Rabbit monoclonal antibody mAb8 of anti-mouse immunoglobulin G2b subtype (IgG2b) and application thereof

Abstract

The invention belongs to the technical field of immunodetection, and discloses a specific monoclonal antibody mAb8 of rabbit anti-mouse immunoglobulin G2b subtype (IgG2b), the specific monoclonal antibody mAb8 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 disclosed by the invention can be combined with mouse immune globulin G2b subtype (IgG2b) in a high-specificity manner, can be applied to immunodetection, and lays a foundation for development and application of engineering antibodies.

Description

Technical Field

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

[0002] Antibodies are widely used in life science research and the pharmaceutical field, mainly for detecting, quantifying, and localizing specific proteins, so as to study their roles in biological pathways and diseases. Currently, the most widely used antibody species in the fields of scientific research and medicine include human antibodies, murine antibodies, and rabbit antibodies. Human antibodies are mainly used for the development of therapeutic antibodies to avoid immune rejection reactions; murine antibodies are commonly used in laboratory research because they are easy to prepare and have low costs. Rabbit antibodies usually have high affinity and specificity and are suitable for a variety of experimental techniques.

[0003] Currently, anti-murine IgG and anti-human IgG labeled antibodies are very 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. 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 polyclonal antibodies, and then apply them after purification. However, for developing antibodies with different types of specificities for IgG heavy and light chains, polyclonal antibodies are far from enough, and monoclonal antibodies need to be prepared, especially monoclonal antibodies against different subtypes of IgG. 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, and the ratio of κ chain:λ chain is about 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-mIgG2b specific monoclonal antibodies have 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 for mIgG2b. Summary of the Invention

[0005] In order to overcome the above technical problems, this application provides a rabbit monoclonal antibody mAb8 against mIgG2b with good specificity and high affinity 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.

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

[0007] 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 QGVYNVDY (SEQ ID NO.1), RTS (SEQ ID NO.2), and LGSYDD (SEQ ID NO.3) respectively.

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

[0009] In certain embodiments, the full length of the heavy chain variable region is 108 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, 6, and 6 respectively. HCDR1, HCDR2, and HCDR3 are 25aa-32aa, 50aa-55aa, and 92aa-97aa respectively, and their amino acid sequences are GFTISNYD (SEQ ID NO.4), ISNNGN (SEQ ID NO.5), and AREISG (SEQ ID NO.6) respectively.

[0010] In certain embodiments, the light chain variable region comprises the 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%, 99% identity; the heavy chain variable region comprises the 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%, 99% identity.

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

[0012] On the other hand, the present application also provides a biological material, which comprises a polynucleotide, a vector, or a cell. The polynucleotide encodes the rabbit monoclonal antibody mAb8 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 mAb8 or an antigen-binding fragment.

[0013] On the other hand, the present application also provides a method for preparing the rabbit monoclonal antibody mAb8 or an antigen-binding fragment, which comprises culturing the cell; optionally, the cell is prepared by transforming a polynucleotide encoding the rabbit monoclonal antibody mAb8 or an antigen-binding fragment into the cell. The polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprises co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0014] In certain embodiments, the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a 293 cell or a CHO cell.

[0015] On the other hand, the present application also provides the use of the rabbit monoclonal antibody mAb8 or an antigen-binding fragment or the biological material in any of the following: 1) Detecting murine immunoglobulin G2b subtype (IgG2b) for non-diagnostic and non-therapeutic purposes; 2) Preparing a product for detecting murine immunoglobulin G2b subtype (IgG2b); 3) Purifying murine immunoglobulin G2b subtype (IgG2b); 4) Prepare a product for purifying murine immunoglobulin G2b subtype (IgG2b).

[0016] On the other hand, the present application also provides a detection reagent or a detection kit, which comprises the rabbit monoclonal antibody mAb8 or an antigen-binding fragment thereof, or the biological material as described above.

[0017] In some embodiments, the detection kit is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, etc.

[0018] Compared with the prior art, the mIgG2b rabbit monoclonal antibody of the present application can bind to mIgG2b with high specificity and has a high affinity, and its affinity constant Ka can reach 7×10 8 L / mol. The monoclonal antibody of the present application can bind to murine immunoglobulin G2b subtype (IgG2b) with high specificity. The present invention also relates to the application of the anti-murine 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 a foundation for the development and application of engineered antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is an electrophoresis diagram of the full-length amplification products of the heavy and light chains of the rabbit monoclonal antibody mAb8, and M is the DNA molecular weight Marker.

[0020] Figure 2 It is a Western blot detection result diagram of the rabbit monoclonal antibody mAb8 specifically recognizing the natural mIgG2b antibody.

[0021] Figure 3 It is an ELISA detection result diagram of the rabbit monoclonal antibody mAb8 specifically recognizing the mIgG2b antibody.

[0022] Figure 4 It is a Western blot detection result diagram of the horseradish peroxidase-labeled rabbit monoclonal antibody mAb8 specifically recognizing the natural mIgG2b antibody. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] 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.

[0024] Example 1 Preparation of Mouse IgG2b Rabbit Monoclonal Antibody 1) Preparation of Immunogen Purchase a third-party mIgG2b antibody as the immunogen.

[0025] 2) Animal Immunization The purchased mIgG2b antibody is emulsified with complete Freund's adjuvant and used to immunize New Zealand white rabbits weighing about 2 kg by subcutaneous injection. The immunization dose is 500 μg / rabbit. The second immunization is carried out two weeks later, emulsified with incomplete Freund's adjuvant, and the immunization dose is 250 μg / rabbit. After two immunizations, tail blood is taken to measure the serum titer by gradient dilution using the ELISA method; based on the standard that the OD450 at an ELISA titer of 128,000 is greater than 1.0, it is determined whether to collect PBMCs or continue immunization according to the results, and the rabbit with the highest antibody titer is selected for PBMCs collection.

[0026] 3) Isolation of PBMCs, Sorting of Antigen-Specific B Cells, and Cloning and Recombination The rabbit is fixed supine on the operating table, the hair on the heart area is removed, the skin is disinfected with alcohol, and the most obvious heart beat is selected for puncture with a 50 ml syringe. After the needle penetrates the heart, blood will flow into the syringe. After obtaining the required amount of blood, the needle is quickly withdrawn, and the whole blood in the syringe is transferred into a sterile 50 ml tube, mixed with an equal volume of PBS, and slowly added drop by drop above the lymphocyte separation solution. Centrifuge at 400 × g at room temperature for 30 minutes. After centrifugation, the liquid surface is 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. Carefully aspirate the mononuclear cell layer and wash it with PBS to remove platelets and lymphocyte separation solution to obtain rabbit PBMCs.

[0027] Continue to sort antigen-specific B cells from rabbit PBMCs for culture, and the supernatant of the cultured B cells is 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 are amplified from the cDNA of the corresponding positive clone, and a rabbit monoclonal antibody expression vector is constructed by cloning and recombination methods, and the sequence is determined by sequencing. The results of the amplified full-length PCR products are as Figure 1 .

[0028] 4) Preparation and Purification of Monoclonal Antibody To obtain a rabbit monoclonal antibody that recognizes human mIgG2b protein, the genes of the heavy and light chains of the rabbit monoclonal antibody were loaded onto an expression vector, and the plasmid was transfected into HEK293 cells; after 120 - 144 hours of transfection, the culture supernatant containing the recombinant rabbit monoclonal antibody that recognizes human mIgG2b protein was obtained. 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 determined by the BCA method, then aliquoted and freeze-dried, and the purified antibody was named rabbit monoclonal antibody mAb8 against murine IgG2b.

[0029] Example 2 Specificity Identification of Rabbit Monoclonal Antibody mAb8 Against mIgG2b 1) Western blot Identification of Rabbit Monoclonal Antibody mAb8 Western blot (WB) was used for detection. Two samples were selected for each of the subtypes mIgG1, mIgG2a, mIgG2b, and mIgG3, and 100 ng of each protein was loaded for SDS-PAGE, followed by WB detection after membrane transfer.

[0030] The results showed that rabbit monoclonal antibody mAb8 could specifically recognize mIgG2b and did not recognize mIgG1, mIgG2a, or mIgG3. The results are shown in Figure 2 .

[0031] 2) ELISA Identification of Rabbit Monoclonal Antibody mAb8 The enzyme-linked immunosorbent assay (ELISA) plates were coated with mIgG1, mIgG2a, mIgG2b, and mIgG3 antibodies and incubated overnight at 4°C; the next day, the ELISA plates were 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 MAB8 with 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 plates were 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 plates were 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 enzyme-linked immunosorbent assay reader.

[0032] The results showed that rabbit monoclonal antibody mAb8 could specifically recognize mIgG2b and did not recognize mIgG1, mIgG2a, or mIgG3. The results are shown in Figure 3 .

[0033] Example 3 Affinity Identification of Rabbit Monoclonal Antibody mAb8 Against mIgG2b The affinity constant (Ka) was determined by non-competitive ELISA.

[0034] Coating: Dilute the antigen with carbonate buffer to concentrations of 1, 0.5, 0.1, 0.05 μg / mL, add 100 μL per well to a 96-well ELISA plate for coating respectively, and incubate at 4 °C for 24 h; Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL per well, and incubate at 37 °C for 2 h; Adding monoclonal antibody: Wash the plate 4 times with PBST, serially dilute the rabbit monoclonal antibody with carbonate buffer starting from 100 μg / mL, add 100 μL to each well, and incubate at 37 °C for 2 h; Adding enzyme-labeled secondary antibody: Wash the plate 4 times with PBST, add 100 μL of HRP-labeled goat anti-rabbit Ig secondary antibody diluted 1:10000 to each well, and place at 37 °C for 30 min; Color development and termination: Wash the plate 4 times with PBST, add 100 μL of substrate chromogenic solution to each well, and react at 37 °C in the dark for 15 min; 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.

[0035] 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 T3 sandwich monoclonal antibody mAb3 is 7×10 8 L / mol.

[0036] Example 4 Analysis of the variable region genes and amino acid sequences of rabbit monoclonal antibody mAb8 Using the recombinant plasmid of mAb8 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 perform sequencing using the sequencer ABI 3730. The nucleotide sequences of the light chain and heavy chain variable regions of rabbit monoclonal antibody mAb8 are obtained by sequencing.

[0037] 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 and heavy chain respectively. The light chain amino acid sequence of rabbit monoclonal antibody mAb8 is shown as SEQ ID NO.7, and the heavy chain amino acid sequence is shown as SEQ ID NO.8.

[0038] The full length of the light chain variable region is 106 amino acids. The numbers of amino acids in the 4 domains of FR are 26, 17, 36, and 10 respectively. The numbers of amino acids in 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 QGVYNVDY (SEQ ID NO.1), RTS (SEQ ID NO.2), and LGSYDD (SEQ ID NO.3).

[0039] The full length of the heavy chain variable region is 108 amino acids. The numbers of amino acids in the 4 domains of FR are 24, 17, 36, and 11 respectively. The numbers of amino acids in the 3 domains of HCDR are 8, 6, and 6 respectively. HCDR1, HCDR2, and HCDR3 are 25aa - 32aa, 50aa - 55aa, and 92aa - 97aa respectively, and their amino acid sequences are GFTISNYD (SEQ ID NO.4), ISNNGN (SEQ ID NO.5), and AREISG (SEQ ID NO.6).

[0040] Identification of HRP - labeled rabbit monoclonal antibody mAb8 as a secondary antibody in Example 5 (1) HRP - labeled rabbit monoclonal antibody mAb8 1. Rabbit monoclonal antibody mAb8 is dissolved in sodium bicarbonate solution with pH 9.6; 2. A certain mass of HRP is dissolved in deionized water, sodium periodate is added and reacted for 30 min, ethylene glycol is added and reacted for another 30 min, and then dialyzed overnight; 3. A certain amount of sodium borohydride is weighed and dissolved in deionized water, sodium borohydride is added to the cross - linked antibody - HRP solution and reacted for 2 h, and then dialyzed overnight; 4. The obtained HRP - labeled antibody is added with an equal amount of glycerol and stored at - 20 °C.

[0041] (2) Western blot identification of HRP - labeled rabbit monoclonal antibody mAb8 Western blot (WB) detection is used. 100 ng of mIgG2b antibody is taken for SDS - PAGE. After membrane transfer, HRP - labeled rabbit monoclonal antibody mAb8 is incubated for WB detection.

[0042] The results show that HRP - labeled rabbit monoclonal antibody mAb8 can well recognize the Fc region of mIgG2b, 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 mAb8 Coat the ELISA plate with goat anti-mouse IgG and incubate overnight at 4°C. Take out the ELISA plate the next day, wash it once with PBST, block it with 1% BSA solution at 37°C for 2 hours, and wash it 3 times with PBST. Add 100 μl of mIgG2b antibody with a concentration of 0.2 μg / ml to each well and incubate at 37°C for 1 hour. After incubation, take out the ELISA plate, wash it 3 times with PBST, add HRP-labeled mAb8 as the detection antibody, serially dilute it 7-fold starting from 1 μg / ml, and incubate at 37°C for 1 hour. After incubation, take out the ELISA plate, wash it 5 times with PBST, add TMB substrate, and develop color at 37°C for 10 min. Add the stop solution after taking it out and measure the OD450 reading on an ELISA reader. The results are shown in Table 1.

[0044] Table 1 ELISA detection results of HRP-labeled rabbit anti-mIgG2b mAb8 specifically recognizing mIgG2b antibody The results showed that HRP-labeled rabbit anti-mIgG2b mAb8 as the secondary antibody could detect the mIgG2b antibody signal well, which was better than the commercial goat anti-mouse IgG polyclonal antibody (as the secondary antibody, it could detect mIgG1, mIgG2a, mIgG2b, and mIgG3 simultaneously).

[0045] This specific embodiment is only an explanation of the present application and does not limit the present application. Those skilled in the art can make modifications to this embodiment without creative contributions according to their needs after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A rabbit monoclonal antibody mAb8 or an antigen-binding fragment thereof against mouse immunoglobulin G2b subtype (IgG2b), characterized in that: The rabbit monoclonal antibody comprises a light chain variable region, wherein the light chain variable region comprises LCDR1-3, wherein LCDR1 comprises an amino acid sequence QGVYNVDY, wherein LCDR2 comprises an amino acid sequence RTS, and wherein LCDR3 comprises an amino acid sequence LGSYDD.

2. The rabbit monoclonal antibody mAb8 or antigen-binding fragment according to claim 1, characterized in that: The rabbit monoclonal antibody mAb8 or the antigen-binding fragment also includes a heavy chain variable region, wherein the heavy chain variable region includes HCDR1-3, wherein the HCDR1 includes the amino acid sequence GFTISNYD, the HCDR2 includes the amino acid sequence ISNNGN, and the HCDR3 includes the amino acid sequence AREISG.

3. The rabbit monoclonal antibody mAb8 or 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 is at least 90% identical to 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 is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

8.

4. The rabbit monoclonal antibody mAb8 or 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 mAb8 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 mAb8 or the antigen-binding fragment according to any one of claims 1 to 4.

6. The method for preparing the rabbit monoclonal antibody mAb8 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 mAb8 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 mAb8 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 G2b subtype (IgG2b) for non-diagnostic and therapeutic purposes; 2) Preparation of products for detecting mouse immunoglobulin G2b subtype (IgG2b); 3) Used to purify mouse immunoglobulin G2b subtype (IgG2b); 4) Preparation of products for purification of mouse immunoglobulin G2b subtype (IgG2b).

9. A detection reagent or a detection kit, characterized in that: The detection reagent or detection kit comprises the rabbit monoclonal antibody mAb8 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) and immunoprecipitation, etc.

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