A rabbit monoclonal antibody against Ki-67 and a preparation method thereof and application of an immunohistochemical reagent

Abstract

The application discloses an anti-Ki-67 rabbit monoclonal antibody and a preparation method and application of an immunohistochemical reagent, characterized by comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 shown in the amino acid sequence of SEQ ID NO:1, HCDR2 shown in the amino acid sequence of SEQ ID NO:2 and HCDR3 shown in the amino acid sequence of SEQ ID NO:3; and the light chain variable region comprising LCDR1 shown in the amino acid sequence of SEQ ID NO:4, LCDR2 shown in the amino acid sequence of SEQ ID NO:5 and LCDR3 shown in the amino acid sequence of SEQ ID NO:6, the preparation method comprising the following steps: transfecting cells by using an expression vector containing a coding gene nucleic acid molecule, continuing to culture the cells after transfection, collecting cell supernatant and purifying to obtain the anti-Ki-67 rabbit monoclonal antibody, and application in immunohistochemical staining (IHC) or immunocytochemical staining (ICC), and the advantages are that the affinity is extremely high, the specificity is strong, the sensitivity is high, the stability is strong, and the IHC or ICC staining background is lower.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and immunodetection technology, specifically relating to an anti-Ki-67 rabbit monoclonal antibody, its preparation method, and the application of immunohistochemical reagents. Background Technology

[0002] Ki-67 (cell proliferation-associated antigen) is a highly phosphorylated nucleoprotein strictly limited to expression during the G1, S, G2, and M phases of the cell cycle, and not expressed in G0 phase (quiescent phase) cells. Therefore, Ki-67 is widely recognized as the "gold standard" biomarker for assessing cell proliferation status and has irreplaceable clinical value in the clinicopathological molecular subtyping, malignancy grading, prognostic assessment, and treatment selection of various malignant tumors, including gastric cancer, cervical cancer, breast cancer, neuroendocrine tumors, and lymphoma.

[0003] Furthermore, existing anti-Ki-67 antibodies suffer from several drawbacks in practical applications, including high working concentrations (typically diluted between 1:100 and 1:200), low yields from industrial recombinant expression, and rapid titer decay due to polymer aggregation and precipitation in dilution systems without animal-derived serum albumin (BSA). These shortcomings severely limit the high-throughput application of fully automated immunohistochemical staining platforms and increase the risk of false negatives or false positives. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a recombinant anti-Ki-67 rabbit monoclonal antibody with extremely high affinity, strong specificity, high sensitivity and strong stability, as well as a high-yield recombinant preparation method and an immunohistochemical kit product with extremely high stability under ultra-high dilution formulation.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an anti-Ki-67 rabbit monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6.

[0006] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0007] Furthermore, the heavy chain variable region and / or light chain variable region, while maintaining the core CDR binding activity, contain one or more conserved substitutions of amino acid residues in their framework region (FR region); and the equilibrium dissociation constant K of the anti-Ki-67 rabbit monoclonal antibody binding to human Ki-67 protein is... D Less than 1×10 -10 M.

[0008] Furthermore, the DNA sequence encoding the heavy chain variable region of the anti-Ki-67 rabbit monoclonal antibody is the nucleotide sequence shown in SEQ ID NO:9, and the DNA sequence encoding the light chain variable region is the nucleotide sequence shown in SEQ ID NO:10.

[0009] The present invention also provides a method for preparing the above-mentioned anti-Ki-67 rabbit monoclonal antibody, comprising the following steps: Construct a recombinant expression vector that simultaneously contains the heavy chain variable region coding gene and the light chain variable region coding gene, or construct a heavy chain expression vector and a light chain expression vector that separately contain the heavy chain variable region coding gene and the light chain variable region coding gene; transfect the recombinant expression vector, or the heavy chain expression vector and the light chain expression vector together, into mammalian host cells (preferably CHO cells or HEK293 cells) for eukaryotic expression, collect the culture supernatant and purify it by Protein A affinity chromatography to obtain the anti-Ki-67 rabbit monoclonal antibody.

[0010] The present invention also provides the application of the above-mentioned anti-Ki-67 rabbit monoclonal antibody in immunohistochemical staining (IHC) or immunocytochemical staining (ICC) reagents.

[0011] The present invention further provides the application of the above-mentioned anti-Ki-67 rabbit monoclonal antibody in the preparation of a detection biological product for detecting the expression level of Ki-67 in clinical samples, wherein the clinical samples are formalin-fixed paraffin-embedded tissue sections (FFPE) or liquid-based cytology slides.

[0012] The present invention also provides a Ki-67 detection kit, which includes the aforementioned anti-Ki-67 rabbit monoclonal antibody, primary antibody dilution buffer, primary antibody post-reagent, HRP enzyme-labeled secondary antibody, catalase blocking solution, DAB concentrate, DAB buffer and hematoxylin; The primary antibody dilution buffer contains a non-protein polymeric stabilizer (such as a combination of polyethylene glycol or polyvinylpyrrolidone of a specific molecular weight) and is free of any animal-derived serum albumin (BSA-free); the clinical working dilution ratio of the anti-Ki-67 rabbit monoclonal antibody in the primary antibody dilution buffer is 1:1000 to 1:5000.

[0013] Compared with the prior art, the advantages of the present invention are as follows: First, a revolutionary breakthrough has been achieved in antibody binding performance and staining process: through screening and recombinant engineering of specific rabbit CDR sequences, the rabbit monoclonal antibody obtained by this invention exhibits ultra-high affinity and extremely strong specificity for the human Ki-67 epitope at the sub-pM level, and can achieve the ideal effect of deep staining of cell nuclei and zero staining of interstitial background in both Western Blot and IHC / ICC detection.

[0014] Secondly, this invention offers significant advantages in the commercial production and storage stability of in vitro diagnostic (IVD) reagents. Thanks to its unexpectedly high affinity, the antibody maintains perfect colorimetric sensitivity even at ultra-high dilution ratios of 1:1000 to 1:5000, with consumption only a fraction of that of commercially available conventional antibodies, significantly reducing industrial production costs. Simultaneously, addressing the inherent tendency of rabbit antibodies to aggregate, this invention innovatively incorporates a proprietary non-protein polymeric stabilizer system, endowing the reagent with excellent formulation stability. In accelerated aging tests at 37°C, the recombinant rabbit monoclonal antibody maintained its potency for two weeks, fully meeting the stringent quality control requirements of cold chain or room temperature transportation in clinical testing. Attached Figure Description

[0015] Figure 1 Western blotting image of anti-Ki-67 rabbit monoclonal antibody as primary antibody; Figure 2 Microscopic observation of HeLa gastric cancer tissue and cervical cancer cells fixed in formalin and embedded in paraffin after immunohistochemical detection using anti-Ki-67 rabbit monoclonal antibody as primary antibody. The left side shows anti-Ki-67 rabbit monoclonal antibody, and the right side shows other commercially available antibodies. Figure 3 The results are from the thermally accelerated stability assay of Ki-67 rabbit monoclonal antibody, where A represents tonsils and B represents HeLa cells. Detailed Implementation

[0016] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: Antigen design, immunization, and specific single B-cell clone screening The full-length sequence of human Ki-67 protein was analyzed, and a short peptide with a specific spatial conformation containing the core hydrophilic region was synthesized using a solid-phase peptide synthesis method as an immunogen. The immunogen was mixed with Freund's complete adjuvant and subcutaneously immunized New Zealand white rabbits at multiple sites. After four booster immunizations, peripheral venous blood was collected from the rabbits, and serum titers were determined using ELISA. Rabbits with titers greater than 1:200,000 were selected. Rabbit spleens and peripheral blood mononuclear cells (PBMCs) were aseptically and painlessly removed. Using a fluorescently labeled Ki-67 antigen probe, single memory B cells specifically secreting anti-Ki-67 antibodies were precisely captured using fluorescence activated cell sorting (FACS) flow cytometry. These single B cells were seeded into 96-well plates for short-term co-culture. The culture supernatant was simultaneously subjected to micro-ELISA screening and FFPE tissue section IHC pre-screening to identify a strongly positive clonal cell line with an extremely clean background.

[0018] Example 2: Antibody gene cloning, sequencing, and vector construction Total RNA was extracted from single cells of the above-mentioned superior single B cells and reverse transcribed into cDNA using a reverse transcription kit. Using degenerate primers targeting the variable region of the rabbit antibody (heavy chain variable region primers, VH-F: CAGTCCCTGGAGGAAAGCGGCGGA, VH-R: GGAGCTCACGGTCACCAGTGTGCC; light chain variable region primers: VL-F: GCCTTGTGATGACCCAGACCCCAA, VL-R: CTTCACCACCACCTCGGTTCCGCCG), gene fragments of the antibody heavy chain variable region (VH) and light chain variable region (VL) were amplified by PCR. The correctly sequenced VH and VL nucleotide sequences (as shown in SEQ ID NO: 9 and 10, respectively) were codon-optimized and then inserted into eukaryotic expression vectors (such as pCDNA3.4) containing the rabbit constant regions (CH and CL), respectively, using molecular cloning techniques to construct complete heavy chain recombinant expression vectors and light chain recombinant expression vectors.

[0019] The nucleotide sequence of the heavy chain variable region (VH) is as follows: SEQ ID NO:9 CAGTCCCTGGAGGAAAGCGGCGGACGGCTGGTGAAGCCCGATGAAACTCTGACCCTGACCTGTACCGTGTCCGGCATCGATCTGAGCAGCAACACCATCTACTGGGTGAGGCAGGCCCCGGGCAACGGCCTGGAGTGGATCGGCTACATCGACAGCAGCGGCAATACCTATTAC GCCTCCTGGGCCAAGTCCAGAAGCACCATCACCAGAAATACCAACGAGAACACCGTGACACTGAAGATGACCTCTCTGACCGCCGCCGACACCGCCACCTACTTCTGCGCCCGGGAATTCTTCTCAGACAACTCCATCTGGGGCCCTGGCACACTGGTGACCGTGAGCTCCTAA.

[0020] The nucleotide sequence of the light chain variable region (VL) is as follows: SEQ ID NO:10 GCCATTGTGATGACCCAGACCCCAAGCTCTAAGAGCGTGCCCGTGGGCGACACCGTGACTATCAACTGCCAGGCCTCCGAGAGCGTGTACAACAACAACCTGGTGAGCTGGTTTCAGCAGAAACCCGGACAGCCACCCAAGCTGCTGATCTACAACGCTAGCACCAGGGC CTCTGGAGTGCCAAGCAGATTTAGCGGATCTGGCAGTGGCACCCAGTTCACCCTGACCATCAGCGACGTGGTGTGTGATGACGCCGCCTCTTACTACTGCGTGGGCTACACCGCCAACAAGGAGGACGGCGTGGCCTTTGGCGGCGGAACCGAGGTGGTGGTGAAGTGA.

[0021] Translating the base sequence into an amino acid sequence yields the amino acid sequences of the heavy chain variable region and the light chain variable region of the Ki-67 monoclonal antibody.

[0022] The amino acid sequence of the heavy chain variable region, SEQ ID NO:7, is as follows: QSLEESGGRLVKPDETLTLTCTVSGIDLSSNTIYWVRQAPGNGLEWIGYIDSSGNTYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAREFFSDNSIWGPGTLVTVSS.

[0023] The amino acid sequence of the light chain variable region, SEQ ID NO:8, is as follows: AIVMTQTPSSKSVPVGDTVTINCQASESVYNNNLVSWFQQKPGQPPKLLIYNASTRASGVPSRFSGSGSGTQFTLTISDVVCDDAASYYCVGYTANKEDGVAFGGGTEVVVK.

[0024] The amplified gene fragments were subjected to high-quality bidirectional sequencing. After sequence alignment and CDR region analysis, the amino acid sequences of the three complementarity-determining regions (HCDR1, HCDR2, HCDR3) of the heavy chain variable region were determined as shown in SEQ ID NO:1-3, and the amino acid sequences of the three complementarity-determining regions (LCDR1, LCDR2, LCDR3) of the light chain variable region were determined as shown in SEQ ID NO:4-6.

[0025] Heavy chain variable regions CDR: HCDR1: GIDLSSNTIY; HCDR2: YIDSSGNTYYASWAKS; HCDR3: EFFSDNSI.

[0026] Light chain variable region CDR: LCDR1: QASESVYNNNLVS; LCDR2: NASTRAS; LCDR3: VGYTANKEDGVA.

[0027] The complete amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0028] Example 3: Eukaryotic expression and purification of recombinant rabbit monoclonal antibody CHO-S (Chinese hamster ovary cells) cells in the logarithmic growth phase were cultured in suspension at a constant temperature, and the cell density was adjusted to 102. 6The constructed heavy chain expression vector and light chain expression vector were mixed at a 1:1 mass ratio and co-transfected into CHO-S cells using polyethyleneimine (PEI) transfection reagent. The cells were cultured continuously for 7 days at 37℃, 5% CO2, and 120 rpm shaking. On day 7, the cell suspension was collected, centrifuged at 4000 rpm for 30 minutes to remove cell debris, and the clear culture supernatant was collected. The supernatant was filtered through a 0.22 μm filter and loaded into a pre-equilibrated Protein A affinity chromatography column. Specific elution was performed using glycine buffer (pH 3.5), followed by neutralization, dialysis with PBS, and sterile filtration through a 0.22 μm filter to obtain highly purified anti-Ki-67 recombinant rabbit monoclonal antibody (clone number: LR089).

[0029] Experimental Results and Verification of Technical Effects Experiment 1: Specificity Analysis Experimental subjects: Human tumor cell lines (HeLa) that highly express Ki-67 were selected.

[0030] Experimental Methods: Total protein was extracted from cells and subjected to SDS-PAGE electrophoresis, then transferred to a PVDF membrane. The Ki-67 recombinant rabbit monoclonal antibody (LR089) prepared in this invention was diluted 1:2000 as the primary antibody and incubated at room temperature for 1 hour. After washing the membrane, HRP-labeled goat anti-rabbit IgG secondary antibody was added. The membrane was then visualized using DAB or ECL.

[0031] Experimental results: (e.g.) Figure 1 As shown, in the electrophoresis lanes of HeLa tumor cell lines, the Ki-67 recombinant rabbit monoclonal antibody (LR089) of this invention can accurately and exclusively recognize a specific single band located at approximately 370 kDa (which is basically consistent with the theoretical molecular weight of 378 kDa), with no other bands or non-specific bands, proving that the antibody has extremely high and single specificity.

[0032] Experiment 2: Precise determination of kinetic affinity (SPR method) The binding kinetics constants of the rabbit monoclonal antibody of this invention to the recombinant Ki-67 protein were determined using a Biacore T200 surface plasmon resonance spectrometer. The antibody was immobilized on the surface of a CM5 chip, and serially diluted Ki-67 protein was injected into the mobile phase.

[0033] Experimental results: According to the system software fitting, the binding rate constant of the anti-Ki-67 rabbit monoclonal antibody of this invention is 4.2 × 10⁻⁶. 5 M -1 ·s -1 Dissociation rate constant 1.5 × 10 -5 s -1 The final calculated affinity equilibrium dissociation constant (K) DThe value is 3.57 × 10 -11 M exhibits extremely strong position-locking guidance capability.

[0034] Experiment 3: Comparative Study of IHC in Clinical FFPE Tissue and ICC in Liquid-Based Cellular Data (Sensitivity and Specificity Detection) Experimental samples: Formalin-fixed paraffin-embedded clinical pathological tissue samples of gastric cancer; Liquid-based cytology slide of cervical cancer cells (HeLa).

[0035] Experimental Groups: Experimental group: Ki-67 recombinant rabbit monoclonal antibody (LR089) kit of this invention; Control group: Commercially available clinical-grade anti-Ki-67 rabbit monoclonal antibody (Clone SP6).

[0036] Test method: 1) Use the Laview-300E fully automated immunohistochemical staining machine and its matching secondary antibody system reagents.

[0037] 2) Heating: to melt the wax on the slices.

[0038] 3) Dewaxing: Place the glass slide into the dewaxing solution three times, 100% alcohol three times, and cleaning solution three times in sequence to complete the dewaxing.

[0039] 4) Antigen retrieval: After dewaxing, add antigen retrieval solution containing EDTA, incubate at 100°C for 20 minutes, and then cool to room temperature.

[0040] 5) Blocking: Add peroxide blocking solution to block endogenous peroxidase.

[0041] 6) Add primary antibody: Add primary antibody, incubate at room temperature for 30 minutes, and then perform three rounds of washing.

[0042] 7) After adding the primary antibody: Add the primary antibody and immunohistochemical reagent, and incubate at room temperature for 10 minutes. Then perform three rounds of washing.

[0043] 8) Add secondary antibody: Add the secondary antibody and immunohistochemical reagent, and incubate at room temperature for 10 minutes. Then perform three rounds of washing.

[0044] 9) Add DAB staining solution: Add freshly prepared DAB staining solution, incubate at room temperature for 10 minutes, and then perform three rounds of washing.

[0045] 10) Add counterstaining reagent: Add hematoxylin solution, incubate for 5 minutes, then rinse with washing solution to complete the reverse blueing process.

[0046] 11) Dehydration and clearing: Soak in 70%, 85%, 95%, 100%, and 100% graded alcohols sequentially for 3 minutes each time; clear with xylene twice for 5 minutes each time.

[0047] 12) Mounting: Mount the sample with neutral resin.

[0048] Experimental results: like Figure 2 Microscopic observation showed that the Ki-67 recombinant rabbit monoclonal antibody (LR089) exhibited significantly better signal intensity than the commercially available Ki-67 rabbit monoclonal antibody (Clone SP6) after IHC and ICC staining of gastric cancer tissue and cervical cancer cells HeLa, respectively. In addition to improving sensitivity, it also showed a lower background, thus providing a more reliable staining effect for diagnosis, especially for positive samples with low abundance of infection, which can avoid false negatives and other misdiagnoses.

[0049] Experiment 4: Accelerated aging stability test of primary antibody dilution formulation Formula A: The detection reagents include anti-Ki-67 rabbit monoclonal antibody, primary antibody post-response reagent, HRP enzyme-labeled secondary antibody, catalase blocking solution, DAB concentrate, DAB buffer and hematoxylin, using PBS buffer containing 1% BSA.

[0050] Formula B: Same as Formula A, except that the PBS buffer containing 1% BSA is replaced with a macromolecular stabilizer containing 0.5 wt% polyvinylpyrrolidone (PVP).

[0051] Both groups of reagents were placed in a 37°C constant temperature chamber for accelerated aging and degradation tests. Samples were taken in the second week for IHC / ICC colorimetric titer determination.

[0052] Experimental results: such as Figure 3 China A and Figure 3 As shown in B.

[0053] Formula A: After being placed at 37°C for 2 weeks, its IHC / ICC color development was significantly weakened and background staining appeared. This was because the rabbit monoclonal antibody underwent charge association and aggregation, which led to a significant drop in potency and the appearance of background staining to varying degrees.

[0054] Formulation B: After being stored at 37°C for two weeks, the IHC / ICC staining intensity remained essentially the same as on day 0, and the signal-to-noise ratio did not change significantly, both meeting clinical diagnostic requirements. This demonstrates that the kit formulation of this invention, combined with this specific antibody structure, possesses excellent shelf-life stability.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against Ki-67 rabbits, characterized in that: It includes a heavy chain variable region and a light chain variable region. The complementary determinant region (CDR) sequences of the heavy chain variable region are HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:

3. The complementary determinant region (CDR) sequences of the light chain variable region are LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:

6.

2. The anti-Ki-67 rabbit monoclonal antibody according to claim 1, characterized in that: The full-length amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the full-length amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The anti-Ki-67 rabbit monoclonal antibody according to claim 2, characterized in that: The DNA sequence encoding the heavy chain variable region is the nucleotide sequence shown in SEQ ID NO:9, and the DNA sequence encoding the light chain variable region is the nucleotide sequence shown in SEQ ID NO:

10.

4. The use of any one of the anti-Ki-67 rabbit monoclonal antibodies according to claims 1-3 in immunohistochemical (IHC) staining and immunocytochemical (ICC) staining reagents.

5. The use of any one of the anti-Ki-67 rabbit monoclonal antibodies according to claims 1-3 in the preparation of a Ki-67 detection kit, characterized in that: The kit includes anti-Ki-67 rabbit monoclonal antibody, primary antibody post-antibody reagent, HRP-labeled secondary antibody, catalase blocking solution, DAB concentrate, DAB buffer, and hematoxylin.

6. The application according to claim 5, characterized in that: The kit also includes a primary antibody dilution buffer containing a non-protein polymeric stabilizer and free of animal-derived serum albumin; the working dilution ratio of the anti-Ki-67 rabbit monoclonal antibody in the primary antibody dilution buffer is 1:1000 to 1:5000.

Images