Rabbit monoclonal antibodies against mouse immunoglobulin G3 subtype (IgG3) and their applications

Abstract

This invention relates to the field of biotechnology and discloses a rabbit-specific monoclonal antibody OTIR8B8 against mouse immunoglobulin G3 subtype (IgG3). The antibody is produced by sorting and culturing specific B cells and then performing molecular clonal recombination. The immunogen of the rabbit monoclonal antibody OTIR8B8 is a natural mouse IgG3 (mIgG3) antibody. The amino acid sequence of the OTIR8B8 antibody light chain (VL) is shown in SEQ ID NO. 1; the amino acid sequence of the OTIR8B8 antibody heavy chain (VH) is shown in SEQ ID NO. 2. The VL region of the OTIR8B8 antibody includes three antigenic determinants: CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO. 3-5, respectively. The VH region of the OTIR8B8 antibody also includes three antigenic determinants: CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO. 6-8, respectively. This invention also relates to the application of rabbit monoclonal antibodies specific to mouse immunoglobulin G3 (IgG3) in immunoassay tools, including but not limited to chemiluminescence, fluorescence, and colorimetric detection for primary antibodies, suitable for various applications such as cell imaging, flow cytometry, Western blotting, and immunohistochemistry, and also provides a foundation for the preparation of engineered antibodies in the next step.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to rabbit monoclonal antibodies against mouse immunoglobulin G3 subtype (IgG3) and their applications in immune detection. Background Technology

[0002] Affinity conjugates / antibodies are among the most commonly used tools in life sciences, used to study proteins and their functions in various biological pathways and diseases. Currently, the most widely used antibody species in scientific research and medicine include murine antibodies, human antibodies, and rabbit antibodies. These antibodies can specifically recognize targets of interest, and then the signal is displayed using enzyme-labeled secondary antibodies or fluorescein-labeled secondary antibodies. Anti-mouse IgG and anti-human IgG labeled antibodies are currently widely used in the market and can be applied to enzyme-linked immunosorbent assays (ELISA), Western blotting, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation. The most commonly used labeling methods include horseradish peroxidase (HRP), alkaline phosphatase, biotin, and fluorescein.

[0003] Currently, the main method for preparing anti-mouse IgG antibodies is to directly immunize animals such as rabbits, sheep, or donkeys with IgG immunoglobulin to produce immunosweet polyclonal antibodies, which are then purified and used. However, polyclonal antibodies are far from sufficient for developing specific antibodies against different types of IgG heavy and light chains; monoclonal antibodies, especially monoclonal antibodies targeting different subtypes of mIgG, are needed.

[0004] Mouse IgG monoclonal antibody heavy chains include five subtypes: IgG1, IgG2a, IgG2b, IgG2c, and IgG3. Light chains include two types: κ chains and λ chains, with a κ chain:λ chain ratio of approximately 20:1. Currently, most antibodies on the market are polyclonal, such as goat polyclonal antibodies, sheep polyclonal antibodies, and donkey polyclonal antibodies; monoclonal antibodies are rare, especially rabbit anti-mIgG3 specific monoclonal antibodies.

[0005] In developing the anti-mIgG3 antibody, we encountered many difficulties in obtaining a monoclonal antibody that specifically recognizes mIgG3 and does not cross-react with mIgG1, mIgG2a, mIgG2b, mIgG2c, human IgG1 (hIgG1), human IgG2 (hIgG2), human IgG3 (hIgG3), human IgG4 (hIgG4), RatIgG, and Rabbit IgG (RabIgG). During this period, we underwent multiple sorting optimizations and extensive screenings, ultimately obtaining the rabbit monoclonal antibody OTIR8B8, as claimed in this invention.

[0006] Antibodies that specifically recognize the anti-mIgG3 subtype, including naked and labeled antibodies, have a wide market application and can be used in enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation. However, the core of these applications is the use of monoclonal antibodies that specifically bind to mIgG3, and their performance directly determines the sensitivity and specificity of the entire detection. Therefore, finding highly specific and sensitive anti-mIgG3 antibodies is crucial for establishing methods for detecting mIgG3. A search of patent websites in China, the United States, EPO, and WIPO, using "mIgG3" or "mIgG3+monoclonal antibody" or "antibody" as keywords, yielded no patents related to monoclonal antibodies with high affinity and specific binding to mIgG3. No immunoassay kits using the rabbit monoclonal antibody OTIR8B8 to detect mIgG3 were also found. Using "mIgG3" or "mIgG3+monoclonal antibody" or "antibody" as keywords, no similar literature related to the content of this invention was found on PubMed. A search of the National Medical Products Administration website revealed that no testing reagents related to mIgG3 have been registered. Summary of the Invention

[0007] The purpose of this invention is to provide the anti-mIgG3 rabbit monoclonal antibody OTIR8B8 with high specificity and affinity and its application in immunoassay tools, including but not limited to chemiluminescence, fluorescence and colorimetric detection for primary antibodies, suitable for various applications such as cell imaging, flow cytometry, protein immunoblotting and immunohistochemistry, and also to provide a foundation for the preparation of engineered antibodies in the next step.

[0008] The rabbit monoclonal antibody is rabbit monoclonal antibody OTIR8B8.

[0009] The rabbit monoclonal antibody OTIR8B8 uses natural mIgG3 antibody as an immunogen. It is obtained by immunizing New Zealand white rabbits, collecting peripheral blood mononuclear cells (PMBCs) from the immunized rabbits, sorting and culturing specific B cells, screening, and using molecular clonal recombination technology.

[0010] The rabbit monoclonal antibody OTIR8B8 has a light chain variable region (VL) containing 108 amino acids, and its amino acid sequence is shown in SEQ ID NO. 1; the heavy chain (VH) of the antibody contains 109 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2.

[0011] The rabbit monoclonal antibody OTIR8B8 comprises three antigenic determinants in its VL region: CDR1, CDR2, and CDR3, with corresponding regions of 27aa-34aa, 52aa-54aa, and 91aa-98aa, respectively. Its amino acid sequences are shown in SEQ ID No. 3-5.

[0012] The rabbit monoclonal antibody OTIR8B8 comprises three antigenic determinants in its VH region: CDR1, CDR2, and CDR3, with corresponding regions of 25aa-32aa, 50aa-56aa, and 93aa-98aa, respectively. Its amino acid residue sequences are shown in SEQ ID No. 6-8.

[0013] The rabbit monoclonal antibody OTIR8B8 described above can bind with high specificity to mIgG3 and can be used as a primary antibody or various labeled secondary antibodies by methods known to those skilled in the art. In particular, it can be used as an enzyme-labeled secondary antibody or a fluorescein-labeled secondary antibody for immunohistochemical detection, Western blotting detection, and flow cytometry detection. Attached Figure Description

[0014] Figure 1 Electrophoresis diagram of the full-length amplification products of the heavy and light chains of rabbit monoclonal antibody OTIR8B8, where M is the DNA molecular weight marker;

[0015] Figure 2 Figure 1. Western blot results of rabbit monoclonal antibody OTIR8B8 specifically recognizing natural mIgG3 antibody;

[0016] Figure 3 Figure 1 shows the results of linear ELISA detection of rabbit monoclonal antibody OTIR8B8 specifically recognizing mIgG3 antibody.

[0017] Figure 4 The image shows the ELISA results of cross-reactivity detection of rabbit monoclonal antibody OTIR8B8 with IgG from other species.

[0018] Figure 5 Western blot results of horseradish peroxidase-labeled rabbit monoclonal antibody OTIR8B8 specifically recognizing natural mIgG3 antibody;

[0019] Figure 6 The image shows the ELISA results of horseradish peroxidase-labeled rabbit monoclonal antibody OTIR8B8 specifically recognizing mIgG3 antibody. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0021] Example 1: Preparation of mIgG3 rabbit monoclonal antibody

[0022] I. Preparation of mIgG3 Immunogen

[0023] Purchase Rockland mIgG3 antibody as an immunogen

[0024] II. Animal Immunization

[0025] The purchased mIgG3 antibodies were emulsified with complete Freund's adjuvant and administered subcutaneously to approximately 2 kg New Zealand White rabbits 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. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. The OD450 at an ELISA titer of 128,000 was considered to be greater than 1.0. Based on the results, it was determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers were selected for PBMC collection.

[0026] III. PBMC Isolation, Specific B Cell Sorting, and Clonal Recombination: Rabbits were fixed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. A 50ml syringe was used to puncture the area with the strongest heartbeat. Blood flowed into the syringe immediately after the needle pierced 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 to 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 (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 further sorted from the rabbit PBMCs for culture. Positive clones were screened using antigen-coated ELISA plates using the cultured B cell supernatant. Cells from positive clones were collected, lysed, and RNA was extracted 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. A rabbit monoclonal antibody expression vector was constructed using clonal recombination, and the sequence was confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0027] IV. Preparation and Purification of Monoclonal Antibodies To obtain rabbit monoclonal antibodies recognizing human mIgG3 protein, this invention loads the heavy and light chain genes of rabbit monoclonal antibodies into an expression vector, and transfects the plasmid into HEK293 cells. After 120-144 hours of transfection, the culture supernatant contains recombinant rabbit monoclonal antibodies recognizing human mIgG3 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, and then aliquoted and lyophilized.

[0028] Example 2: Identification of anti-mIgG3 rabbit monoclonal antibody OTIR8B8

[0029] I. Western blot identification of rabbit monoclonal antibody OTIR8B8

[0030] Western blot (WB) was used for detection. Two different samples were selected for each subtype of mIgG1, mIgG2a, mIgG2b, and mIgG3. 100 ng of each protein was loaded for SDS-PAGE, and then transferred to a membrane for WB detection.

[0031] The results showed that the rabbit monoclonal antibody OTIR8B8 specifically recognized mIgG3, but not mIgG1, mIgG2a, or mIgG2b. The antibody dilution ratio was 1:5000-10000, 1 mg / ml. See attached results. Figure 2 .

[0032] II. ELISA Identification of Rabbit Monoclonal Antibody OTIR8B8

[0033] Antibodies against mIgG1, mIgG2a, mIgG2b, mIgG3, mIgG3 Fc, and mIgG3 F(ab')2 were coated onto ELISA plates and incubated overnight at 4°C. The next day, the plates were removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. 100 μl of rabbit monoclonal antibody OTIR8B8 was added to each well at concentrations of 10, 3, 1, 0.3, and 0 ng / mL, and incubated at 37°C for 1 hour. After incubation, the plates were removed, washed three times with PBST, and HRP-labeled goat anti-rabbit secondary antibody was added as the detection antibody. The plates were incubated at 37°C for 1 hour. After incubation, the plates were removed, washed five times with PBST, and TMB substrate was added. The plates were then developed at 37°C for 10 minutes. Stop solution was added, and the OD450 reading was measured using a microplate reader. Cross-recognition between rabbit monoclonal antibody OTIR8B8 and other species IgGs was detected using the same method.

[0034] The results showed that the rabbit monoclonal antibody OTIR8B8 specifically recognized mIgG3, specifically the Fc fragment, but not mIgG1, mIgG2a, or mIgG2b, with a titer reaching 1.5*10.7 The results are shown Figure 3 . Figure 4 The results indicate that the rabbit monoclonal antibody OTIR8B8 can specifically recognize mIgG3 and does not cross-recognize with IgG from other species, including human IgG (hIgG1, hIgG2, hIgG3, hIgG4), RatIgG, and RabIgG.

[0035] Example 3: Analysis of the variable region gene and amino acid sequence of rabbit monoclonal antibody OTIR8B8

[0036] Using the recombinant plasmid of the OTIR8B8 antibody as a DNA template, sequencing primers for the light chain variable region and heavy chain variable region were designed based on the vector sequences at the 5' ends of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequences of the light and heavy chain variable regions of the rabbit monoclonal antibody OTIR8B8 were obtained by sequencing.

[0037] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light and heavy chains were sequenced and analyzed. The light chain amino acid sequence of the rabbit monoclonal antibody OTIR8B8 is shown in SEQ ID NO.1, and the heavy chain amino acid sequence is shown in SEQ ID NO.2. The full length of VL is 108 amino acids. The number of amino acids in the four domains of its FR are 26, 17, 36, and 10, respectively. The number of amino acids in the three domains of CDR are 8, 3, and 8, respectively. The regions of CDR1, CDR2, and CDR3 are 27aa-34aa, 52aa-54aa, and 91aa-98aa, respectively, and their amino acid sequences are QSVYNNNW, KAS, and QGTYSGGT, respectively.

[0038] Analysis revealed that the rabbit monoclonal antibody OTIR8B8 VH has a full length of 109 amino acids. The number of amino acids in the four domains of its FR is 24, 17, 36 and 11, respectively. The number of amino acids in the three domains of its CDR is 8, 7 and 6, respectively. CDR1, CDR2 and CDR3 are 25aa-32aa, 50aa-56aa and 93aa-98aa, respectively. Their amino acid sequences are GFSLSSYA, INAGNRP and SRGDNL, respectively.

[0039] Example 4: Identification of HRP-labeled rabbit monoclonal antibody OTIR8B8 as a secondary antibody

[0040] I. HRP-labeled rabbit monoclonal antibody OTIR8B8:

[0041] 1. Rabbit monoclonal antibody OTIR8B8 was dissolved in sodium bicarbonate solution at pH 9.6;

[0042] 2. Dissolve a certain mass of HRP in deionized water, add sodium periodate and react for 30 min, add ethylene glycol and continue reacting for 30 min, then dialyze overnight;

[0043] 3. Weigh a certain amount of sodium borohydride and dissolve it in deionized water. Add sodium borohydride to the cross-linked antibody-HRP solution, react for 2 hours, and dialyze overnight.

[0044] 4. Add an equal amount of glycerol to the obtained HRP-labeled antibody and store at -20 degrees Celsius.

[0045] II. Identification:

[0046] 1. Western blot identification of HRP-labeled rabbit monoclonal antibody OTIR8B8

[0047] Western blot (WB) was used for detection. 100 ng of mIgG3 antibody was subjected to SDS-PAGE, transferred to a membrane, and then incubated with HRP-labeled rabbit monoclonal antibody OTIR8B8 for WB detection.

[0048] The results showed that the HRP-labeled rabbit monoclonal antibody OTIR8B8 could effectively recognize the mIgG3 Fc region, with a molecular weight of approximately 50 kDa. The antibody dilution ratio was 1:5000-10000, 1 mg / ml. See the results below. Figure 5 .

[0049] III. Identification of Rabbit Monoclonal Antibody OTIR8B8 by ELISA

[0050] Goat anti-mouse IgG was coated onto an ELISA plate and incubated overnight at 4°C. The next day, the plate was removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. 100 μL of mIgG3 antibody (0.2 μg / mL) was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was removed, washed three times with PBST, and HRP-labeled OTIR8B8 was added as the detection antibody, serially diluted seven times starting at 1 μg / mL, and incubated at 37°C for 1 hour. After incubation, the plate was removed, washed five times with PBST, and TMB substrate was added, with color development at 37°C for 10 minutes. Finally, stop solution was added, and the OD450 reading was measured using an ELISA reader.

[0051] The results showed that HRP-labeled rabbit anti-mIgG3 OTIR8B8, as a secondary antibody, could effectively detect mIgG3 antibody signals, achieving a dilution ratio of 36K-18K at 1 mg / ml. This was superior to commercially available goat anti-mouse IgG polyclonal antibody (which, as a secondary antibody, can simultaneously detect mIgG1, mIgG2a, mIgG2b, and mIgG3). See attached figures. Figure 6 . sequence list <110> Wuxi Aorui Dongyuan Biotechnology Co., Ltd. <120> Rabbit monoclonal antibodies against mouse immunoglobulin G3 subtype (IgG3) and their applications <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 108 <212> PRT <213> Oryctolagus cuniculus <400> 1 Ala Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn 20 25 30 Asn Trp Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Arg Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Thr Tyr Ser Gly 85 90 95 Gly Thr Phe Gly Gly Gly Thr Glu Val Val Val Glu 100 105 <210> 2 <211> 109 <212> PRT <213> Oryctolagus cuniculus <400> 2 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 Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Phe Ile Asn Ala Gly Asn Arg Pro 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 Ser Arg Gly Asp 85 90 95 Asn Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 <210> 3 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 3 Gln Ser Val Tyr Asn Asn Asn Trp 1 5 <210> 4 <211> 3 <212> PRT <213> Oryctolagus cuniculus <400> 4 Lys Ala Ser 1 <210> 5 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 5 Gln Gly Thr Tyr Ser Gly Gly Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 6 Gly Phe Ser Leu Ser Ser Tyr Ala 1 5 <210> 7 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 7 Ile Asn Ala Gly Asn Arg Pro 1 5 <210> 8 <211> 6 <212> PRT <213> Oryctolagus cuniculus <400> 8 Ser Arg Gly Asp Asn Leu 1 5

Claims

1. A specific rabbit monoclonal antibody against mouse immunoglobulin G3 subtype (IgG3), characterized in that, The specific rabbit monoclonal antibody is rabbit monoclonal antibody OTIR8B8, which includes a light chain variable region and a heavy chain variable region. The light chain variable region includes three antigenic determinants: CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID No. 3-5, respectively. The heavy chain variable region includes three antigenic determinants: CDR1, CDR2, and CDR3, and the amino acid residue sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID No. 6-8, respectively.

2. The specific rabbit monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

2.

3. The application of the specific rabbit monoclonal antibody against mouse immunoglobulin G3 subtype (IgG3) as described in any one of claims 1-2 in the preparation of an immunoassay tool for mouse immunoglobulin G3 subtype (IgG3), characterized in that, The immunoassay tools are used for chemiluminescence, fluorescence and colorimetric detection of primary antibodies, cell imaging, flow cytometry, Western blotting of proteins, and immunohistochemistry.

Images