Anti-CDX2 monoclonal antibody and application thereof

Abstract

The invention discloses an anti-CDX2 monoclonal antibody and an application thereof. The anti-CDX2 monoclonal antibody disclosed by the invention is a rabbit monoclonal antibody, has extremely high specificity, can be specifically combined with human CDX2 protein, and has an affinity constant as high as 2.66 * 10 < 10 >. The anti-CDX2 monoclonal antibody disclosed by the invention can be used for detecting human CDX2 protein by methods such as an immunohistochemical method, an immunoblotting method, an enzyme-linked immunosorbent assay or a flow cytometry.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an anti-CDX2 monoclonal antibody and applications thereof. Background Art

[0002] The gene encoding the caudal type homeobox 2 (CDX2) protein is a member of the caudal homeobox transcription factor gene family. This protein is a master regulator of intestinal-specific genes involved in cell growth and differentiation. This protein also plays a role in early embryonic development of the intestine. Aberrant expression of this gene is associated with intestinal inflammation and tumorigenesis.

[0003] Detection of CDX2 protein expression can help detect or diagnose some intestinal inflammation or tumors.

[0004] For example, the invention application with publication number CN106906288A discloses that the detection of leukemia CDX2 gene expression level is used for clinical early diagnosis of leukemia and monitoring of minimal residual disease (MRD).

[0005] However, immunohistochemistry and other tests are often required in clinical applications. In this case, appropriate antibodies against CDX2 protein are needed.

[0006] The invention application with publication number CN113265000A discloses a monoclonal antibody against CDX2 protein, its cell line, preparation method, and application. The antibody is prepared by chemically synthesizing amino acids 84-100 at the C-terminus of CDX2 protein as an antigenic peptide and coupling it to KLH to obtain an immunogen. Mice were immunized with the CDX2-KLH protein, and after cell fusion, screening, and subcloning, a mouse hybridoma cell line 12B6 that efficiently secretes anti-CDX2 protein monoclonal antibodies was obtained, as well as the anti-CDX2 protein monoclonal antibodies secreted by this cell line. However, in this prior art, the affinity constant of the antibody is 9.6×10 8 , the affinity constant is low.

[0007] The development of antibodies with higher affinity constants will facilitate the detection of CDX2 protein. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention provides an anti-CDX2 monoclonal antibody and its application.

[0009] The present invention first provides an anti-CDX2 monoclonal antibody, wherein the CDR-H1 region of the heavy chain is SYAMG; the CDR-H2 region is IISSSGGTEYASWARG; and the CDR-H3 region is RSSGVSTL; CDR-L1 region of the light chain: QSSQSVWKNNYFS; CDR-L2 region: ETSRLES; CDR-L3 region: LGGYDCTTADCDA.

[0010] Preferably, the HFR1 region of the heavy chain is: QSLEESGGRLVTPGTPLTLTCTVSGFSLS; HFR2 region is: WFRQAPGKGLEYIG; HFR3 region is: RFTISKTSSTTVDLKVTGPTTEDTATYFCSR; HFR4 region is: WGQGTLVTVSS; LFR1 region of the light chain: AQVLTQTPSSVSAAVGGTVTINC; LFR2 region: WYQQKPGQPPKLLIY; LFR3 region: GVPSRFKGSGSGTQFTLTISGIQCDDAATYYC; LFR4 region: FGGGTEVVVKGDP.

[0011] More preferably, the amino acid sequence of the heavy chain is shown as SEQ ID No. 2; and the amino acid sequence of the light chain is shown as SEQ ID No. 11.

[0012] The present invention further provides a gene sequence encoding the anti-CDX2 monoclonal antibody.

[0013] The present invention further provides the use of the anti-CDX2 monoclonal antibody in preparing a kit for immunoassay of human CDX2 protein, wherein the immunoassay is immunohistochemistry (HIC), immunoblotting (WB), enzyme-linked immunosorbent assay (ELISA), or flow cytometry.

[0014] The present invention further provides a kit for immunoassay of human CDX2 protein, comprising the anti-CDX2 monoclonal antibody. The immunoassay is performed by immunohistochemistry, immunoblotting, enzyme-linked immunosorbent assay, or flow cytometry. The kit also includes other reagents required for the corresponding immunoassay method.

[0015] Beneficial effects of the present invention: The anti-CDX2 monoclonal antibody of the present invention is a rabbit monoclonal antibody with extremely high specificity and can specifically bind to human CDX2 protein with an affinity constant of up to 2.66×10 10 The anti-CDX2 monoclonal antibody of the present invention can be used for detecting human CDX2 protein by immunohistochemistry, immunoblotting, enzyme-linked immunosorbent assay or flow cytometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the WB test result diagram.

[0017] Figure 2 This is the HIC test result of colon tissue.

[0018] Figure 3 This is the HIC test result of placenta tissue. DETAILED DESCRIPTION

[0019] Example 1: Preparation of anti-CDX2 monoclonal antibodies The amino acid sequence (SEQ ID No. 1) of human CDX2 protein (SwissProt ID: Q99626; NCBI No.: NP_001256.4) was selected, which is isoform 1 of human CDX2 protein with a full length of 313 aa.

[0020] The experimental rabbits were immunized with recombinant human CDX2 full-length protein as the immunogen by subcutaneous injection on the back, with 0.1 mg of immunogen used for each immunization, and a total of 4 immunizations were performed.

[0021] Blood was collected from experimental rabbits and peripheral blood mononuclear cells (PBMCs) were isolated. B cells in the PBMCs were labeled and screened, and the genetic information of B cells expressing CDX2 antibodies was obtained for amplification and recombination. The genetic information that met the requirements was screened and loaded into the vector and then transfected into the vector cells. The antibodies secreted by the vector cells were cultured and purified to obtain monoclonal antibodies.

[0022] The specific sequence is as follows: (1) The full length of the heavy chain amino acid sequence is shown in SEQ ID No. 2, and the specific sequences of each region are as follows: HFR1 area: QSLEESGGRLVTPGTPLTLTCTVSGFSLS; CDR-H1 region: SYAMG; HFR2 region: WFRQAPGKGLEYIG; CDR-H2 region: IISSSGGTEYASWARG; HFR3 area: RFTISKTSSTTVDLKVTGPTTEDTATYFCSR; CDR-H3 region: RSSGVSTL; HFR4 region: WGQGTLVTVSS; Constant region (Tail): GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRT ARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK.

[0023] (2) The full length of the light chain amino acid sequence is shown in SEQ ID No. 11, and the specific sequences of each region are as follows: LFR1 region: AQVLTQTPSSVSAAVGGTVTINC; CDR-L1 region: QSSQSVWKNNYFS; LFR2 area: WYQQKPGQPPKLLIY; CDR-L2 region: ETSRLES; LFR3 area: GVPSRFKGSGSGTQFTLTISGIQCDDAATYYC; CDR-L3 region: LGGYDCTTADCDA; LFR4 region: FGGGTEVVVKGDP; Constant region (Tail): VAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0024] Example 2: Affinity constant detection The detection steps are as follows: (1) Use recombinant human CDX2 full-length protein to coat the ELISA plate at a coating concentration of 2 μg / mL, 100 μL / well, and coat overnight at 4°C.

[0025] (2) The next day, rinse three times with PBST buffer.

[0026] (3) Then add 200 μL of blocking solution to each well, block at 37°C for 2 hours, and rinse again with PBST buffer three times.

[0027] (4) The prepared anti-CDX2 monoclonal antibody was used as the primary antibody, first diluted to 100 ng / mL (625-fold dilution) with buffer, and then diluted in 3-fold steps for a total of 10 gradients. The last well was used as a blank control well (only buffer was added), and the volume added to each well was 100 μL.

[0028] (5) The secondary antibody was HRP-labeled goat anti-rabbit diluted 1:20,000, 100 μL per well, incubated at 37°C for 1 hour, and rinsed three times with PBST buffer.

[0029] (6) Add 100 μL TMB color development solution to each well and color for 10 minutes, then add stop solution to terminate the reaction.

[0030] (7) Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 450 nm. Draw a curve of OD values ​​versus antibody dilution factors, find the dilution factor A corresponding to half the maximum binding OD value, and calculate the affinity constant of the monoclonal antibody using the affinity constant calculation formula.

[0031] The affinity constant calculation formula is: affinity constant ≈ 150,000 × A / original monoclonal antibody concentration.

[0032] Finally, the affinity constant of the monoclonal antibody in this application was calculated to be 2.66×10 10 .

[0033] Example 3: WB detection (1) The recombinant human CDX2 full-length protein was used as the test sample for electrophoresis. The recombinant human CDX2 full-length protein was diluted with Caco-2 cell lysate, and the amount of recombinant protein loaded in each lane was 10 μg.

[0034] (2) Then transfer to PVDF membrane.

[0035] (3) Block with 2.5% skim milk for 60 minutes.

[0036] (4) Wash and add anti-CDX2 monoclonal antibody prepared at an antibody concentration of 5 μg / mL and incubate for 60 minutes.

[0037] (5) After washing, add HRP-labeled goat anti-rabbit secondary antibody and incubate for 60 minutes.

[0038] (6) After cleaning, add 2 mL of ECL developer, develop at room temperature for 5 minutes, expose in an imager for 30 seconds, and take a picture.

[0039] Test results such as Figure 1As shown, the molecular weight of the recombinant human CDX2 full-length protein is about 40 KDa. The figure shows a specific band near 40 KDa, and the band is clear without any other bands, indicating that the prepared anti-CDX2 monoclonal antibody can be used to detect human CDX2 protein.

[0040] Example 4: IHC detection Paraffin sections of human colon and placental tissue were prepared from Hangzhou First People's Hospital. Sections were deparaffinized, hydrated, and fixed (using Tris / EDTA buffer, pH 9.0, heat-fixed). Sections were then incubated with 0.3 μg / mL anti-CDX2 monoclonal antibody at room temperature for 1 hour, rinsed three times with PBS, incubated with secondary antibody at room temperature for 15 minutes, rinsed three times with PBS, and developed with freshly prepared DAB colorimetric solution for 3-10 minutes. Sections were counterstained with hematoxylin for 25 seconds and then blued with PBS for 30 seconds. Sections were then dehydrated using a gradient of 85% alcohol for 3 minutes, 95% alcohol for 3 minutes, 100% alcohol for 3 minutes, and finally cleared with xylene for 3 minutes before mounting with neutral gum.

[0041] like Figure 2 Shown are the results of immunohistochemical staining of colon tissue. Figure 3 The immunohistochemical staining results of placental tissue are shown. As can be seen from the figure, the positive staining results have moderate intensity, correct positioning, and no nonspecific staining, which meets the requirements. This shows that the anti-CDX2 monoclonal antibody of the present application can be used for immunohistochemical detection of CDX2 protein.

Claims

1. An anti-CDX2 monoclonal antibody, characterized in that Heavy chain CDR-H1 region: SYAMG; CDR-H2 region: IISSSGGTEYASWARG; CDR-H3 region: RSSGVSTL; CDR-L1 region of the light chain: QSSQSVWKNNYFS; CDR-L2 region: ETSRLES; CDR-L3 region: LGGYDCTTADCDA.

2. The anti-CDX2 monoclonal antibody according to claim 1, characterized in that HFR1 region of the heavy chain: QSLEESGGRLVTPGTPLTLTCTVSGFSLS; HFR2 region: WFRQAPGKGLEYIG; HFR3 region: RFTISKTSSTTVDLKVTGPTTEDTATYFCSR; HFR4 region: WGQGTLVTVSS; LFR1 region of the light chain: AQVLTQTPSSVSAAVGGTVTINC; LFR2 region: WYQQKPGQPPKLLIY; LFR3 region: GVPSRFKGSGSGTQFTLTISGIQCDDAATYYC; LFR4 region: FGGGTEVVVKGDP.

3. The anti-CDX2 monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain is shown in SEQ ID No. 2; the amino acid sequence of the light chain is shown in SEQ ID No.

11.

4. A gene sequence encoding the anti-CDX2 monoclonal antibody according to any one of claims 1 to 3.

5. Use of the anti-CDX2 monoclonal antibody according to any one of claims 1 to 3 in the preparation of a kit for immunodetection of human CDX2 protein.

6. The use according to claim 5, characterized in that Immunoassays include immunohistochemistry, immunoblotting, enzyme-linked immunosorbent assay, or flow cytometry.

7. A kit for immunodetection of human CDX2 protein, characterized in that: The invention comprises the anti-CDX2 monoclonal antibody according to any one of claims 1 to 3.

8. The kit for immunoassay of human CDX2 protein according to claim 7, characterized in that: Immunoassays include immunohistochemistry, immunoblotting, enzyme-linked immunosorbent assay, or flow cytometry.

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