Preparation of rabbit monoclonal antibody of anti-human integrase interaction molecule 1 (INI1) and application thereof

Abstract

The application relates to the field of biotechnology, and discloses a rabbit anti-human integrase interacting molecule 1 (INI1) rabbit monoclonal antibody OTIR4G9, and amino acid sequences of antibody light chain variable regions (VL) and heavy chain variable regions (VH) are shown in SEQ ID NO. 4 and SEQ ID NO. 5. The application also provides a preparation method of the anti-INI1 rabbit monoclonal antibody, wherein the antibody is obtained by screening specific B cells after immunizing a New Zealand white rabbit with an INI1 polypeptide and then being produced by molecular cloning and recombination. The application provides an anti-INI1 rabbit monoclonal antibody which can be used in an immunodetection tool for detecting an INI1 protein, and the tool comprises an immunohistochemical detection kit and other labeled tissue cell kits, test papers and the like. The monoclonal antibody has high specificity and sensitivity, can specifically combine with the INI1 protein, and significantly improves the specificity and reliability of INI1 protein immunodetection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method and application of a rabbit monoclonal antibody against integrase interacting molecule 1 (INI1) specifically binding to integrase interacting molecule 1 for immunoassay. Background Technology

[0002] The INI-1 gene, located at 22q11.2 and also known as hSNF5, SMARCB1, and BAF47, is a core subunit of the SWI / SNF complex. It plays a role in ATP-dependent chromatin remodeling, thereby regulating gene expression and being associated with various cellular functions, including repairing damaged DNA and regulating cell growth. In 1998, using gene localization methods, it was first clearly demonstrated in malignant rhabdomyosarcoma that the loss of INI1 protein expression due to bilateral allele inactivation was a characteristic alteration of MRT. SMARCA4 (BRG1), S MARCA2 (BRM), and SMARCB1 (INI1) belong to the chromatin remodeling complex family. Mutations or deletions of these genes lead to gene inactivation, which is reflected in the loss of corresponding protein expression by immunohistochemical detection. These protein loss can occur alone or in combination in different tumors. It is commonly seen in highly aggressive, poorly prognostic malignant tumors, and histologically, the tumor cells often exhibit rhabdomyosarcoma-like cell characteristics. The most common cause of INI1 expression loss is malignant rhabdoid tumors and epithelioid sarcomas. Secondly, INI1 expression loss can also be seen in some epithelioid malignant peripheral nerve sheath tumors, malignant pigmented nerve sheath tumors, epithelioid nerve sheath tumors, poorly differentiated chordomas, and a few extraosseous myxoid chondrosarcomas and soft tissue myoepithelial tumors.

[0003] Current research indicates that INI1 expression patterns differ across various tumors. Abnormal INI1 protein expression can be categorized into three modes: complete loss, mosaic expression, and decreased expression. In complete loss, INI1 protein expression is negative in tumor cells, but positive in internal controls such as stromal cells and vascular endothelial cells. Currently, INI1 monoclonal antibodies are commonly used in pathological diagnosis for malignant rhabdomyosarcoma and related diseases.

[0004] Immunohistochemical pathology, based on the protein level, can further determine the tissue origin, primary site, pathological type, and residual marginal cancer cells of a tumor. Besides its diagnostic role, it also guides prognosis. At its core, immunohistochemical pathology requires highly specific and sensitive antibodies, especially monoclonal antibodies. With the deepening research on the disease and the INI1 protein, the application scope of anti-INI1 monoclonal antibodies is constantly expanding. Simultaneously, with the widespread use of automated instruments, the differences in detection conditions across various testing platforms, and the rapid development of pathological AI, the requirements for antibody quality are becoming increasingly stringent. However, there is currently a lack of high-quality anti-INI1 monoclonal antibodies; therefore, the development of highly specific, highly sensitive anti-INI1 monoclonal antibodies suitable for different detection platforms is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a rabbit monoclonal antibody that specifically binds to the INI1 protein, and its application in the preparation of an immunoassay tool for detecting the INI1 protein.

[0006] The present invention provides a rabbit monoclonal antibody against INI1, namely OTIR4G9. The antibody light chain variable region (VL) contains 108 amino acids, and its sequence is shown in SEQ ID NO.4; the heavy chain variable region (VH) contains 116 amino acids, and its sequence is shown in SEQ ID NO.5.

[0007] The rabbit monoclonal antibody specifically recognizes the INI1 protein.

[0008] The inventors provide a method for preparing the antibody, which involves synthesizing three polypeptides and conjugating them to the carrier protein KLH as immunogens for use in immunizing New Zealand white rabbits. Peripheral blood mononuclear cells (PBMCs) are obtained from the immunized animals, and specific B cells are sorted, then molecularly cloned and transfected into mammalian cells using a recombinant vector. Cells are cultured to obtain a supernatant containing secreted antibodies, which is then purified by affinity chromatography to obtain a rabbit monoclonal antibody against INI1.

[0009] This invention also provides the application of anti-INI1 rabbit monoclonal antibody in the preparation of immunoassay tools for detecting INI1 protein. The immunoassay tools may include reagent kits, chips, or test strips, etc.

[0010] This invention also provides an immunohistochemical detection kit, comprising an anti-INI1 rabbit monoclonal antibody, capable of detecting the expression status of INI1 protein in tissue cells. The tissues include, but are not limited to, normal tissues: tonsils, thyroid gland, spleen, and stomach; tumor tissues: lung cancer, breast cancer, thyroid tumors, lymphoma, and tumor tissues lacking INI1 expression. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0012] Figure 1 IHC results of anti-INI1 rabbit monoclonal antibody OTIR4G9 in normal tissues.

[0013] Figure 2 IHC results of anti-INI1 rabbit monoclonal antibody OTIR4G9 on INI1-positive tumor tissue.

[0014] Figure 3 IHC results of the anti-INI1 rabbit monoclonal antibody OTIR4G9 on INI1-deficient tumor tissue. Detailed Implementation

[0015] This invention discloses a method for preparing anti-INI1 rabbit monoclonal antibodies, a method for immunoassay, and their applications. The technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0016] Example 1: Preparation of anti-INI1 rabbit monoclonal antibody

[0017] 1. Preparation of antigens

[0018] The INI1 protein sequence NP_003064.2, containing 385 amino acids, was obtained from the NCBI website and used as the standard sequence. Amino acid sequences at positions 114-131, 246-262, and 357-379 were selected for synthesis and conjugated to the carrier protein KLH as immunogens.

[0019] 2. Animal immunization

[0020] The synthesized INI1 peptide was mixed in a 1:1:1 ratio and emulsified with complete Freund's adjuvant. New Zealand white rabbits weighing approximately 2 kg were immunized subcutaneously at a dose of 500 μg per rabbit. A second immunization was performed two weeks later, using incomplete Freund's adjuvant emulsification at a dose of 250 μg per rabbit. After three immunizations, tail blood was collected and serum titers were determined using serial dilutions with ELISA and IHC. The criteria for determining whether to collect PBMCs or continue immunization were based on the OD450 greater than 1.0 at an ELISA titer of 128000 and the intensity of nuclear staining signal on tissues detected by IHC.

[0021] 3. PBMC isolation, specific B cell sorting, and clonal recombination

[0022] New Zealand white rabbits whose serum titers met the standards after immunization were fixed supine on the operating table. The fur around the heart was shaved, and the skin was disinfected with alcohol swabs. A 50ml syringe was used to puncture the heart at the site of strongest heartbeat. Blood flowed into the syringe immediately after the needle entered the heart. The needle was quickly withdrawn, and the whole blood was transferred to a sterile 50ml tube. This was mixed with an equal volume of PBS and slowly added dropwise onto the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., a mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, yielding rabbit PBMCs. Antigen-specific B cells were then sorted and cultured, and positive clones were screened using antigen-coated ELISA plates. Positive clones were lysed and the lysate was collected. RNA was extracted from the lysate and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. Rabbit monoclonal antibody expression vectors were constructed using clonal recombination methods, and the sequences were determined by sequencing.

[0023] 4. Preparation and purification of monoclonal antibodies

[0024] To obtain a rabbit monoclonal antibody that specifically recognizes the human INI1 protein, this invention loads the heavy and light chain genes of the rabbit monoclonal antibody into an expression vector, and transfects the recombinant plasmid into HEK293 cells. After 120-144 hours of transfection, the culture supernatant contains the recombinant rabbit monoclonal antibody recognizing the human INI1 protein. The cell suspension is collected, the supernatant is obtained by centrifugation, and the antibody is purified by affinity chromatography. The concentration of the purified monoclonal antibody is determined by the BCA method.

[0025] Example 2: Analysis of the variable region gene and amino acid sequence of rabbit monoclonal antibody OTIR4G9

[0026] Using the recombinant plasmid of the OTIR4G9 antibody as a DNA template, sequencing primers for the light and heavy chain variable regions were designed based on the 5' vector sequences of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody OTIR4G9 was obtained by sequencing. The sequencing results of the light and heavy chains were analyzed using IMGT / V-QUEST analysis software at http: / / www.imgt.org. The amino acid sequence of the light chain of the rabbit monoclonal antibody OTIR4G9 is shown in SEQ ID NO.4, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.5. The light chain variable region (VL) is 108 amino acids long. Its four FR domains have 26, 17, 36, and 10 amino acids respectively, and its three CDR domains have 8, 3, and 8 amino acids respectively. The regions of CDR1, CDR2, and CDR3 are 27-34 aa, 52-54 aa, and 91-98 aa respectively, with amino acid sequences of: QSVYNNND; QAS; LGGYDDDV. The heavy chain variable region (VH) contains 116 amino acids. Its four FR domains have 24, 17, 36, and 11 amino acids respectively, and its three CDR domains have 8, 7, and 13 amino acids respectively, corresponding to amino acid regions of 25-32 aa, 50-56 aa, and 93-105 aa, with sequences of: GFSLSSYY; SSSSGST; ARVEDDDYGDTYA.

[0027] Example 3: IHC detection of rabbit monoclonal antibody OTIR4G9

[0028] 1. Experimental Methods

[0029] (1) Take each tissue block fixed in formalin and embed it in paraffin. Use a Leica tissue slicer to slice the tissue with a thickness of 4 μm.

[0030] (2) Dewaxing and hydration: analytical grade xylene for 10 min × 3 times, anhydrous ethanol for 10 min × 3 times, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and deionized water for 3 min × 3 times.

[0031] (3) Add antigen retrieval solution [1mM EDTA, 10mM Tris buffer (pH 8.0)] and autoclave for 2.5 min. When the autoclave temperature drops to about 90℃, open the autoclave, remove the specimen, and then allow it to cool naturally to room temperature. Immerse in deionized water for 3 min × 3 times.

[0032] (4) Inactivate endogenous peroxidase in tissues with 3% hydrogen peroxide and let stand at room temperature for 10 min. Soak in deionized water for 5 min × 3 times.

[0033] (5) Add anti-INI rabbit monoclonal antibody OTIR4G9 (0.51 μg / ml) and place in a humidified chamber. Incubate at 37°C for 60 min. Wash with PBST (0.1% Tween-20) for 5 min × 3 times.

[0034] (6) Add ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer (Catalog No. PV-8000) and incubate at 37°C for 30 min. Wash with PBS for 5 min × 3 times.

[0035] (7) Develop color with DAB solution for 5 minutes. Wash with distilled water.

[0036] (8) Counterstain cell nuclei with hematoxylin for 2 min, rinse with distilled water, and differentiate with 1% hydrochloric acid. Rinse 3 times with distilled water and let stand at room temperature for 1 min.

[0037] (9) Dehydration and clearing: 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 3×5 min; xylene for 3×5 min, neutral resin for mounting.

[0038] (10) Microscopic examination.

[0039] 2. Experimental Results:

[0040] The rabbit monoclonal antibody OTIR4G9 against INI1 was used as the primary antibody to detect INI1 expression in normal tissues, tumor tissues with INI1 expression deficiency, and other tumor tissues using the IHC method. The results are as follows:

[0041] (1) IHC results in normal tissues such as tonsils, thyroid gland, spleen, and stomach are as follows: Figure 1 As shown, the results indicate that various cells in these four tissues exhibited varying degrees of nuclear staining, meaning that INI1 was expressed in different cell types across different tissues. This is consistent with previous reports.

[0042] (2) Results of staining in INI1-positive lung cancer, breast cancer, lymphoma, and thyroid tumors, as shown in... Figure 2 As shown, the results indicate that INI1 is expressed in both tumor cells and stromal cells. In the same sample, the positive signal on tumor cells is stronger than that on stromal cells, and the cells with active proliferation have stronger expression, which is consistent with the function of INI1.

[0043] (3) Staining results in tumor tissues lacking INI1 expression are as follows: Figure 3 The left image shows the result under low magnification, and the right image is a magnified view within the box in the left image. The results show that the tumor cells are unstained and negative, while the arrow indicates that the nuclei of the vascular endothelial cells used as internal controls are positive.

[0044] In summary, the independently developed rabbit monoclonal antibody OTIR4G9 against INI1 protein showed nuclear positivity in various normal and tumor tissues according to IHC detection, and negative on tumor cells in some tumor tissues lacking expression. Meanwhile, it showed positivity in vascular endothelial and stromal cells used as internal controls. This indicates that the anti-INI1 protein monoclonal antibody OTIR4G9 has good specificity, and the antibody concentration of only 0.51 μg / ml already showed good staining effect, indicating that this clone has high sensitivity and can fully meet the needs of pathologists for anti-INI1 monoclonal antibodies. sequence list <110> Wuxi Aorui Dongyuan Biotechnology Co., Ltd. <120> Preparation and Uses of Rabbit Monoclonal Antibody Against Human Integrase Interacting Molecule 1 (INI1) <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> PRT <213> Homo sapiens <400> 1 Thr Glu Pro Pro Thr Tyr Leu Arg Glu Gln Lys Ala Lys Arg Asn Ser 1 5 10 15 Gln Trp <210> 2 <211> 17 <212> PRT <213> Homo sapiens <400> 2 Glu Ser Tyr Pro Thr Asp Ser Ile Leu Glu Asp Gln Ser Asp Gln Arg 1 5 10 15 Val <210> 3 <211> twenty three <212> PRT <213> Homo sapiens <400> 3 Thr Asp Ala Glu Met Glu Lys Lys Ile Arg Asp Gln Asp Arg Asn Thr 1 5 10 15 Arg Arg Met Arg Arg Leu Ala 20 <210> 4 <211> 108 <212> PRT <213> Oryctolagus cuniculus <400> 4 Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn 20 25 30 Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 Leu Ile Tyr Gln Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Ala Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp 85 90 95 Asp Val Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 <210> 5 <211> 116 <212> PRT <213> Oryctolagus cuniculus <400> 5 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Tyr 20 25 30 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ser Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val Glu 85 90 95 Asp Asp Asp Tyr Gly Asp Thr Tyr Ala Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115

Claims

1. A rabbit monoclonal antibody against INI1, characterized in that, The anti-INI1 rabbit monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

5. The light chain variable region contains 108 amino acids and three antigenic determinants: CDR1, CDR2, and CDR3, with regions of 27-34 aa, 52-54 aa, and 91-98 aa, respectively. The heavy chain variable region contains 116 amino acids and three antigenic determinants: CDR1, CDR2, and CDR3, with regions of 25-32 aa, 50-56 aa, and 93-105 aa, respectively.

2. The anti-INI1 rabbit monoclonal antibody according to claim 1, characterized in that, The anti-INI1 rabbit monoclonal antibody specifically recognizes the INI1 protein.

3. The use of the anti-INI1 rabbit monoclonal antibody according to claim 1 in the preparation of an immunoassay tool for detecting INI1 protein.

4. The application according to claim 3, characterized in that, The immune detection tools include reagent kits, chips, or test strips.

5. The use of the anti-INI1 rabbit monoclonal antibody according to claim 1 in the preparation of a kit for labeling tissue cells and tumor cells, characterized in that, The tissue is the tonsils, thyroid gland, spleen, or stomach; the tumor is lung cancer, breast cancer, thyroid tumor, or lymphoma.

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