay of 5 hybridoma cell lines;

[0026] Figure 4 This is an affinity map of 5 antibodies detected by SPR.

[0027] Figure 5 This is a paired detection chart of 5 antibodies;

[0028] Figure 6 This is a time-resolved microsphere immunochromatographic assay for detecting ST2 antigen. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0030] Example 1:

[0031] 1) Expression of ST2 antigen:

[0032] The ST2 antigen sequence was synthesized into the pCDNA3.4 eukaryotic expression vector (amino acid sequence shown in SEQ NO: 1). The plasmid was transformed into DH5α competent cells for preservation. The preserved glycerol-containing bacteria were cultured overnight at 180 rpm, and the cells were collected by centrifugation at 8000 rpm for 10 min for plasmid extraction. One day before transfection, cells were seeded at 1-1.5 × 10^6 / ml and cultured at 37℃, 125 rpm, and 5% CO2. Cell counts were performed before transfection, and the cell count reached 2.5-3 × 10^6 cells / ml. 6 / ml, viability ≥96% is sufficient for transfection; transfection is performed using PEI max transfection reagent, 2ml of feed solution is added 20hr after transfection; 4 days after expression, centrifuge at 8000rpm for 6min, collect the supernatant for purification; after centrifugation, discard the precipitate, take the supernatant, purify 100ml of culture medium supernatant directly with 1ml Ni Smart Beads, pack the packing material into a gravity column tube; equilibrate with 20 column volumes of equilibration buffer, which can be PBS buffer or Tris buffer; after equilibration, load the sample; after loading, wash again with 20 column volumes of equilibration buffer; collect the elution in separate tubes, the elution buffer is 250mM imidazole, collect one column volume per tube, collect a total of 5 tubes; dialyze the sample into PBS solution overnight.

[0033] 2) ST2 antibody hybridoma cell screening

[0034] Mice were immunized with the recombinant ST2 antigen. The antigen was diluted with PBS and then mixed with adjuvant at a volume ratio of 1:1. BALB / c mice were subcutaneously injected with 0.2 mL of the mixture. Two weeks after the first immunization, booster immunizations were performed, for a total of four booster immunizations. Blood was collected from the tail vein for titer determination. ELISA was performed using 100 ng of the recombinant ST2 antigen, coated overnight at 4°C. After discarding the coating solution, the mice were blocked at 37°C for 1 hour and washed five times with PBST. Then, 100 μL of mouse serum at different dilutions was added. After washing five times with PBST, 100 μL of a 1:5000 diluted goat anti-rabbit secondary antibody was added to each well and incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of TMB was added directly for color development. Finally, 0.25 M HCl was added to terminate the reaction. Results are shown in the figure. Figure 1 The serum was diluted at a ratio of 1:102400, and the OD450 was around 1.2, indicating that the antibody titer was qualified.

[0035] In a laminar flow hood, the spleens of the immunized mice to be fused were removed, transferred to a cell sieve, ground, and diluted with serum-free medium. The cells were then preheated and activated in an incubator. Similarly, the thymus of healthy mice was removed, transferred to a prepared cell sieve, ground, and the cell pellet was resuspended in 1 ml of serum for later use.

[0036] Prepare PEG and serum-free culture medium and preheat them in an incubator. Remove the prepared SP2 / 0 cells and spleen cells from the incubator and mix them in a 50ml centrifuge tube at a ratio of SP2 / 0:B cells = 1:5, mixing thoroughly. Centrifuge the suspension and discard the supernatant. Add PEG to initiate cell fusion, and after 1 minute of fusion, add serum-free culture medium to terminate the reaction. Centrifuge again and discard the supernatant. Resuspend the pellet in HAT medium and evenly seed the cells into 96-well plates using a multipipeline.

[0037] After culturing the fused cells for 4-5 days, the medium was changed, and all the medium was aspirated with a pipette. Then, 20% HAT medium was added, and the cells were placed in an incubator for further culture. When the cells in the wells reached about 80% confluence, ELISA was performed to confirm the viability of the five hybridoma cell lines 1D2, 5C6, 1C3, 120, and 120-1.

[0038] 3) ST2 antibody sequence analysis

[0039] Five ELISA-positive clones were used for gene retrieval using degenerate primers. The retrieved sequences, analyzed using the Kabat database, all conformed to the characteristics of antibody sequences. The amino acid sequence of the heavy chain variable region of antibody 1D2 is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of antibody 5C6 is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5; the amino acid sequence of the heavy chain variable region of antibody 1C3 is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of antibody 120 is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9; the amino acid sequence of the heavy chain variable region of antibody 120-1 is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11.

[0040] 4) Recombinant expression and purification of ST2 antibody

[0041] The extracted full-length gene sequence was synthesized into a pCDNA3.4 vector containing mouse IgG1. The plasmid was transformed into DH5α competent cells for preservation. The preserved glycerol-containing bacteria were cultured overnight at 180 rpm, and the cells were collected by centrifugation at 8000 rpm for 10 min for plasmid extraction. One day before transfection, cells were seeded at 1-1.5 × 10^6 / ml and cultured at 37℃, 125 rpm, and 5% CO2. Cell counting was performed before transfection; cells were considered ready when the cell count reached 2.5-3 × 10^6 / ml and the viability was ≥96%. Transfection was performed using PEImax transfection reagent, and 2 ml of feed solution was added 20 hours after transfection. Four days after expression, the cells were centrifuged at 8000 rpm for 6 min, and the supernatant was collected for purification. After centrifugation, the precipitate was discarded, and the supernatant was collected. 200 ml of the supernatant was purified using 2 ml of rProtein At. 2 ml of rProtein At was then... The column was packed with AT packing material and equilibrated with 20 column volumes of equilibration buffer (PBS or Tris buffer). After equilibration, the sample was slowly loaded. After loading, the column was washed with 20 column volumes of equilibration buffer. After equilibration, elution was performed and collected in aliquots. The eluent was 0.1 M Glycine (pH = 3.0), and 2 ml of eluent was collected from each aliquot. After elution, the pH was adjusted to neutral with 2 M Tris (pH = 8.0). The purity and concentration were determined by electrophoresis. The aliquots were dialyzed overnight into 1×PBS (pH = 7.4). The SDS-PAGE electrophoresis images of the three antibody expression lines are shown below. Figure 2 As can be seen from the figure, the non-reducing band of the five antibodies is around 180 kDa, the reduced band is around 55 kDa for the heavy chain and around 25 kDa for the light chain.

[0042] 5) ELISA detection of the affinity of antibodies 1D2, 5C6, 1C3, 120, and 120-1.

[0043] ELISA was performed by coating 100 ng of ST2 recombinant protein overnight at 4°C. The coating solution was discarded, and the sample was washed five times with PBST, followed by blocking at 37°C for 1 hour. Then, 100 μl of ST2 recombinant antibody at different dilutions was added to each well (5 antibody strains, starting at 1000 ng / ml, 100 μl per well), followed by five-fold serial dilutions. A negative control well was also included. After washing five times with PBST, 100 μl of goat anti-rabbit secondary antibody diluted 1:5000 was added to each well, and the sample was incubated at 37°C for 1 hour. After washing five times with PBST, 100 μl of TMB was added directly for color development. Finally, 0.25 M HCl was added to terminate the reaction. Results are shown in [link to results]. Figure 3 All five recombinant antibodies exhibited high ELISA activity.

[0044] 6) SPR assay to determine the affinity of 5 antibodies

[0045] The affinity of five ST2 antibodies was detected using an SPR multifunctional detection device with a NiNTA chip. First, the ST2 antigen was diluted to 30 μg / ml, and 50 μL was pipetted into the corresponding well of the NiNTA chip. The cap was closed, and the start button was pressed for ligand immobilization for 5 minutes. After immobilization, the ST2 sample was discarded, dried, and the diluted antibody was simultaneously added to the target chip wells at a concentration gradient using a multi-channel pipette (50 μL / well). The cap was closed, and the start button was quickly pressed to initiate the analyte-ligand binding kinetics detection process for 5 minutes. After this, the antibody sample was discarded, dried, and 150 μL of dilution buffer was added to the experimental wells. The cap was closed, and the start button was pressed for analyte dissociation for 10 minutes. Finally, data were statistically analyzed, and the results are shown below. Figure 4 The affinity of 1C3 is 4×10^-9 M, the affinity of 1D2 is 4×10^-7 M, the affinity of 5C6 is 3×10^-7 M, the KD of HM120 is 3.6810^-10 M, and the affinity of HM120-1 is 1.6×10^-9 M.

[0046] 7) SPR detection of paired antibodies from 5 strains

[0047] Dilute the ST2 antigen to 20 μg / ml, and pipette 50 μL into the corresponding well of the Ni NTA chip. Cap the chip and click the start button to perform ligand fixation for 5 minutes. After the fixation program finishes, discard the ST2 sample, blot dry, add 50 μL of the diluted first ST2 antibody, cap the chip, and then quickly click the start button on the software to perform the analyte-ligand binding detection process for 5 minutes. After the analyte-ligand binding detection process finishes, discard the antibody sample, blot dry, add 50 μL of the diluted second ST2 antibody, cap the chip, and then quickly click the start button on the software to perform the analyte-ligand binding detection process for 5 minutes. After the analyte-ligand binding detection process finishes, discard the antibody sample. Finally, perform paired data analysis. Results are shown in [link to results]. Figure 5 1C3 and HM120-1, 5C6 exhibit different epitopes.

[0048] 8) Time-resolved microsphere immunochromatography for the detection of ST2 antigen

[0049] Commercially available ST2 capture and detection antibodies were purchased. Time-resolved microsphere immunochromatography (RT-CIG) was prepared using the same method. Specifically, the capture antibody and rabbit anti-chicken IgY were coated onto an NC membrane, with the detection antibody as the T line and the rabbit anti-chicken IgY as the C line, and dried overnight at 45°C. Gold-sprayed 1C3 antibody was then conjugated to time-resolved microspheres, with 1 mg of time-resolved microspheres conjugated to 0.1 mg of antibody. After conjugation, the microspheres were sprayed onto the conjugation pad and dried overnight at 45°C. The NC membrane, gold pad, sample pad, and absorbent paper were assembled, cut into strips, and loaded into the immunochromatographic cartridge for blank and antigen testing. The sample volume for both blank and antigen solutions was 80 μL, and the detection time was 10 min. Finally, the T / C values ​​were statistically analyzed, and the results are shown below. Figure 6 When HM120-1 and 1C3 were used as paired antibodies for ST2 detection, the correlation was 0.9977, and the detection range was 1-250 ng / ml. In contrast, the correlation of commercially available ST2 paired antibodies for ST2 detection was 0.9943, and the detection lower limit was lower than that of the HM120-1 and 1C3 group. This indicates that the antibody of the present invention has higher sensitivity and is superior to commercially available antibodies.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

[0051] sequence list

[0052] SEQ ID NO: 1

[0053] KFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNP

[0054] IDHHS

[0055] SEQ ID NO:2

[0056] EVQLQQSGAELVKPGASVKLSCTVSGFNIKDTYIHWVKQRPEQGLEWIGRIDPANDNTKYDPKFQDKATISSDTSSNAAFLQLNSLTSEDTAVYYCADGSPFNYFSLDYWGQGTSVTVSS

[0057] SEQ ID NO:3

[0058] DILMTQSTSSLSASLGDRVTISCRASQDISKYLNWYQQKADGTVKLLIYYTSRLDSGVPSRFSGSGSGTDYSLTITNLEQEDIATYFCQQGNTLPWTFGGGTKLELK

[0059] SEQ ID NO:4

[0060] QVQLQQSGPELKKPGETVKISCKTSGYTFTSSGINWVKQAPGKGLKWMGWINTYTRQPTYTADFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATLPYYAMDYWGQGTSVTVSS

[0061] SEQ ID NO:5

[0062] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSAHVPYTFGGGTKLEIK

[0063] SEQ ID NO:6

[0064] QVQLKQSGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWVGFITYSAYTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRYDYDFDYWGQGTTLTVSS

[0065] SEQ ID NO:7

[0066] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRIRKNNLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGGGTKLELK

[0067] SEQ ID NO:8

[0068] EVKLMESGGGLVQPGGSLKLSCAASGFTFNIYTMSWVRQTPEKRLEWVAYINNGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCARQVTYFDYWGQGTTVTVSS

[0069] SEQ ID NO:9

[0070] DVQITQTPASLAASVGETVTITCRASENIYYTLAWYQQKQGKSPQLLIYNANTLEDGVPSRFSGSGSGTQYSMKINSTQPEDTATYFCQQSYGVPWTFGGGTKLELK

[0071] SEQ ID NO:10

[0072] QVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARDYGPYYFNYWGQGTTLTVSS

[0073] SEQ ID NO:11

[0074] DIQMTQSPSSLSASLGERVSLTCRASQDIGFYLSWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPPTFGGGTRLEIK

Claims

1. A monoclonal antibody against human ST2, characterized in that, The monoclonal antibody is 1D2, and the amino acid sequence of the heavy chain variable region of the 1D2 antibody is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO:

3.

2. A monoclonal antibody against human ST2, characterized in that, The monoclonal antibody is 5C6, and the amino acid sequence of the heavy chain variable region of the 5C6 antibody is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO:

5.

3. A monoclonal antibody against human ST2, characterized in that, The monoclonal antibody is 1C3, and the amino acid sequence of the heavy chain variable region of the 1C3 antibody is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO:

7.

4. A monoclonal antibody against human ST2, characterized in that, The monoclonal antibody is 120, and the amino acid sequence of the heavy chain variable region of antibody 120 is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO:

9.

5. A monoclonal antibody against human ST2, characterized in that, The monoclonal antibody is 120-1, and the amino acid sequence of the heavy chain variable region of antibody 120-1 is SEQ ID NO:10, and the amino acid sequence of the light chain variable region is SEQ ID NO:

11.

6. A carrier, characterized in that, The monoclonal antibody containing the anti-human ST2 as described in any one of claims 1-5.

7. The use of a monoclonal antibody containing the anti-human ST2 as described in any one of claims 1-5 in the preparation of an ST2 antigen detection reagent.

Images