Rabbit monoclonal antibody mAb8 against murine immunoglobulin G2b subtype (IgG2b) and uses thereof

Abstract

The application belongs to the technical field of immune detection, and discloses a specific monoclonal antibody mAb8 of rabbit anti-mouse immunoglobulin G2b subtype (IgG2b), which comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1-3, the LCDR1 comprises an amino acid sequence QGVYNVDY, the LCDR2 comprises an amino acid sequence RTS, and the LCDR3 comprises an amino acid sequence LGSYDD; the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises an amino acid sequence GFTISNYD, the HCDR2 comprises an amino acid sequence ISNNGN, and the HCDR3 comprises an amino acid sequence AREISG. The monoclonal antibody can be combined with the mouse immunoglobulin G2b subtype (IgG2b) with high specificity, can be applied to immune detection, and lays a foundation for development and application of engineering antibodies.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, specifically to the rabbit monoclonal antibody mAb8 against mouse immunoglobulin G2b subtype (IgG2b) and its applications. Background Technology

[0002] Antibodies have a wide range of applications in life science research and medicine, primarily used to detect, quantify, and locate specific proteins to study their roles in biological pathways and diseases. Currently, the most widely used antibody species in scientific research and medicine include human antibodies, mouse antibodies, and rabbit antibodies. Human antibodies are mainly used for the development of therapeutic antibodies to avoid immune rejection reactions; mouse antibodies are commonly used in laboratory research due to their ease of preparation and low cost. Rabbit antibodies typically possess high affinity and specificity, making them suitable for various experimental techniques.

[0003] Currently, anti-mouse IgG and anti-human IgG 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. The most commonly used labeling methods include horseradish peroxidase (HRP), alkaline phosphatase, biotin, and fluorescein. The main method for preparing anti-mouse IgG antibodies is to directly immunize rabbits, sheep, or donkeys with IgG immunoglobulin to produce immunosweet polyclonal antibodies, which are then purified for application. However, polyclonal antibodies are far from sufficient for developing specific antibodies against different types of IgG heavy and light chains; monoclonal antibodies, especially those targeting different IgG subtypes, are needed. Mouse IgG monoclonal antibody heavy chains include four subtypes: IgG1, IgG2a, IgG2b, and IgG3, while light chains include two types: κ and λ chains, with a κ chain:λ chain ratio of approximately 20:1. Currently, most of the antibodies on the market are polyclonal antibodies, such as goat polyclonal antibodies, sheep polyclonal antibodies, and donkey polyclonal antibodies. There are very few monoclonal antibodies, especially rabbit anti-mIgG2b specific monoclonal antibodies, which have not been reported.

[0004] To meet the demand for mIgG2b-specific monoclonal antibodies, there is an urgent need to develop monoclonal antibodies with high specificity and high sensitivity against mIgG2b. Summary of the Invention

[0005] To overcome the aforementioned technical problems, this application provides mAb8, a rabbit monoclonal antibody against mIgG2b with high specificity and affinity, and its application in immunoassay, including but not limited to chemiluminescence, fluorescence, and colorimetric detection as a primary antibody. It is 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.

[0006] On one hand, this application provides a rabbit monoclonal antibody mAb8 or antigen-binding fragment against mouse immunoglobulin G2b subtype (IgG2b), wherein the rabbit monoclonal antibody includes a light chain variable region, the light chain variable region includes LCDR1-3, wherein LCDR1 includes the amino acid sequence QGVYNVDY (SEQ ID NO.1), LCDR2 includes the amino acid sequence RTS (SEQ ID NO.2), and LCDR3 includes the amino acid sequence LGSYDD (SEQ ID NO.3).

[0007] In some embodiments, the light chain variable region has a total length of 106 amino acids, with the number of amino acids in the four domains of its FR being 26, 17, 36, and 10, respectively, and the number of amino acids in the three domains of its LCDR being 8, 3, and 6, respectively. The regions of LCDR1, LCDR2, and LCDR3 are 27aa-34aa, 52aa-54aa, and 91aa-96aa, respectively, and their amino acid sequences are QGVYNVDY (SEQ ID NO.1), RTS (SEQ ID NO.2), and LGSYDD (SEQ ID NO.3), respectively.

[0008] In some embodiments, the rabbit monoclonal antibody mAb8 or antigen-binding fragment further includes a heavy chain variable region comprising HCDR1-3, wherein HCDR1 comprises the amino acid sequence GFTISNYD (SEQ ID NO.4), HCDR2 comprises the amino acid sequence ISNNGN (SEQ ID NO.5), and HCDR3 comprises the amino acid sequence AREISG (SEQ ID NO.6).

[0009] In some embodiments, the total length of the heavy chain variable region is 108 amino acids, with the number of amino acids in the four domains of its FR being 24, 17, 36 and 11, the number of amino acids in the three domains of HCDR being 8, 6 and 6, and the number of amino acids in HCDR1, HCDR2 and HCDR3 being 25aa-32aa, 50aa-55aa and 92aa-97aa, respectively. Their amino acid sequences are GFTISNYD (SEQ ID NO.4), ISNNGN (SEQ ID NO.5) and AREISG (SEQ ID NO.6), respectively.

[0010] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:7, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:8, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0011] In some embodiments, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody, and antibody minimum recognition unit; preferably, the remaining sequence of the antibody is derived from one or more species including rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

[0012] On the other hand, this application also provides a biological material comprising a polynucleotide, a carrier, or a cell, wherein the polynucleotide encodes the rabbit monoclonal antibody mAb8 or an antigen-binding fragment; the carrier carries the polynucleotide; the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the rabbit monoclonal antibody mAb8 or an antigen-binding fragment.

[0013] On the other hand, this application also provides a method for preparing the rabbit monoclonal antibody mAb8 or antigen-binding fragment, including culturing the cells; optionally, the cells are prepared by transforming a polynucleotide encoding a rabbit monoclonal antibody mAb8 or antigen-binding fragment into the cells, the polynucleotide including a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0014] In some embodiments, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.

[0015] On the other hand, this application also provides the use of the rabbit monoclonal antibody mAb8 or antigen-binding fragment or the biological material described herein in any of the following:

[0016] 1) Non-diagnostic and non-therapeutic target detection of mouse immunoglobulin G2b subtype (IgG2b);

[0017] 2) Prepare a product for detecting mouse immunoglobulin G2b subtype (IgG2b);

[0018] 3) Used for purifying mouse immunoglobulin G2b subtype (IgG2b);

[0019] 4) Prepare products for purifying mouse immunoglobulin G2b subtype (IgG2b).

[0020] On the other hand, this application also provides a detection reagent or detection kit, which contains the rabbit monoclonal antibody mAb8 or antigen-binding fragment, or the biological material.

[0021] In some embodiments, the detection kit is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation.

[0022] Compared to existing technologies, the rabbit monoclonal antibody against mIgG2b in this application can bind with high specificity to mIgG2b and has high affinity, with an affinity constant Ka reaching 7 × 10⁻⁶. 8 L / mol. The monoclonal antibody of this application can bind with high specificity to mouse immunoglobulin G2b subtype (IgG2b). This invention also relates to the application of anti-mouse immunoglobulin G2b subtype (IgG2b) specific rabbit monoclonal antibody 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, laying the foundation for the development and application of engineered antibodies. Attached Figure Description

[0023] Figure 1 This is an electrophoresis diagram of the full-length heavy and light chain amplification products of rabbit monoclonal antibody mAb8, where M is the DNA molecular weight marker.

[0024] Figure 2 The Western blot results of rabbit monoclonal antibody mAb8 specifically recognizing natural mIgG2b antibody are shown in the figure.

[0025] Figure 3 The image shows the ELISA results of rabbit monoclonal antibody mAb8 specifically recognizing mIgG2b antibody.

[0026] Figure 4 The Western blot results of horseradish peroxidase-labeled rabbit monoclonal antibody mAb8 specifically recognizing natural mIgG2b antibody are shown in the figure. Detailed Implementation

[0027] 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.

[0028] Example 1: Preparation of mouse IgG2b rabbit monoclonal antibody

[0029] 1) Preparation of immunogens

[0030] Purchase third-party mIgG2b antibodies as immunogens.

[0031] 2) Animal immunization

[0032] The purchased mIgG2b antibody was 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.

[0033] 3) PBMC isolation, specific B cell sorting, and clonal recombination

[0034] The rabbit was placed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. The area with the most prominent heartbeat was selected and punctured with a 50ml syringe. Blood flowed into the syringe immediately after the needle entered the heart. The needle was quickly withdrawn after obtaining the required amount of blood. The whole blood in the syringe was transferred into a sterile 50ml tube and mixed with an equal volume of PBS. The mixture was then 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 (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, thus obtaining rabbit PBMCs.

[0035] Antigen-specific B cells were further sorted from rabbit PBMCs and cultured. Positive clones were screened from the B cell supernatant using antigen-coated ELISA plates. 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 their sequences were confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0036] 4) Preparation and purification of monoclonal antibodies

[0037] To obtain a rabbit monoclonal antibody recognizing human mIgG2b protein, the heavy and light chain genes of the rabbit monoclonal antibody were loaded into an expression vector, and the plasmid was transfected into HEK293 cells. After 120-144 hours of transfection, the culture supernatant contained the recombinant rabbit monoclonal antibody recognizing human mIgG2b protein. The cell suspension was collected, the supernatant was obtained by centrifugation, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then aliquoted, lyophilized, and named the purified antibody mouse IgG2b rabbit monoclonal antibody mAb8.

[0038] Example 2: Specificity identification of anti-mIgG2b rabbit monoclonal antibody mAb8

[0039] 1) Identification of rabbit monoclonal antibody mAb8 by Western blot

[0040] Western blot (WB) was used for detection. Two 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.

[0041] The results showed that the rabbit monoclonal antibody mAb8 specifically recognized mIgG2b, but not mIgG1, mIgG2a, or mIgG3. (See attached results). Figure 2 .

[0042] 2) Rabbit monoclonal antibody mAb8 ELISA identification

[0043] Antibodies against mIgG1, mIgG2a, mIgG2b, and mIgG3 were 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 rabbit monoclonal antibody MAB8 was added to each well at concentrations of 10, 3, 1, 0.3, and 0 ng / mL, respectively, and incubated at 37°C for 1 hour. After incubation, the plate was removed, washed three times with PBST, and HRP-labeled goat anti-rabbit secondary antibody was added as the detection antibody. The plate was incubated at 37°C for 1 hour. After incubation, the plate was removed, washed five times with PBST, and TMB substrate was added. The plate was incubated at 37°C for 10 minutes. Finally, stop solution was added, and the OD450 reading was measured using an ELISA reader.

[0044] The results showed that the rabbit monoclonal antibody mAb8 specifically recognized mIgG2b, but not mIgG1, mIgG2a, or mIgG3. (See attached results). Figure 3 .

[0045] Example 3: Affinity identification of anti-mIgG2b rabbit monoclonal antibody mAb8

[0046] Affinity constant (Ka) was determined using a non-competitive ELISA method.

[0047] Coating: Dilute the antigen with carbonate buffer to concentrations of 1, 0.5, 0.1, and 0.05 μg / mL, add 100 μL / well to a 96-well microplate and coat accordingly. Incubate at 4°C for 24 h.

[0048] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h;

[0049] Add monoclonal antibody: Wash the plate 4 times with PBST, serially dilute the rabbit monoclonal antibody with carbonate buffer starting at 100 μg / mL, add 100 μL to each well, and incubate at 37°C for 2 h;

[0050] Add enzyme-labeled secondary antibody: Wash the plate 4 times with PBST, add 100 μL of HRP enzyme-labeled goat anti-rabbit Ig secondary antibody diluted 1:10000 to each well, and incubate at 37℃ for 30 min;

[0051] Color development and termination: Wash the plate 4 times with PBST, add 100 μL of substrate color development solution to each well, and react at 37℃ in the dark for 15 min; add 50 μL of 1.0 mol / L H2SO4 stop solution to each well to terminate the reaction;

[0052] Detection: The absorbance value at a wavelength of 450 nm (A450nm) was measured.

[0053] Plotting the logarithm of antibody concentration on the x-axis and OD value on the y-axis, an S-shaped curve was obtained. The calculated affinity constant Ka for the T3 sandwich monoclonal antibody mAb3 was 7 × 10⁻⁶. 8 L / mol.

[0054] Example 4: Analysis of the variable region gene and amino acid sequence of the rabbit monoclonal antibody mAb8. Using the recombinant plasmid of the mAb8 antibody as a DNA template, sequencing primers for the light chain and heavy chain variable regions 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 mAb8 were obtained through sequencing.

[0055] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light chain and heavy chain were sequenced and analyzed. The light chain amino acid sequence of the rabbit monoclonal antibody mAb8 is shown in SEQ ID NO.7, and the heavy chain amino acid sequence is shown in SEQ ID NO.8.

[0056] The light chain variable region is 106 amino acids in length. 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 LCDR are 8, 3 and 6, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-34aa, 52aa-54aa and 91aa-96aa, respectively. Their amino acid sequences are QGVYNVDY (SEQ ID NO.1), RTS (SEQ ID NO.2) and LGSYDD (SEQ ID NO.3), respectively.

[0057] The heavy chain variable region is 108 amino acids in length. The number of amino acids in the four domains of its FR are 24, 17, 36 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 6 and 6, respectively. HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-55aa and 92aa-97aa, respectively. Their amino acid sequences are GFTISNYD (SEQ ID NO.4), ISNNGN (SEQ ID NO.5) and AREISG (SEQ ID NO.6), respectively.

[0058] Example 5: Identification of HRP-labeled rabbit monoclonal antibody mAb8 as a secondary antibody

[0059] (1) HRP-labeled rabbit monoclonal antibody mAb8

[0060] 1. Rabbit monoclonal antibody mAb8 was dissolved in sodium bicarbonate solution at pH 9.6;

[0061] 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;

[0062] 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.

[0063] 4. Add an equal amount of glycerol to the obtained HRP-labeled antibody and store at -20 degrees Celsius.

[0064] (2) Western blot identification of HRP-labeled rabbit monoclonal antibody mAb8

[0065] Western blot (WB) was used for detection. 100 ng of mIgG2b antibody was subjected to SDS-PAGE, transferred to a membrane, and then incubated with HRP-labeled rabbit monoclonal antibody mAb8 for WB detection.

[0066] The results showed that the HRP-labeled rabbit monoclonal antibody mAb8 could effectively recognize the mIgG2b Fc region, with a molecular weight of approximately 50 kDa. (See attached results). Figure 4 .

[0067] (3) Identification by HRP-labeled rabbit monoclonal antibody mAb8 ELISA

[0068] 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 mIgG2b 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 mAb8 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. Stop solution was added, and the OD450 reading was measured using an ELISA reader. The results are shown in Table 1.

[0069] Table 1. ELISA results of HRP-labeled rabbit anti-mIgG2b mAb8-specific mIgG2b antibody.

[0070]

[0071] The results showed that HRP-labeled rabbit anti-mIgG2b mAb8, as a secondary antibody, could effectively detect mIgG2b antibody signals, which was superior to commercially available goat anti-mouse IgG polyclonal antibody (which, as a secondary antibody, could simultaneously detect mIgG1, mIgG2a, mIgG2b, and mIgG3).

[0072] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A rabbit monoclonal antibody mAb8 or antigen-binding fragment against mouse immunoglobulin G2b isotype, characterized in that, The rabbit monoclonal antibody mAb8 or antigen-binding fragment includes a light chain variable region, which includes LCDR1-3, wherein the amino acid sequence of LCDR1 is QGVYNVDY, the amino acid sequence of LCDR2 is RTS, and the amino acid sequence of LCDR3 is LGSYDD; the rabbit monoclonal antibody mAb8 or antigen-binding fragment also includes a heavy chain variable region, which includes HCDR1-3, wherein the amino acid sequence of HCDR1 is GFTISNYD, the amino acid sequence of HCDR2 is ISNNGN, and the amino acid sequence of HCDR3 is AREISG.

2. The rabbit monoclonal antibody mAb8 or antigen-binding fragment according to claim 1, characterized in that, The light chain variable region includes the amino acid sequence shown in SEQ ID NO:7; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:

8.

3. The rabbit monoclonal antibody mAb8 or antigen-binding fragment according to claim 1 or 2, characterized in that, The antigen-binding fragment is selected from one of F(ab')2, Fab', Fab, Fv, scFv and dsFv; the remaining sequence of the antibody is derived from one of the following species: rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey and human.

4. A biomaterial, characterized in that, The biological material includes a polynucleotide, a carrier, or a cell, wherein the polynucleotide encodes the rabbit monoclonal antibody mAb8 or antigen-binding fragment as described in any one of claims 1-3; the carrier carries the polynucleotide; the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the rabbit monoclonal antibody mAb8 or antigen-binding fragment as described in any one of claims 1-3.

5. The method for preparing the rabbit monoclonal antibody mAb8 or antigen-binding fragment according to any one of claims 1-3, characterized in that, Includes culturing the cells as described in claim 4; The cells are prepared by converting cells with a polynucleotide encoding a rabbit monoclonal antibody mAb8 or an antigen-binding fragment, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

6. The preparation method according to claim 5, characterized in that, The cells in question are eukaryotic cells.

7. The preparation method according to claim 6, characterized in that, The cells in question are mammalian cells.

8. The preparation method according to claim 7, characterized in that, The cells are either 293 cells or CHO cells.

9. The use of the rabbit monoclonal antibody mAb8 or antigen-binding fragment according to any one of claims 1-3 or the biological material according to claim 4 in any of the following: 1) Non-diagnostic and non-therapeutic target detection of mouse immunoglobulin G2b subtype; 2) Prepare products for detecting mouse immunoglobulin G2b subtypes; 3) Used for purifying mouse immunoglobulin G2b isotype; 4) Prepare products for purifying mouse immunoglobulin G2b subtype.

10. A detection reagent or detection kit, characterized in that, The detection reagent or detection kit contains the rabbit monoclonal antibody mAb8 or antigen-binding fragment as described in any one of claims 1-3, or the biological material as described in claim 4.

11. The detection reagent or detection kit according to claim 10, characterized in that, The detection kit is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining, flow cytometry, and immunoprecipitation.

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