Human immunoglobulin G2 subtype (IgG2) rabbit monoclonal antibody and application thereof

Abstract

The invention belongs to the technical field of immunodetection, and discloses a specific monoclonal antibody mAb48 of rabbit anti-human immunoglobulin G2 subtype (IgG2), the specific monoclonal antibody mAb48 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 EDIGYG, the LCDR2 comprises an amino acid sequence GAS, and the LCDR3 comprises an amino acid sequence QQGFS; the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises an amino acid sequence GFSLSNYE, the HCDR2 comprises an amino acid sequence ISSSGST, and the HCDR3 comprises an amino acid sequence ARNNNN. The monoclonal antibody disclosed by the invention can be combined with human immune globulin G2 subtype (IgG2) in a high-specificity manner, can be applied to immunodetection, and provides a basis for the preparation of engineering antibodies in the next step.

Description

Technical Field

[0001] The present application relates to the field of immune detection technology, and in particular to human immunoglobulin G2 subtype (IgG2) rabbit monoclonal antibody and its application. Background Art

[0002] Affinity binders / antibodies are one of the most commonly used tools in life sciences 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 human antibodies, mouse antibodies, and rabbit antibodies. These antibodies can specifically recognize the target of interest, and then display the signal through enzyme-labeled secondary antibodies or fluorescein-labeled secondary antibodies. At present, anti-human IgG and anti-mouse IgG labeled antibodies are widely used in the market, and can be used in enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemistry staining (IHC), flow cytometry (FCM) and immunoprecipitation, etc. The most commonly used labeling methods include horseradish peroxidase (HRP), alkaline phosphatase, biotin, fluorescein, etc.

[0003] At present, the main method for preparing anti-human IgG antibodies is to directly immunize rabbits, sheep, donkeys and other animals with IgG immunoglobulins to produce immunological clear polyclonal antibodies, which are then purified and used. However, in the development of different types of specific antibodies for IgG heavy chains and light chains, polyclonal antibodies are far from enough, and monoclonal antibodies need to be prepared, especially monoclonal antibodies against different subtypes of hIgG.

[0004] The heavy chain of human IgG monoclonal antibody includes four subtypes: IgG1, IgG2, IgG3, and IgG4, and the light chain includes two types: κ chain and λ chain, with the ratio of κ chain to λ chain being about 6:4. Currently, most of the antibodies on the market are polyclonal antibodies, such as goat polyclonal antibodies, sheep polyclonal antibodies, and donkey polyclonal antibodies, and there are very few monoclonal antibodies, especially rabbit anti-hIgG2 specific monoclonal antibodies.

[0005] In order to meet the demand for hIgG2-specific monoclonal antibodies, it is urgently necessary to develop highly specific and highly sensitive monoclonal antibodies against hIgG2. Summary of the invention

[0006] In order to overcome the above technical problems, the present application provides an anti-hIgG2 rabbit monoclonal antibody mAb48 with good specificity and high affinity and its application in immunoassay, including but not limited to chemiluminescence, fluorescence and colorimetric detection of 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 engineering antibodies in the next step.

[0007] On the one hand, the present application provides an anti-human immunoglobulin G2 subtype (IgG2) rabbit monoclonal antibody mAb48 or an antigen-binding fragment, wherein the rabbit monoclonal antibody comprises a light chain variable region, wherein the light chain variable region comprises LCDR1-3, wherein the LCDR1 comprises the amino acid sequence EDIGYG (SEQ ID NO.1), the LCDR2 comprises the amino acid sequence GAS (SEQ ID NO.2), and the LCDR3 comprises the amino acid sequence QQGFS (SEQ ID NO.3).

[0008] Furthermore, the rabbit monoclonal antibody mAb48 or 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 GFSLSNYE (SEQ ID NO.4), the HCDR2 includes the amino acid sequence ISSSGST (SEQ ID NO.5), and the HCDR3 includes the amino acid sequence ARNNNN (SEQ ID NO.6).

[0009] Furthermore, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO.7.

[0010] Furthermore, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO.8.

[0011] On the other hand, the present application also provides an isolated polynucleotide encoding the rabbit monoclonal antibody mAb48 or the antigen-binding fragment.

[0012] On the other hand, the present application also provides a recombinant expression vector, which contains the polynucleotide.

[0013] On the other hand, the present application also provides a host cell, wherein the host cell contains the polynucleotide or the recombinant expression vector.

[0014] On the other hand, the present application also provides the use of the rabbit monoclonal antibody mAb48 or the antigen-binding fragment, wherein the use is any of the following: 1) Used to detect human immunoglobulin G2 subtype; 2) A detection reagent or kit for preparing human immunoglobulin G2 subtype.

[0015] On the other hand, the present application also provides a detection reagent or detection kit for detecting human immunoglobulin G2 subtypes, wherein the detection reagent or detection kit contains the rabbit monoclonal antibody mAb48 or the antigen-binding fragment.

[0016] Furthermore, the detection kit is used for double antibody sandwich ELISA detection or chemiluminescence detection kit, and the rabbit monoclonal antibody mAb48 or antigen-binding fragment is the detection antibody.

[0017] Compared with the prior art, the hIgG2 rabbit monoclonal antibody of the present application can bind to hIgG2 with high specificity and high affinity, and its affinity constant Ka can reach 9×10 8 L / mol. The monoclonal antibody of the present application can bind to human immunoglobulin G2 subtype (IgG2) with high specificity. The present invention also relates to the application of anti-human immunoglobulin G2 subtype (IgG2) specific rabbit monoclonal antibody in immunoassay tools, including but not limited to chemiluminescence, fluorescence and colorimetric detection of 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 engineering antibodies in the next step. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the electrophoresis diagram of the full-length amplified products of the heavy chain and light chain of the rabbit monoclonal antibody mAb48, and M is the DNA molecular weight marker.

[0019] Figure 2 This is a Western blot test result of rabbit monoclonal antibody mAb48 specifically recognizing natural hIgG2 antibody. The samples in lanes 1-4 are hIgG1, hIgG2, hIgG3, and hIgG4, respectively.

[0020] Figure 3 This is the ELISA test result of rabbit monoclonal antibody mAb48 specifically recognizing hIgG2 antibody.

[0021] Figure 4 This is a Western blot test result showing horseradish peroxidase-labeled rabbit monoclonal antibody mAb48 specifically recognizing natural hIgG2 antibody. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by manufacturers.

[0023] Example 1 Preparation of human IgG2 rabbit monoclonal antibody 1) Preparation of immunogen The hIgG2 antibody from abcam was purchased as the immunogen.

[0024] 2) Animal immunization The hIgG2 antibody purchased above was emulsified with complete Freund's adjuvant, and New Zealand white rabbits weighing about 2 kg were immunized by subcutaneous injection, with an immunization dose of 500 μg / rabbit. The second immunization was performed two weeks later, emulsified with incomplete Freund's adjuvant, with an immunization dose of 250 μg / rabbit. After two immunizations, tail blood was collected and serum titer was determined by gradient dilution using the ELISA method; based on the OD450 greater than 1.0 when the ELISA titer was 128,000, it was determined whether to collect PBMCs or continue immunization based on the results, and the rabbit with the highest antibody titer was selected for PBMCs collection.

[0025] 3) PBMCs isolation, specific B cell sorting, and cloning recombination The rabbit was fixed on the operating table in supine position, the hair at the heart area was trimmed, the skin was disinfected with alcohol, and the most obvious heartbeat was selected for puncture with a 50ml syringe. Blood flowed into the syringe after the needle pierced the heart. After obtaining the required amount of blood, the needle was quickly pulled out, and the whole blood in the syringe was transferred to a sterile 50ml tube, mixed with an equal amount of PBS, and then slowly added drop by drop to the lymphocyte separation solution. Centrifuged at room temperature at 400×g for 30 minutes. 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 to obtain rabbit PBMCs.

[0026] Antigen-specific B cells were further isolated from rabbit PBMCs for culture. The supernatant of the cultured B cells was screened for positive clones using an antigen-coated ELISA plate. The full-length sequences of the naturally paired rabbit monoclonal antibody light and heavy chains were amplified from the cDNA of the corresponding positive clones, 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 product are shown in Figure 2. Figure 1 .

[0027] 4) Preparation and purification of monoclonal antibodies In order to obtain rabbit monoclonal antibodies that recognize human hIgG2 protein, the heavy chain and light chain genes of rabbit monoclonal antibodies were loaded on the expression vector, and the plasmid was transfected into HEK293 cells; after transfection for 120-144 hours, the culture supernatant was obtained to contain recombinant rabbit monoclonal antibodies that recognize human hIgG2 protein. The cell suspension was collected, the supernatant was centrifuged, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then the purified antibody was aliquoted and lyophilized. The purified antibody was named human IgG2 rabbit monoclonal antibody mAb48.

[0028] Example 2 Specificity Identification of Anti-hIgG2 Rabbit Monoclonal Antibody mAb48 1) Western blot identification of rabbit monoclonal antibody mAb48 Western blot (WB) was used for detection. One sample was selected for each subtype of hIgG1, hIgG2, hIgG3, and hIgG4, and 100 ng of each protein was loaded for SDS-PAGE, and WB detection was performed after transfer.

[0029] The results showed that the rabbit monoclonal antibody mAb48 could specifically recognize hIgG2, but not hIgG1, hIgG3, and hIgG4. Figure 2 .

[0030] 2) Rabbit monoclonal antibody mAb48 ELISA identification The ELISA plate was coated with hIgG1, hIgG2, hIgG3, and hIgG4 antibodies at 4°C overnight; the next day, the ELISA 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 MAB48 was added to each well, with concentrations of 10, 3, 1, 0.3, and 0 ng / ml, respectively, and incubated at 37°C for 1 hour; after the incubation, the ELISA plate was removed, washed three times with PBST, and HRP-labeled goat anti-rabbit secondary antibodies were added as detection antibodies, respectively, and incubated at 37°C for 1 hour; after the incubation, the ELISA plate was removed, washed five times with PBST, and TMB substrate was added, and color was developed at 37°C for 10 minutes. After removal, stop solution was added, and OD450 readings were measured on an ELISA reader.

[0031] The results showed that the rabbit monoclonal antibody mAb48 could specifically recognize hIgG2, but not hIgG1, hIgG3, and hIgG4. Figure 3 .

[0032] Example 3 Affinity Identification of Anti-hIgG2 Rabbit Monoclonal Antibody mAb48 The affinity constant (Ka) was determined by non-competitive ELISA.

[0033] Coating: Dilute the antigen with carbonate buffer to a concentration of 1, 0.5, 0.1, and 0.05 μg / mL, add 100 μL / well to a 96-well ELISA plate for coating, and incubate at 4°C for 24 h; Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h; Adding monoclonal antibody: Wash the plate 4 times with PBST, dilute the rabbit monoclonal antibody starting at 100 μg / mL with carbonate buffer, add 100 μL to each well, and incubate at 37°C for 2 h; Add enzyme-labeled secondary antibody: wash the plate 4 times with PBST, add 100 μL of 1:10000 diluted HRP enzyme-labeled goat anti-rabbit Ig secondary antibody 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 color development solution to each well, and react at 37°C in the dark for 15 min; add 50 μL of 1.0 mol / L H2SO4 stop solution to each well to terminate the reaction; Detection: Measure the absorbance value at a wavelength of 450nm (A450nm).

[0034] The S-shaped curve was drawn with the logarithm of antibody concentration as the horizontal axis and the OD value as the vertical axis. After calculation, the affinity constant Ka of the monoclonal antibody mAb3 in the T3 sandwich method was 9×10 8 L / mol.

[0035] Example 4 Analysis of variable region genes and amino acid sequences of rabbit monoclonal antibody mAb48 The recombinant plasmid of mAb48 antibody was used as a DNA template, and the sequencing primers of the light chain variable region and the heavy chain variable region were designed according to the vector sequences at the 5' end of the light chain and the heavy chain on the template, and sequencing was performed using a sequencer ABI 3730. The nucleotide sequences of the light chain and heavy chain variable regions of the rabbit monoclonal antibody mAb48 were obtained by sequencing.

[0036] Using the Internet, IMGT / V-QUEST analysis software was used at http: / / www.imgt.org to analyze the sequencing results of the nucleotide sequences of the light chain and the heavy chain, respectively, and the light chain amino acid sequence of the rabbit monoclonal antibody mAb48 was obtained as shown in SEQ ID NO.7, and the heavy chain amino acid sequence was shown in SEQ ID NO.8.

[0037] The total length of the light chain variable region is 104 amino acids, the amino acid numbers of the four FR domains are 26, 17, 36 and 11 respectively, the amino acid numbers of the three LCDR domains are 6, 3 and 5 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-93aa respectively, and their amino acid sequences are EDIGYG (SEQ ID NO.1), GAS (SEQID NO.2) and QQGFS (SEQ ID NO.3), respectively.

[0038] The total length of the heavy chain variable region is 112 amino acids, the amino acid numbers of the four domains of FR are 25, 17, 38 and 10 respectively, the amino acid numbers of the three domains of HCDR are 8, 7 and 6 respectively, HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-101aa respectively, and their amino acid sequences are GFSLSNYE (SEQ ID NO.4), ISSSGST (SEQ ID NO.5), ARNNNN (SEQ ID NO.6) Example 5 Identification of HRP-labeled rabbit monoclonal antibody mAb48 as a secondary antibody (1) HRP-labeled rabbit monoclonal antibody mAb48 1. Rabbit monoclonal antibody mAb48 was dissolved in sodium bicarbonate solution at pH 9.6; 2. Take a certain amount of HRP and dissolve it in deionized water. Add sodium periodate to react for 30 minutes. Add ethylene glycol to continue the reaction for 30 minutes. Dialyze overnight. 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. 4. Add the obtained HRP-labeled antibody to an equal amount of glycerol and store at -20 degrees Celsius.

[0039] (2) Western blot identification of HRP-labeled rabbit monoclonal antibody mAb48 Western blot (WB) was used for detection. 100 ng of hIgG2 antibody was taken for SDS-PAGE, and after transfer to the membrane, it was incubated with HRP-labeled rabbit monoclonal antibody mAb48 for WB detection.

[0040] The results showed that the HRP-labeled rabbit monoclonal antibody mAb48 could well recognize the hIgG2 Fc region with a molecular weight of about 55KD. Figure 4 .

[0041] (3) ELISA identification of HRP-labeled rabbit monoclonal antibody mAb48 The ELISA plate was coated with goat anti-mouse IgG and incubated at 4°C overnight. The next day, the ELISA plate was removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed 3 times with PBST. 100ul of hIgG2 antibody was added to each well at a concentration of 0.2ug / ml and incubated at 37°C for 1 hour. After the incubation, the ELISA plate was removed, washed 3 times with PBST, and HRP-labeled mAb48 was added as the detection antibody, with 7 dilution gradients starting from 1ug / ml, and incubated at 37°C for 1 hour. After the incubation, the ELISA plate was removed, washed 5 times with PBST, and TMB substrate was added, and color was developed at 37°C for 10min. After removal, the stop solution was added and the OD450 reading was measured on the ELISA reader. The results are shown in Table 1.

[0042] Table 1 ELISA test results of HRP-labeled rabbit anti-hIgG2 mAb48 specifically recognizing hIgG2 antibody The results showed that HRP-labeled rabbit anti-hIgG2 mAb48 could detect hIgG2 antibody signals well as a secondary antibody, which was superior to the commercial goat anti-mouse IgG polyclonal antibody (which could simultaneously detect hIgG1, hIgG2, hIgG3, and hIgG4 as a secondary antibody).

[0043] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A rabbit monoclonal antibody mAb48 or an antigen-binding fragment thereof against human immunoglobulin G2 subtype (IgG2), 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 EDIGYG, wherein LCDR2 comprises an amino acid sequence GAS, and wherein LCDR3 comprises an amino acid sequence QQGFS.

2. The rabbit monoclonal antibody mAb48 or the antigen-binding fragment according to claim 1, characterized in that: The rabbit monoclonal antibody mAb48 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 GFSLSNYE, the HCDR2 includes the amino acid sequence ISSSGST, and the HCDR3 includes the amino acid sequence ARNNNN.

3. The rabbit monoclonal antibody mAb48 or the antigen-binding fragment according to claim 1, characterized in that: The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO.

7.

4. The rabbit monoclonal antibody mAb48 or the antigen-binding fragment according to claim 1, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO.

8.

5. An isolated polynucleotide, characterized in that The isolated polynucleotide encodes the rabbit monoclonal antibody mAb48 or the antigen-binding fragment thereof according to any one of claims 1-4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector contains the polynucleotide according to claim 5.

7. A host cell, characterized in that The host cell contains the polynucleotide according to claim 5 or the recombinant expression vector according to claim 6.

8. Use of the rabbit monoclonal antibody mAb48 or the antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The application is any of the following, 1) Used to detect human immunoglobulin G2 subtype; 2) A reagent or kit for preparing a detection reagent of human immunoglobulin G2 subtype.

9. A detection reagent or detection kit for detecting human immunoglobulin G2 subtype, characterized in that: The detection reagent or detection kit contains the rabbit monoclonal antibody mAb48 or the antigen-binding fragment according to any one of claims 1 to 4.

10. The detection reagent or detection kit according to claim 6, characterized in that: The detection kit is used for double antibody sandwich ELISA detection or chemiluminescence detection kit, and the rabbit monoclonal antibody mAb48 or antigen binding fragment is the detection antibody.

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