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

Abstract

The present invention belongs to the field of immunoassay technology and discloses a rabbit anti-human immunoglobulin G2 subtype (IgG2) specific monoclonal antibody mAb48. The monoclonal antibody comprises a light chain variable region and a heavy chain variable region. The light chain variable region includes LCDR1-3, wherein LCDR1 includes the amino acid sequence EDIGYG, the LCDR2 includes the amino acid sequence GAS, and the LCDR3 includes the amino acid sequence QQGFS; the heavy chain variable region includes HCDR1-3, wherein HCDR1 includes the amino acid sequence GFSLSNYE, the HCDR2 includes the amino acid sequence ISSSGST, and the HCDR3 includes the amino acid sequence ARNNNN. The monoclonal antibody of the present application can bind to the human immunoglobulin G2 subtype (IgG2) with high specificity and can be used in immunoassays, providing a basis for the preparation of engineered 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 among the most commonly used tools in the life sciences, used to study proteins and their functions in various biological pathways and diseases. Currently, the most widely used antibody species in scientific research and medicine include human, mouse, and rabbit antibodies. These antibodies can specifically recognize the target of interest, and the signal is then visualized using enzyme-labeled or fluorescein-conjugated secondary antibodies. Currently, anti-human IgG and anti-mouse IgG labeled antibodies are widely used in enzyme-linked immunosorbent assays (ELISA), Western blots, immunohistochemistry (IHC), flow cytometry (FCM), and immunoprecipitation. Commonly used labeling methods include horseradish peroxidase (HRP), alkaline phosphatase, biotin, and fluorescein.

[0003] Currently, the primary method for producing anti-human IgG antibodies is to directly immunize rabbits, sheep, donkeys, or other animals with IgG immunoglobulins to produce polyclonal antibodies, which are then purified for use. However, to develop antibodies specific for different types of IgG heavy and light chains, polyclonal antibodies are far from sufficient. Monoclonal antibodies, particularly those targeting different subtypes of hIgG, are required.

[0004] Human IgG monoclonal antibody heavy chains include four subtypes: IgG1, IgG2, IgG3, and IgG4. Light chains include both κ and λ chains, with a κ:λ ratio of approximately 6:4. Currently, the market primarily consists of polyclonal antibodies, such as goat, sheep, and donkey polyclonal antibodies. Monoclonal antibodies are rare, and in particular, rabbit anti-hIgG2-specific monoclonal antibodies are still lacking.

[0005] In order to meet the demand for hIgG2-specific monoclonal antibodies, there is an urgent need to develop monoclonal antibodies with high specificity and high sensitivity 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 immunoassays, 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 a rabbit monoclonal antibody mAb48 or an antigen-binding fragment thereof against human immunoglobulin G2 subtype (IgG2), 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, 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 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 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 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 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 containing the polynucleotide.

[0013] On the other hand, the present application also provides a host cell, which 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 antigen-binding fragment thereof, wherein the use is any of the following:

[0015] 1) Used to detect human immunoglobulin G2 subtype;

[0016] 2) A detection reagent or kit for preparing human immunoglobulin G2 subtype.

[0017] 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.

[0018] Furthermore, the detection kit is used for double antibody sandwich ELISA detection or chemiluminescence detection, and the rabbit monoclonal antibody mAb48 or antigen-binding fragment is the detection antibody.

[0019] 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 The monoclonal antibody of the present application can bind to the human immunoglobulin G2 subtype (IgG2) with high specificity. The present invention also relates to the use 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. It is 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

[0020] Figure 1 Figure 3 is the electrophoresis diagram of the full-length amplified products of the heavy and light chains of the rabbit monoclonal antibody mAb48. M is the DNA molecular weight marker.

[0021] Figure 2 This is the 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.

[0022] Figure 3 This is the ELISA test result of rabbit monoclonal antibody mAb48 specifically recognizing hIgG2 antibody.

[0023] Figure 4 This is the Western blot detection result of horseradish peroxidase-labeled rabbit monoclonal antibody mAb48 specifically recognizing natural hIgG2 antibody. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, conditions described in laboratory manuals, or conditions recommended by the manufacturer.

[0025] Example 1 Preparation of human IgG2 rabbit monoclonal antibody

[0026] 1) Preparation of immunogen

[0027] The hIgG2 antibody from abcam was purchased as the immunogen.

[0028] 2) Animal immunization

[0029] The hIgG2 antibody purchased above was emulsified in complete Freund's adjuvant and administered subcutaneously to immunize approximately 2 kg New Zealand white rabbits at a dose of 500 μg per rabbit. Two weeks later, a second immunization was performed using incomplete Freund's adjuvant at a dose of 250 μg per rabbit. After both immunizations, tail blood was collected and serum titers were determined by serial dilution ELISA. The OD450 value (OD450) was greater than 1.0 at an ELISA titer of 128,000, and the decision to collect PBMCs or continue immunization was made based on the results. PBMCs were collected from rabbits with the highest antibody titers.

[0030] 3) PBMCs isolation, specific B cell sorting, and cloning and recombination

[0031] The rabbit was fixed in supine position on the operating table, the hair at the heart area was trimmed, the skin was disinfected with alcohol, and a 50ml syringe was used to puncture the most obvious heartbeat. Blood flowed into the syringe after the needle penetrated 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. After mixing with an equal amount of PBS, the tube was slowly added dropwise to the lymphocyte separation solution. The tube was centrifuged at room temperature at 400×g for 30 minutes. After centrifugation, the liquid surface was divided into four layers from top to bottom: a yellow plasma layer, a white film layer (mononuclear cell layer), a separation solution layer, and a 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.

[0032] Antigen-specific B cells were further isolated from rabbit PBMCs and cultured. 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. A rabbit monoclonal antibody expression vector was constructed by cloning and recombination methods, and the sequence was confirmed by sequencing. The results of the amplified full-length PCR product are shown in Figure 2. Figure 1 .

[0033] 4) Preparation and purification of monoclonal antibodies

[0034] To generate a rabbit monoclonal antibody that recognizes human hIgG2, 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 recombinant rabbit monoclonal antibody that recognizes human hIgG2. The cell suspension was harvested, centrifuged, and the supernatant was purified by affinity chromatography. The purified antibody concentration was determined by the BCA assay, and the purified antibody was aliquoted and lyophilized. The purified antibody was designated human IgG2 rabbit monoclonal antibody mAb48.

[0035] Example 2 Specificity Identification of Anti-hIgG2 Rabbit Monoclonal Antibody mAb48

[0036] 1) Western blot identification of rabbit monoclonal antibody mAb48

[0037] Western blot (WB) was used for detection. One sample of each hIgG1, hIgG2, hIgG3, and hIgG4 subtype was selected, and 100 ng of each protein was loaded on SDS-PAGE. After transfer to a membrane, WB was performed.

[0038] The results showed that the rabbit monoclonal antibody mAb48 could specifically recognize hIgG2, but not hIgG1, hIgG3, and hIgG4. Figure 2 .

[0039] 2) Rabbit monoclonal antibody mAb48 ELISA identification

[0040] ELISA plates were coated with hIgG1, hIgG2, hIgG3, and hIgG4 antibodies at 4°C overnight. The next day, the plates were removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. Rabbit monoclonal antibody MAB48 (100 μl) 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 plates were removed, washed three times with PBST, and HRP-conjugated goat anti-rabbit secondary antibodies were added as detection antibodies, respectively, and incubated at 37°C for 1 hour. After incubation, the plates were removed, washed five times with PBST, and TMB substrate was added for color development at 37°C for 10 minutes. Stop solution was added, and OD450 readings were measured on a microplate reader.

[0041] The results showed that the rabbit monoclonal antibody mAb48 could specifically recognize hIgG2, but not hIgG1, hIgG3, and hIgG4. Figure 3 .

[0042] Example 3 Affinity Identification of Anti-hIgG2 Rabbit Monoclonal Antibody mAb48

[0043] The affinity constant (Ka) was determined by non-competitive ELISA.

[0044] 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.

[0045] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h;

[0046] Adding monoclonal antibodies: Wash the plate four times with PBST, dilute the rabbit monoclonal antibody starting at 100 μg / mL in carbonate buffer, add 100 μL to each well, and incubate at 37°C for 2 h.

[0047] Add enzyme-labeled secondary antibody: wash the plate four 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;

[0048] Color development and termination: Wash the plate four 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;

[0049] Detection: Measure the absorbance value at a wavelength of 450 nm (A450nm).

[0050] The logarithm of the antibody concentration was used as the horizontal axis and the OD value was used as the vertical axis to draw an S-shaped curve. After calculation, the affinity constant Ka of the monoclonal antibody mAb3 in the T3 sandwich method was 9×10 8 L / mol.

[0051] Example 4 Analysis of the Gene and Amino Acid Sequences of the Variable Regions of Rabbit Monoclonal Antibody mAb48: Using the recombinant plasmid of the mAb48 antibody as a DNA template, sequencing primers for the light 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 mAb48 were obtained through sequencing.

[0052] The nucleotide sequences of the light chain and heavy chain were sequenced and analyzed using IMGT / V-QUEST analysis software on the Internet at http: / / www.imgt.org. The light chain amino acid sequence of the rabbit monoclonal antibody mAb48 was shown in SEQ ID NO. 7, and the heavy chain amino acid sequence was shown in SEQ ID NO. 8.

[0053] The total length of the light chain variable region is 104 amino acids, with the four FR domains containing 26, 17, 36 and 11 amino acids respectively, the three LCDR domains containing 6, 3 and 5 amino acids respectively, the LCDR1, LCDR2 and LCDR3 regions containing 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.

[0054] The heavy chain variable region is 112 amino acids in length. The amino acid numbers of the four FR domains are 25, 17, 38, and 10, respectively. The amino acid numbers of the three HCDR domains are 8, 7, and 6, respectively. HCDR1, HCDR2, and HCDR3 are 26aa-33aa, 51aa-57aa, and 96aa-101aa, respectively. Their amino acid sequences are GFSLSNYE (SEQ ID NO. 4), ISSSGST (SEQ ID NO. 5), and ARNNNN (SEQ ID NO. 6).

[0055] Example 5 Identification of HRP-labeled rabbit monoclonal antibody mAb48 as a secondary antibody

[0056] (1) HRP-labeled rabbit monoclonal antibody mAb48

[0057] 1. Rabbit monoclonal antibody mAb48 was dissolved in sodium bicarbonate solution at pH 9.6;

[0058] 2. Dissolve a certain amount of HRP in deionized water, add sodium periodate and react for 30 minutes, add ethylene glycol and continue to react for 30 minutes, and dialyze overnight;

[0059] 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.

[0060] 4. Add the obtained HRP-labeled antibody to an equal amount of glycerol and store at -20 degrees Celsius.

[0061] (2) Western blot identification of HRP-labeled rabbit monoclonal antibody mAb48

[0062] Western blot (WB) was used for detection. 100 ng of hIgG2 antibody was subjected to SDS-PAGE, transferred to a membrane, and incubated with HRP-labeled rabbit monoclonal antibody mAb48 for WB detection.

[0063] The results showed that HRP-labeled rabbit monoclonal antibody mAb48 could well recognize the hIgG2 Fc region with a molecular weight of about 55KD. Figure 4 .

[0064] (3) ELISA identification of HRP-labeled rabbit monoclonal antibody mAb48

[0065] ELISA plates were coated with goat anti-mouse IgG overnight at 4°C. The next day, the plates were removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. 100 μl of hIgG2 antibody was added to each well at a concentration of 0.2 μg / ml and incubated at 37°C for 1 hour. After incubation, the plates were removed and washed three times with PBST. HRP-labeled mAb48 was added as the detection antibody in seven serial dilutions starting at 1 μg / ml and incubated at 37°C for 1 hour. After incubation, the plates were removed and washed five times with PBST. TMB substrate was added and color was developed at 37°C for 10 minutes. Stop solution was added and OD450 readings were measured on a microplate reader. The results are shown in Table 1.

[0066] Table 1 ELISA test results of HRP-labeled rabbit anti-hIgG2 mAb48 specifically recognizing hIgG2 antibody

[0067]

[0068] The results showed that HRP-labeled rabbit anti-hIgG2 mAb48 could effectively detect hIgG2 antibody signals 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).

[0069] 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 non-creative modifications to the present embodiment 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 mAb48 against human immunoglobulin G2 subtype (IgG2) or an antigen-binding fragment thereof, characterized in that: The rabbit monoclonal antibody includes a light chain variable region, which includes LCDR1-3, the amino acid sequence of LCDR1 is EDIGYG, the amino acid sequence of LCDR2 is GAS, and the amino acid sequence of LCDR3 is QQGFS; the rabbit monoclonal antibody mAb48 or its antigen-binding fragment also includes a heavy chain variable region, which includes HCDR1-3, the amino acid sequence of HCDR1 is GFSLSNYE, the amino acid sequence of HCDR2 is ISSSGST, and the amino acid sequence of HCDR3 is ARNNNN.

2. The rabbit monoclonal antibody mAb48 or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the light chain variable region 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.

3. An isolated polynucleotide, characterized in that The isolated polynucleotide encodes the rabbit monoclonal antibody mAb48 or an antigen-binding fragment thereof according to any one of claims 1-2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the polynucleotide according to claim 3.

5. A host cell, characterized in that The host cell contains the polynucleotide according to claim 3 or the recombinant expression vector according to claim 4.

6. Use of the rabbit monoclonal antibody mAb48 or an antigen-binding fragment thereof according to any one of claims 1-2, characterized in that: The application is for preparing a detection reagent or kit for human immunoglobulin G2 subtype.

7. 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 an antigen-binding fragment thereof according to any one of claims 1-2.

8. The detection reagent or detection kit according to claim 7, characterized in that The detection kit is used for double-antibody sandwich ELISA detection or chemiluminescence detection, and the rabbit monoclonal antibody mAb48 or its antigen-binding fragment is the detection antibody.

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