Rabbit monoclonal antibody mAb6 against murine immunoglobulin G2a subtype (IgG2a) and uses thereof

Abstract

The application belongs to the technical field of immune detection, and discloses rabbit monoclonal antibody mAb6 against mouse immunoglobulin 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 of the application can be combined with mouse immunoglobulin G2a subtype (IgG2a) with high specificity, can be applied to immune detection, and provides a basis for preparation of a next-step engineering antibody.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay technology, in particular to rabbit monoclonal antibody mAb6 against murine immunoglobulin G2a subtype (mIgG2a) and its application. BACKGROUND

[0002] Affinity binders / 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 medical fields include human antibodies, mouse 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-mouse IgG and anti-human IgG labeled antibodies are widely used in the market, which can be applied to enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation, etc. The most commonly used labeling methods include horseradish peroxidase (HRP), alkaline phosphatase, biotin, and fluorescein, etc.

[0003] Currently, the main method for preparing anti-mouse IgG antibodies is to directly immunize rabbits or sheep or donkeys with IgG immunoglobulin to produce immunological polyclonal antibodies, which are then purified and applied. However, polyclonal antibodies are far from enough for developing antibodies specific to different types of IgG heavy chains and light chains, and it is necessary to prepare monoclonal antibodies, especially monoclonal antibodies specific to different subtypes of IgG.

[0004] Mouse IgG monoclonal antibodies include four subtypes of IgG1, IgG2a, IgG2b, and IgG3, and two types of kappa chain and lambda chain, with a ratio of about 20:1. Currently, most of the antibodies on the market are polyclonal antibodies, such as goat polyclonal antibodies, sheep polyclonal antibodies, and donkey polyclonal antibodies, and there are very few monoclonal antibodies, especially no rabbit anti-mIgG2a specific monoclonal antibody has been reported.

[0005] In order to meet the demand for mIgG2a specific monoclonal antibodies, it is urgent to develop monoclonal antibodies specific to mIgG2a with high specificity and high sensitivity. SUMMARY

[0006] In order to overcome the above technical problems, the present application provides rabbit monoclonal antibody mAb6 specific to mIgG2a with high specificity and high sensitivity and its application in immunoassay, including but not limited to chemiluminescence, fluorescence, and colorimetric detection of primary antibodies, which is suitable for various applications, such as cell imaging, flow cytometry detection, Western blot, and immunohistochemistry, and also provides a basis for the preparation of next-generation engineered antibodies.

[0007] In one aspect, the present application provides a rabbit monoclonal antibody mAb6 or antigen binding fragment against mouse immunoglobulin G2a subtype (IgG2a), the light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO. 1, the LCDR2 comprising the amino acid sequence GTN, and the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO. 3.

[0008] In certain embodiments, the light chain variable region is 106 amino acids in length, the 4 domains of the FRs are 26, 17, 36 and 10 amino acids in length, respectively, the 3 domains of the LCDRs are 8, 3 and 6 amino acids in length, respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-34aa, 52aa-54aa and 91aa-96aa, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are KSVYKNNY (SEQ ID NO. 1), GTN (SEQ ID NO. 2), AGGYID (SEQ ID NO. 3), respectively.

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

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

[0011] In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 7 or an amino acid sequence that is at least 95% identical, preferably at least 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO. 7; and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 or an amino acid sequence that is at least 95% identical, preferably at least 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO. 8.

[0012] In some embodiments, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, diabody, and antibody minimal recognition unit; preferably, the antibody rest of the sequence is derived from one or more of rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

[0013] In another aspect, the present application also provides a biological material comprising a polynucleotide encoding the rabbit monoclonal antibody mAb6 or antigen-binding fragment; a vector carrying the polynucleotide; or a cell carrying the polynucleotide, or containing the vector, or capable of expressing the rabbit monoclonal antibody mAb6 or antigen-binding fragment.

[0014] In another aspect, the present application also provides a method for preparing the rabbit monoclonal antibody mAb6 or antigen-binding fragment, comprising culturing the cell; optionally, the cell is prepared by transforming a polynucleotide encoding the rabbit monoclonal antibody mAb6 or antigen-binding fragment into a cell, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, the transforming comprising 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] In another aspect, the present application also provides use of the rabbit monoclonal antibody mAb6 or antigen-binding fragment or the biological material in any of the following:

[0017] 1) detecting mouse immunoglobulin G2a subtype (IgG2a) for non-diagnostic and therapeutic purposes;

[0018] 2) preparing a product for detecting mouse immunoglobulin G2a subtype (IgG2a);

[0019] 3) purifying mouse immunoglobulin G2a subtype (IgG2a);

[0020] 4) preparing a product for purifying mouse immunoglobulin G2a subtype (IgG2a).

[0021] In another aspect, the present application also provides a detection reagent or a detection kit comprising the rabbit monoclonal antibody mAb6 or antigen-binding fragment, or the biological material.

[0022] In some embodiments, the detection kit is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM) and co-immunoprecipitation, etc.

[0023] Compared with the prior art, the mIgG2a rabbit monoclonal antibody of the present application can be specifically bound to mIgG2a with high specificity and high affinity, and the affinity constant Ka can reach 8x10 8 L / mol. The present application also relates to the application of the anti-murine immunoglobulin G2a subtype (IgG2a) specific rabbit monoclonal antibody in immunodetection tools, including but not limited to chemiluminescence, fluorescence and colorimetric detection for primary antibody, suitable for various applications such as cell imaging, flow cytometry, protein immunoblotting and immunohistochemistry, and also provides a basis for the preparation of next-step engineering antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is an electrophoresis map of the full-length amplification products of the heavy chain and light chain of the rabbit monoclonal antibody mAb6, and M is a DNA molecular weight marker.

[0025] Figure 2 It is a Western blot detection result map of the rabbit monoclonal antibody mAb6 specifically recognizing natural mIgG2a antibody.

[0026] Figure 3 It is an ELISA detection result map of the rabbit monoclonal antibody mAb6 specifically recognizing mIgG2a antibody.

[0027] Figure 4 It is a Western blot detection result map of the horseradish peroxidase-labeled rabbit monoclonal antibody mAb6 specifically recognizing natural mIgG2a antibody. DETAILED DESCRIPTION

[0028] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are generally carried out according to the conventional conditions, the conditions described in the laboratory manual or the conditions suggested by the manufacturer.

[0029] Example 1 Preparation of murine IgG2a rabbit monoclonal antibody

[0030] 1) Preparation of immunogen

[0031] Third-party mIgG2a antibody was purchased as an immunogen.

[0032] 2) Animal immunization

[0033] The above-mentioned purchased mlgG2a antibody was emulsified with complete Freund's adjuvant, and a New Zealand white rabbit weighing about 2 kg was immunized by subcutaneous injection, with an immunization dose of 500 μg per rabbit. Two weeks later, the second immunization was performed with incomplete Freund's adjuvant, with an immunization dose of 250 μg per rabbit. After two immunizations, the tail blood was taken to determine the serum titer by gradient dilution ELISA. According to the standard that the OD450 of ELISA titer 128000 is 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 PBMCs collection.

[0034] 3) PBMCs separation, specific B cell sorting, and cloning recombination

[0035] The rabbit was fixed on the operating table in a supine position, the fur around the heart was removed, the skin was disinfected with alcohol, and a 50 ml syringe was used to puncture the most obvious place of heart beat. After the needle was inserted into the heart, blood flowed into the syringe. After the required amount of blood was obtained, 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 amount of PBS, it was slowly added dropwise to the top of the lymphocyte separation medium, and centrifuged at 400 x 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 (single nucleus cell layer), separation medium layer and red blood cell layer. The single nucleus cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium to obtain rabbit PBMCs.

[0036] The antigen-specific B cells were further sorted from the rabbit PBMCs for culture, and 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 clone, and the rabbit monoclonal antibody expression vector was constructed by cloning recombination method, and the sequence was determined by sequencing. The results of amplification of the full-length PCR product are shown in Figure 1 .

[0037] 4) Preparation and purification of monoclonal antibody

[0038] In order to obtain a rabbit monoclonal antibody recognizing human mlgG2a protein, the rabbit monoclonal antibody heavy chain and light chain genes were loaded into an expression vector, and the plasmid was transfected into HEK293 cells. Recombinant rabbit monoclonal antibody recognizing human mlgG2a protein was obtained in the culture supernatant 120-144 hours after transfection. 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 BCA method, and then it was aliquoted, lyophilized, and the purified antibody was named as mouse IgG2a rabbit monoclonal antibody mAb6.

[0039] Example 2 Specificity identification of anti-mlgG2a rabbit monoclonal antibody mAb6

[0040] 1) Rabbit monoclonal antibody mAb6 Western blot identification

[0041] Western blot (WB) was used for detection. Two samples of each subtype of mlgGl, mlgG2a, mlgG2b, mlgG3 were selected, and 100 ng of each protein was subjected to SDS-PAGE, and then WB detection was performed after membrane transfer.

[0042] The results showed that the rabbit monoclonal antibody mAb6 could specifically recognize mlgG2a, but not mlgGl, mlgG2b, and mlgG3. The results are shown in Figure 2 .

[0043] 2) Rabbit monoclonal antibody mAb6 ELISA identification

[0044] mIgGl, mlgG2a, mlgG2b, and mlgG3 antibodies were coated on the enzyme-labeled plate at 4°C overnight. The next day, the enzyme-labeled plate was taken out, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed 3 times with PBST. 100 μl of rabbit monoclonal antibody MAB6100 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 enzyme-labeled plate was taken out, washed 3 times with PBST, and HRP-labeled goat anti-rabbit secondary antibody was added as a detection antibody, and incubated at 37°C for 1 hour. After incubation, the enzyme-labeled plate was taken out, washed 5 times with PBST, and TMB substrate was added, and color development was performed at 37°C for 10 min. After taking out, the stop solution was added, and the OD450 reading was measured on the enzyme-labeled instrument.

[0045] The results showed that the rabbit monoclonal antibody mAb6 could specifically recognize mlgG2a, but not mlgGl, mlgG2b, and mlgG3. The results are shown in Figure 3 .

[0046] Example 3 Affinity identification of anti-mlgG2a rabbit monoclonal antibody mAb6

[0047] The non-competitive ELISA method was used to determine the affinity constant (Ka).

[0048] Coating: The antigen was diluted with carbonate buffer to a concentration of 1, 0.5, 0.1, and 0.05 μg / mL, and added to a 96-well enzyme-labeled plate at 100 μL / well for coating, and incubated at 4°C for 24 h.

[0049] Blocking: The plate was washed 4 times with PBST, and BSA solution was added at 200 μL / well, and incubated at 37°C for 2 h.

[0050] Add monoclonal antibody: Wash the plate 4 times with PBST, dilute the rabbit monoclonal antibody with carbonate buffer starting from 100 μg / mL by doubling dilution, add 100 μL to each well, and incubate at 37°C for 2 h.

[0051] Addition of enzyme-labeled secondary antibody: wash the plate with PBST for 4 times, add 100 μL of 1:10000 dilution of HRP enzyme-labeled goat anti-rabbit Ig secondary antibody to each well, and place at 37°C for 30 min;

[0052] Color development and termination: wash the plate with PBST for 4 times, add 100 μL of substrate color development solution to each well, and place at 37°C for 15 min in the dark; add 50 μL of 1.0 mol / L H2SO4 termination solution to each well to terminate the reaction.

[0053] Detection: measure the absorbance value (A450nm) at 450 nm.

[0054] Taking the logarithm of the antibody concentration as the abscissa and the OD value as the ordinate, an S-shaped curve was drawn, and it was calculated that the affinity constant Ka of the anti-mIgG2a rabbit monoclonal antibody mAb6 was 8 x 10 8 L / mol.

[0055] Example 4 Analysis of variable region genes and amino acid sequences of rabbit monoclonal antibody mAb6

[0056] Using the Internet, the nucleotide sequences of the light chain variable region and the heavy chain variable region were analyzed using the IMGT / V-QUEST analysis software at http: / / www.imgt.org, and the amino acid sequence of the light chain variable region of the rabbit monoclonal antibody mAb6 was obtained as shown in SEQ ID NO. 7, and the amino acid sequence of the heavy chain variable region was obtained as shown in SEQ ID NO. 8.

[0057] 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, 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 the amino acid sequences thereof are KSVYKNNY (SEQ ID NO. 1), GTN (SEQ ID NO. 2) and AGGYID (SEQ ID NO. 3) respectively.

[0058] The full length of the heavy chain variable region is 109 amino acids, the amino acid numbers of the four domains of FR are 24, 17, 36 and 11 respectively, the amino acid numbers of the three domains of HCDR are 8, 7 and 6 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-56aa and 93aa-98aa respectively, and the amino acid sequences thereof are RFSLSNYR (SEQ ID NO. 4), IFTRGST (SEQ ID NO. 5), ARGWNS (SEQ ID NO. 6)

[0059] Example 5 Identification of HRP-labeled rabbit monoclonal antibody mAb6 as a secondary antibody

[0060] (1) HRP-labeled rabbit monoclonal antibody mAb6

[0061] 1. Dissolve the rabbit monoclonal antibody mAb6 in a sodium bicarbonate solution at pH 9.6;

[0062] 2. Take a certain mass of HRP and dissolve it in deionized water, add sodium periodate and react for 30 min, then add ethylene glycol and continue to react for 30 min, and dialyze overnight;

[0063] 3. Weigh a certain amount of sodium borohydride and dissolve it in deionized water, then add it to the antibody-HRP solution after crosslinking, react for 2 h, and dialyze overnight;

[0064] 4. Add the obtained HRP-labeled antibody to an equal amount of glycerol-20 degrees Celsius for storage.

[0065] (2) HRP-labeled rabbit monoclonal antibody mAb6 Western blot identification

[0066] Western blot (WB) detection was performed. 100 ng of mIgG2a antibody was subjected to SDS-PAGE, and after transfer, HRP-labeled rabbit monoclonal antibody mAb6 was incubated for WB detection.

[0067] The results showed that the HRP-labeled rabbit monoclonal antibody mAb6 could well recognize the mIgG2a Fc region with a molecular weight of about 50KD, and the results are shown in Figure 4 .

[0068] (3) HRP-labeled rabbit monoclonal antibody mAb6 ELISA identification

[0069] Sheep anti-mouse IgG coated enzyme-labeled plate, 4°C overnight; the next day, remove the enzyme-labeled plate, PBST wash once, 1% BSA solution 37°C blocking 2 hours, PBST wash 3 times; each hole added mIgG2a antibody 100ul, concentration of 0.2ug / ml, 37°C incubation 1 hour; after incubation, remove the enzyme-labeled plate, PBST wash 3 times, add HRP labeled mAb6 as detection antibody, 1ug / ml start dilution 7 gradient, 37°C incubation 1 hour; after incubation, remove the enzyme-labeled plate, PBST wash 5 times, add TMB substrate, 37°C color development 10min. After removal, add stop solution, determine OD450 reading on the enzyme-labeled instrument, the results are shown in Table 1.

[0070] Table 1 ELISA detection results of HRP labeled rabbit anti-mIgG2a mAb6 specific recognition of mIgG2a antibody

[0071]

[0072]

[0073] The results show that HRP labeled rabbit anti-mIgG2a mAb6 as secondary antibody can well detect mIgG2a antibody signal, which is better than commercial sheep anti-mouse IgG polyclonal antibody (as secondary antibody can detect mIgG1, mIgG2a, mIgG2b, mIgG3 at the same time).

[0074] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the 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 mAb6 against mouse immunoglobulin G2a subtype (IgG2a) or its antigen-binding fragment, characterized in that, It includes a light chain variable region and a heavy chain variable region. The light chain variable region includes complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO.1, the amino acid sequence of LCDR2 is GTN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

3. The heavy chain variable region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.4, the amino acid sequence of HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.

6.

2. The rabbit monoclonal antibody mAb6 or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region includes an amino acid sequence as 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; the heavy chain variable region includes an amino acid sequence as 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.

3. The rabbit monoclonal antibody mAb6 or its 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, and bispecific antibodies.

4. A biomaterial, characterized in that, The biological material includes a polynucleotide, a carrier, or a cell, wherein the polynucleotide encodes the rabbit monoclonal antibody mAb6 or its antigen-binding fragment as described in any one of claims 1-3; the carrier carries the polynucleotide; the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the rabbit monoclonal antibody mAb6 or its antigen-binding fragment as described in any one of claims 1-3.

5. The method for preparing the rabbit monoclonal antibody mAb6 or its antigen-binding fragment according to any one of claims 1-3, characterized in that, Includes culturing the cells as described in claim 4; The cells are prepared by converting cells with a polynucleotide encoding a rabbit monoclonal antibody mAb6 or its antigen-binding fragment, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

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

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

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

9. The use of the rabbit monoclonal antibody mAb6 according to any one of claims 1-3 or its antigen-binding fragment, or the biological material according to claim 4, in any of the following: 1) Non-diagnostic and therapeutic target detection of mouse immunoglobulin G2a subtype (IgG2a); 2) Prepare products for detecting mouse immunoglobulin G2a subtype (IgG2a); 3) Used for purifying mouse immunoglobulin G2a subtype (IgG2a); 4) Prepare products for purifying mouse immunoglobulin G2a subtype (IgG2a).

10. A detection reagent or detection kit, characterized in that, The detection reagent or detection kit contains the rabbit monoclonal antibody mAb6 or its antigen-binding fragment as described in any one of claims 1-3, or the biological material as described in claim 4.

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

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