Rabbit monoclonal antibody mAb6 of anti-mouse immunoglobulin G2a subtype (IgG2a) and application thereof

Abstract

The invention belongs to the technical field of immunodetection, and discloses a rabbit monoclonal antibody mAb6 of an anti-mouse immune globulin G2a subtype (IgG2a) and application thereof. The rabbit monoclonal antibody mAb6 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 KSVYKNNY, the LCDR2 comprises an amino acid sequence GTN, and the LCDR3 comprises an amino acid sequence AGGYID; the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises an amino acid sequence RFSLSNYR, the HCDR2 comprises an amino acid sequence IFTRGST, and the HCDR3 comprises an amino acid sequence ARGWNS. The rabbit monoclonal antibody disclosed by the invention can be combined with a mouse immune globulin G2a subtype (IgG2a) in a high-specificity manner, can be applied to immunodetection, and provides a basis for further preparation of engineering antibodies.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and specifically relates to a rabbit monoclonal antibody mAb6 against murine immunoglobulin G2a subtype (mIgG2a) and its applications. Background Art

[0002] Affinity conjugates / antibodies are one of the most commonly used tools in life sciences for studying proteins and their functions in various biological pathways and diseases. Currently, the most widely used antibody species in scientific research and the pharmaceutical field include human antibodies, murine antibodies, and rabbit antibodies. These antibodies can specifically recognize the target of interest, and then display signals through enzyme-labeled secondary antibodies or fluorescent-labeled secondary antibodies. Currently, anti-murine IgG and anti-human IgG labeled antibodies are widely used in the market and can be applied to enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, etc. The most commonly used labeling methods include horseradish peroxidase (HRP), alkaline phosphatase, biotin, fluorescein, etc.

[0003] Currently, the main method for preparing anti-murine IgG antibodies is to directly immunize animals such as rabbits, sheep, or donkeys with IgG immunoglobulin to produce immunological sera polyclonal antibodies, and then apply them after purification. However, for developing antibodies specific to different types of IgG heavy and light chains, polyclonal antibodies are far from sufficient, and monoclonal antibodies need to be prepared, especially monoclonal antibodies against different subtypes of IgG.

[0004] The heavy chain of murine IgG monoclonal antibodies includes four subtypes: IgG1, IgG2a, IgG2b, and IgG3, and the light chain includes two types: κ chain and λ chain, with the ratio of κ chain:λ chain being approximately 20:1. Currently, most of the antibodies in the market are polyclonal antibodies, such as goat polyclonal antibodies, sheep polyclonal antibodies, donkey polyclonal antibodies, etc., and there are few monoclonal antibodies, especially no rabbit anti-mIgG2a specific monoclonal antibodies have been reported.

[0005] To meet the demand for mIgG2a specific monoclonal antibodies, there is an urgent need to develop monoclonal antibodies with high specificity and high sensitivity against mIgG2a. Summary of the Invention

[0006] To overcome the above technical problems, this application provides a rabbit monoclonal antibody mAb6 against mIgG2a with high specificity and high sensitivity and its applications in immunoassay, 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 provides a basis for the preparation of next-generation engineered antibodies.

[0007] On the one hand, the present application provides a rabbit monoclonal antibody mAb6 or an antigen-binding fragment thereof against murine immunoglobulin G2a subtype (IgG2a), wherein the light chain variable region comprises complementary determining regions LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.1, the LCDR2 comprises the amino acid sequence GTN, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.3.

[0008] In certain embodiments, the full length of the light chain variable region is 106 amino acids, the amino acid numbers of the 4 domains of the FR are 26, 17, 36 and 10 respectively, and the amino acid numbers of the 3 domains of the LCDR are 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 KSVYKNNY (SEQ ID NO.1), GTN (SEQ ID NO.2), AGGYID (SEQ ID NO.3).

[0009] In certain embodiments, the heavy chain variable region comprises complementary determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO.4, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.5, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.6.

[0010] In certain embodiments, the full length of the heavy chain variable region is 109 amino acids, the amino acid numbers of the 4 domains of the FR are 24, 17, 36 and 11 respectively, and the amino acid numbers of the 3 domains of the HCDR are 8, 7 and 6 respectively. HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-56aa and 93aa-98aa respectively, and their amino acid sequences are RFSLSNYR (SEQ ID NO.4), IFTRGST (SEQ ID NO.5), ARGWNS (SEQ ID NO.6).

[0011] In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:7, preferably at least 96%, 97%, 98%, 99% identity; the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:8, preferably at least 96%, 97%, 98%, 99% identity.

[0012] In some embodiments, the antigen-binding fragment is one of F(ab’)2, Fab’, Fab, Fv, scFv, dsFv, bispecific antibody, and the minimum antibody recognition unit; preferably, the species from which the remaining sequences of the antibody are derived include one or more of rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

[0013] On the other hand, the present application also provides a biological material, which includes a polynucleotide, a vector, or a cell. The polynucleotide encodes the rabbit monoclonal antibody mAb6 or an antigen-binding fragment; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector, or is capable of expressing the rabbit monoclonal antibody mAb6 or an antigen-binding fragment.

[0014] On the other hand, the present application also provides a method for preparing the rabbit monoclonal antibody mAb6 or an antigen-binding fragment, including culturing the cell; optionally, the cell is prepared by transforming a polynucleotide encoding the rabbit monoclonal antibody mAb6 or an antigen-binding fragment into the cell. The polynucleotide includes 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 cell.

[0015] In some embodiments, the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a 293 cell or a CHO cell.

[0016] On the other hand, the present application also provides the use of the rabbit monoclonal antibody mAb6 or an antigen-binding fragment or the biological material in any of the following: 1) Detecting murine immunoglobulin G2a subtype (IgG2a) for non-diagnostic and non-therapeutic purposes; 2) Preparing a product for detecting murine immunoglobulin G2a subtype (IgG2a); 3) Purifying murine immunoglobulin G2a subtype (IgG2a); 4) Preparing a product for purifying murine immunoglobulin G2a subtype (IgG2a).

[0017] On the other hand, the present application also provides a detection reagent or a detection kit, which contains the rabbit monoclonal antibody mAb6 or an antigen-binding fragment, or the biological material.

[0018] In some embodiments, the detection kit is used for enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western blot), immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, etc.

[0019] Compared with the prior art, the mIgG2a rabbit monoclonal antibody of the present application can specifically bind to mIgG2a with high affinity, and its affinity constant Ka can reach 8×10 8 L / mol. The present invention also relates to the application of anti-mouse immunoglobulin G2a subtype (IgG2a) 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, western blotting and immunohistochemistry, and also provides a basis for the preparation of next-generation engineered antibodies. Description of the Drawings

[0020] Figure 1 Electrophoresis diagram of the full-length amplification products of the heavy and light chains of rabbit monoclonal antibody mAb6, where M is the DNA molecular weight Marker.

[0021] Figure 2 Western blot detection result diagram of rabbit monoclonal antibody mAb6 specifically recognizing native mIgG2a antibody.

[0022] Figure 3 ELISA detection result diagram of rabbit monoclonal antibody mAb6 specifically recognizing mIgG2a antibody.

[0023] Figure 4 Western blot detection result diagram of horseradish peroxidase-labeled rabbit monoclonal antibody mAb6 specifically recognizing native mIgG2a antibody. Detailed Description of the Invention

[0024] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to conventional conditions, the conditions described in laboratory manuals or the conditions recommended by the manufacturers.

[0025] Example 1 Preparation of Mouse IgG2a Rabbit Monoclonal Antibody 1) Preparation of Immunogen Purchase a third-party mIgG2a antibody as the immunogen.

[0026] 2) Animal Immunization The purchased mIgG2a antibody was emulsified with complete Freund's adjuvant and used to immunize New Zealand white rabbits weighing about 2 kg by subcutaneous injection. The immunization dose was 500 μg per rabbit. The second immunization was carried out two weeks later, emulsified with incomplete Freund's adjuvant, and the immunization dose was 250 μg per rabbit. After two immunizations, tail blood was taken and the serum titer was measured by gradient dilution using the ELISA method; based on the standard that the OD450 at an ELISA titer of 128,000 was greater than 1.0, it was determined whether to collect PBMCs or continue immunization according to the results, and the rabbit with the highest antibody titer was selected for PBMC collection.

[0027] 3) Isolation of PBMCs, sorting of specific B cells, cloning and recombination The rabbit was fixed supine on the operating table, the hair on the heart area was removed, the skin was disinfected with alcohol, and the most obvious part of the heartbeat was selected for puncture with a 50 ml syringe. After the needle penetrated the heart, blood rushed into the syringe. After obtaining the required amount of blood, the needle was quickly pulled out, and the whole blood in the syringe was transferred into a sterile 50 ml tube. After mixing with an equal volume of PBS, it was slowly added drop by drop above the lymphocyte separation solution and centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface was divided into four layers from top to bottom: yellow plasma layer, white film layer (i.e., mononuclear cell layer), separation solution layer, and red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation solution, and then rabbit PBMCs could be obtained.

[0028] Antigen-specific B cells were further sorted from rabbit PBMCs for culture, and the supernatant of the cultured B cells was screened for positive clones using an ELISA plate coated with the antigen. The full-length sequences of the heavy and light chains of the naturally paired rabbit monoclonal antibody were amplified from the cDNA of the corresponding positive clone, and a rabbit monoclonal antibody expression vector was constructed by cloning and recombination methods, and the sequence was determined by sequencing. The results of the amplified full-length PCR products were as Figure 1 .

[0029] 4) Preparation and purification of monoclonal antibody To obtain a rabbit monoclonal antibody that recognizes human mIgG2a protein, the heavy and light chain genes of the rabbit monoclonal antibody were loaded on an expression vector, and the plasmid was transfected into HEK293 cells; after transfection for 120 - 144 hours, the culture supernatant contained the recombinant rabbit monoclonal antibody that recognizes human mIgG2a protein. The cell suspension was collected, centrifuged to obtain the supernatant, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was measured by the BCA method, and then aliquoted and freeze-dried. The purified antibody was named rabbit monoclonal antibody mAb6 against mouse IgG2a.

[0030] Example 2 Specific identification of rabbit monoclonal antibody mAb6 against mIgG2a 1) Western blot identification of rabbit monoclonal antibody mAb6 Detection was performed by Western blot (WB). For each of the subtypes mIgG1, mIgG2a, mIgG2b, and mIgG3, 2 samples were selected. 100 ng of each protein was loaded for SDS-PAGE, and WB detection was performed after transfer to the membrane.

[0031] The results showed that the rabbit monoclonal antibody mAb6 could specifically recognize mIgG2a and did not recognize mIgG1, mIgG2b, or mIgG3. The results are shown in Figure 2 .

[0032] 2) ELISA identification of rabbit monoclonal antibody mAb6 The enzyme-linked immunosorbent assay (ELISA) plate was coated with mIgG1, mIgG2a, mIgG2b, and mIgG3 antibodies and incubated overnight at 4 °C. The next day, the ELISA plate was taken out, washed once with PBST, blocked with 1% BSA solution at 37 °C for 2 hours, and then washed 3 times with PBST. 100 μl of rabbit monoclonal antibody MAB6 at concentrations of 10, 3, 1, 0.3, and 0 ng / ml was added to each well and incubated at 37 °C for 1 hour. After incubation, the ELISA plate was taken out, washed 3 times with PBST, and HRP-labeled goat anti-rabbit secondary antibody was added as the detection antibody and incubated at 37 °C for 1 hour. After incubation, the ELISA plate was taken out, washed 5 times with PBST, TMB substrate was added, and the color was developed at 37 °C for 10 min. After taking it out, the stop solution was added, and the OD450 reading was measured on an ELISA reader.

[0033] The results showed that the rabbit monoclonal antibody mAb6 could specifically recognize mIgG2a and did not recognize mIgG1, mIgG2b, or mIgG3. The results are shown in Figure 3 .

[0034] Example 3 Affinity identification of anti-mIgG2a rabbit monoclonal antibody mAb6 The affinity constant (Ka) was determined by non-competitive ELISA.

[0035] Coating: The antigen was diluted with carbonate buffer to concentrations of 1, 0.5, 0.1, and 0.05 μg / mL, and 100 μL / well was added to a 96-well ELISA plate for coating and incubated at 4 °C for 24 h; Blocking: The plate was washed 4 times with PBST, and 200 μL / well of BSA solution was added and incubated at 37 °C for 2 h; Adding monoclonal antibody: The plate was washed 4 times with PBST, and the rabbit monoclonal antibody was serially diluted starting from 100 μg / mL with carbonate buffer. 100 μL was added to each well and incubated at 37 °C for 2 h; Adding enzyme-labeled secondary antibody: The plate was washed 4 times with PBST, and 100 μL of 1:10000 diluted HRP enzyme-labeled goat anti-rabbit Ig secondary antibody was added to each well and incubated at 37 °C for 30 min; Color development and termination: Wash the plate 4 times with PBST. Add 100 μL of substrate color development solution to each well and react for 15 min at 37°C in the dark. Add 50 μL of 1.0 mol / L H2SO4 termination solution to each well to terminate the reaction. Detection: Measure the absorbance value (A450nm) at a wavelength of 450 nm.

[0036] Using the logarithm of the antibody concentration as the abscissa and the OD value as the ordinate, plot an S-shaped curve. After calculation, the affinity constant Ka of the anti-mIgG2a rabbit monoclonal antibody mAb6 is 8×10 8 L / mol.

[0037] Example 4 Analysis of the variable region genes and amino acid sequences of rabbit monoclonal antibody mAb6 Using the recombinant plasmid of mAb6 antibody as the DNA template, design sequencing primers for the light chain variable region and heavy chain variable region according to the 5' end vector sequences of the light chain and heavy chain on the template, and use the sequencer ABI 3730 for sequencing. The nucleotide sequences of the light chain and heavy chain variable regions of rabbit monoclonal antibody mAb6 are obtained by sequencing.

[0038] Using the Internet, on http: / / www.imgt.org, use the IMGT / V-QUEST analysis software to analyze the sequencing results data of the nucleotide sequences of the light chain variable region and heavy chain variable region respectively. The amino acid sequence of the light chain variable region of rabbit monoclonal antibody mAb6 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0039] The full length of the light chain variable region is 106 amino acids. The amino acid numbers of the 4 domains of FR are 26, 17, 36, and 10 respectively. The amino acid numbers of the 3 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, and their amino acid sequences are KSVYKNNY (SEQ ID NO.1), GTN (SEQ ID NO.2), AGGYID (SEQ ID NO.3).

[0040] The full length of the heavy chain variable region is 109 amino acids. The amino acid numbers of the 4 domains of FR are 24, 17, 36, and 11 respectively. The amino acid numbers of the 3 domains of HCDR are 8, 7, and 6 respectively. HCDR1, HCDR2, and HCDR3 are 25aa-32aa, 50aa-56aa, and 93aa-98aa respectively, and their amino acid sequences are RFSLSNYR (SEQ ID NO.4), IFTRGST (SEQ IDNO.5), ARGWNS (SEQ ID NO.6) Identification of HRP-labeled rabbit monoclonal antibody mAb6 as a secondary antibody (1) HRP-labeled rabbit monoclonal antibody mAb6 1. Rabbit monoclonal antibody mAb6 was dissolved in sodium bicarbonate solution with pH 9.6; 2. A certain mass of HRP was dissolved in deionized water, sodium periodate was added and reacted for 30 min, ethylene glycol was added and reacted for another 30 min, and then dialyzed overnight; 3. A certain amount of sodium borohydride was weighed and dissolved in deionized water, sodium borohydride was added to the cross-linked antibody-HRP solution and reacted for 2 h, and then dialyzed overnight; 4. The obtained HRP-labeled antibody was added with an equal amount of glycerol and stored at -20 °C.

[0041] (2) Western blot identification of HRP-labeled rabbit monoclonal antibody mAb6 Western blot (WB) detection was used. 100 ng of mIgG2a antibody was taken for SDS-PAGE, and after transfer to the membrane, HRP-labeled rabbit monoclonal antibody mAb6 was incubated for WB detection.

[0042] The results showed that HRP-labeled rabbit monoclonal antibody mAb6 could well recognize the Fc region of mIgG2a, with a molecular weight of about 50 KD. The results are shown in Figure 4 .

[0043] (3) ELISA identification of HRP-labeled rabbit monoclonal antibody mAb6 The enzyme-linked immunosorbent assay (ELISA) plate was coated with goat anti-mouse IgG and incubated overnight at 4 °C; the ELISA plate was taken out the next day, washed once with PBST, blocked with 1% BSA solution at 37 °C for 2 h, and washed 3 times with PBST; 100 μl of mIgG2a antibody with a concentration of 0.2 μg / ml was added to each well and incubated at 37 °C for 1 h; after incubation, the ELISA plate was taken out, washed 3 times with PBST, HRP-labeled mAb6 was added as the detection antibody and serially diluted 7-fold starting from 1 μg / ml, and incubated at 37 °C for 1 h; after incubation, the ELISA plate was taken out, washed 5 times with PBST, TMB substrate was added, and the color was developed at 37 °C for 10 min. After taking out, the stop solution was added, and the OD450 reading was measured on an ELISA reader. The results are shown in Table 1.

[0044] Table 1 ELISA detection results of HRP-labeled rabbit anti-mIgG2a mAb6 specifically recognizing mIgG2a antibody The results showed that HRP-labeled rabbit anti-mIgG2a mAb6 as the secondary antibody could detect the mIgG2a antibody signal well, which was superior to the commercial goat anti-mouse IgG polyclonal antibody (which could detect mIgG1, mIgG2a, mIgG2b, and mIgG3 simultaneously as the secondary antibody).

[0045] This specific embodiment is only an interpretation of the present application and is not a limitation thereof. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A rabbit monoclonal antibody mAb6 or an antigen-binding fragment thereof against mouse immunoglobulin G2a subtype (IgG2a), characterized in that: The light chain variable region includes complementary determining regions LCDR1, LCDR2 and LCDR3, wherein LCDR1 includes the amino acid sequence shown in SEQ ID NO.1, LCDR2 includes the amino acid sequence GTN, and LCDR3 includes the amino acid sequence shown in SEQ ID NO.

3.

2. The rabbit monoclonal antibody mAb6 or the antigen-binding fragment according to claim 1, characterized in that: The heavy variable region includes complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR1 includes the amino acid sequence shown in SEQ ID NO.4, the HCDR2 includes the amino acid sequence shown in SEQ ID NO.5, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO.

6.

3. The rabbit monoclonal antibody mAb6 or the antigen-binding fragment according to claim 1 or 2, characterized in that: The light chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:7; the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:

8.

4. The rabbit monoclonal antibody mAb6 or the antigen-binding fragment according to claim 1 or 2, characterized in that: The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody and antibody minimum recognition unit; preferably, the species from which the remaining sequence of the antibody originates include one or more of rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey and human.

5. Biomaterial, characterized in that The biological material includes a polynucleotide, a vector or a cell, wherein the polynucleotide encodes the rabbit monoclonal antibody mAb6 or the antigen-binding fragment according to any one of claims 1 to 4; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector, or is capable of expressing the rabbit monoclonal antibody mAb6 or the antigen-binding fragment according to any one of claims 1 to 4.

6. The method for preparing the rabbit monoclonal antibody mAb6 or the antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: Comprising culturing the cell as claimed in claim 5; Optionally, the cell is obtained by transforming a polynucleotide encoding the rabbit monoclonal antibody mAb6 or antigen-binding fragment into the cell, the polynucleotide includes 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 cell.

7. The preparation method according to claim 6, characterized in that: The cell is a eukaryotic cell, preferably a mammalian cell, more preferably a 293 cell or a CHO cell.

8. Use of the rabbit monoclonal antibody mAb6 or the antigen-binding fragment according to any one of claims 1 to 4 or the biomaterial according to claim 5 in any of the following: 1) Detection of mouse immunoglobulin G2a subtype (IgG2a) for non-diagnostic and therapeutic purposes; 2) Preparation of products for detecting mouse immunoglobulin G2a subtype (IgG2a); 3) Used to purify mouse immunoglobulin G2a subtype (IgG2a); 4) Preparation of products for purification of mouse immunoglobulin G2a subtype (IgG2a).

9. A detection reagent or a detection kit, characterized in that: The detection reagent or detection kit comprises the rabbit monoclonal antibody mAb6 or the antigen-binding fragment according to any one of claims 1 to 4, or the biological material according to claim 5.

10. The detection reagent or detection kit according to claim 9, characterized in that: The detection kit is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemistry staining (IHC), flow cytometry (FCM), immunoprecipitation, etc.

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