CN119684465B - Human immunoglobulin G2 subtype (IgG2) rabbit monoclonal antibody and its application - Google Patents
Human immunoglobulin G2 subtype (IgG2) rabbit monoclonal antibody and its applicationInfo
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Abstract
本发明属于免疫检测技术领域，公开了一种兔抗人免疫球蛋白G2亚型（IgG2）的特异性单克隆抗体mAb48，其包含轻链可变区和重链可变区，所述轻链可变区包括LCDR1‑3，所述LCDR1包括氨基酸序列EDIGYG，所述LCDR2包括氨基酸序列GAS，所述LCDR3包括氨基酸序列QQGFS；所述重链可变区包括HCDR1‑3，所述HCDR1包括氨基酸序列GFSLSNYE，所述HCDR2包括氨基酸序列ISSSGST，所述HCDR3包括氨基酸序列ARNNNN。本申请的单克隆抗体可与人免疫球蛋白G2亚型（IgG2）高特异性结合，可以应用于免疫检测中，为下一步工程抗体的制备提供基础。
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
The application relates to the technical field of immunodetection, in particular to a human immunoglobulin G2 subtype (IgG 2) rabbit monoclonal antibody and application thereof.
Background
Affinity conjugates/antibodies are one of the most commonly used tools in life sciences for studying proteins and their functions in various biological pathways and diseases. The most widely applied antibody species in the scientific research field and the medicine field at present comprise human antibodies, murine antibodies and rabbit antibodies. These antibodies can specifically recognize the target of interest and then display a signal via an enzyme-labeled secondary antibody or a fluorescein-labeled secondary antibody. The existing anti-human IgG and anti-mouse IgG labeled antibodies are widely applied to the market, and can be applied to enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western blot), immunohistochemical staining (IHC), flow Cytometry (FCM), co-immunoprecipitation and the like, and the most commonly used labeling methods comprise horseradish peroxidase (HRP), alkaline phosphatase, biotin, fluorescein and the like.
The main method for preparing the anti-human IgG antibody is to directly immunize animals such as rabbits, sheep, donkey and the like with IgG immunoglobulin to generate immune serum polyclonal antibody, and then purify and apply the immune serum polyclonal antibody. However, the development of antibodies specific for different types of IgG heavy and light chains, polyclonal antibodies, is far from sufficient, and monoclonal antibodies, particularly against different subtypes of hIgG, are required.
Human IgG monoclonal antibodies comprise four subtypes, igG1, igG2, igG3, and IgG4, and light chains comprise two types, kappa and lambda, with kappa being about 6:4. At present, polyclonal antibodies such as goat polyclonal antibodies, sheep polyclonal antibodies, donkey polyclonal antibodies and the like are mostly used in the market, and few monoclonal antibodies, particularly rabbit anti-hIgG 2 specific monoclonal antibodies are not found.
To meet the requirements of monoclonal antibodies specific to hIgG2, development of monoclonal antibodies with high specificity and high sensitivity against hIgG2 is urgently needed.
Disclosure of Invention
In order to overcome the technical problems, the application provides the anti-hIgG 2 rabbit monoclonal antibody mAb48 with good specificity and high affinity and the application thereof in immunodetection, including but not limited to chemiluminescence, fluorescence and chromogenic detection for primary antibodies, which are suitable for various applications, such as cell imaging, flow cytometry detection, western immunoblotting and immunohistochemistry, and also provide a basis for the preparation of the next engineering antibody.
In one aspect, the application provides a rabbit monoclonal antibody mAb48 or antigen-binding fragment of an anti-human immunoglobulin G2 subtype (IgG 2), said rabbit monoclonal antibody comprising a light chain variable region comprising LCDR1-3, said LCDR1 comprising amino acid sequence EDIGYG (SEQ ID No. 1), said LCDR2 comprising amino acid sequence GAS (SEQ ID No. 2), and said LCDR3 comprising amino acid sequence QQGFS (SEQ ID No. 3).
Further, the rabbit monoclonal antibody mAb48 or antigen-binding fragment further comprises a heavy chain variable region comprising HCDR1-3, the HCDR1 comprising amino acid sequence GFSLSNYE (SEQ ID No. 4), the HCDR2 comprising amino acid sequence ISSSGST (SEQ ID No. 5), and the HCDR3 comprising amino acid sequence ARNNNN (SEQ ID No. 6).
Further, the light chain variable region comprises an amino acid sequence shown as SEQ ID NO.7 or comprises an amino acid sequence which is obtained by substituting, deleting and/or adding one or more amino acids and/or modifying the tail end of any one or more amino acids in the amino acid sequence shown as SEQ ID NO.7 and has more than 90 percent of homology.
Further, the heavy chain variable region comprises an amino acid sequence shown as SEQ ID NO.8 or comprises an amino acid sequence which is obtained by substituting, deleting and/or adding one or more amino acids and/or modifying the tail end of any one or more amino acids in the amino acid sequence shown as SEQ ID NO.8 and has more than 90 percent of homology.
In another aspect, the application provides isolated polynucleotides encoding the rabbit monoclonal antibody mAb48 or antigen-binding fragment.
In another aspect, the application also provides a recombinant expression vector comprising the polynucleotide.
In another aspect, the application provides a host cell comprising said polynucleotide, or said recombinant expression vector.
In another aspect, the application also provides the use of said rabbit monoclonal antibody mAb48 or antigen-binding fragment, either,
1) For detecting human immunoglobulin G2 subtype;
2) The method is used for preparing detection reagents or kits of the human immunoglobulin G2 subtype.
In another aspect, the application also provides a detection reagent or a detection kit for detecting the human immunoglobulin G2 subtype, wherein the detection reagent or the detection kit contains the rabbit monoclonal antibody mAb48 or the antigen binding fragment.
Further, the detection kit is used for a double-antibody sandwich ELISA detection or a chemiluminescent method, and the rabbit monoclonal antibody mAb48 or antigen binding fragment is a detection antibody.
Compared with the prior art, the hIgG2 rabbit monoclonal antibody can be combined with the hIgG2 in a high specificity way, has high affinity, and the affinity constant Ka can reach 9 multiplied by 10 8 L/mol. The monoclonal antibody of the application can be combined with the high specificity of the human immunoglobulin G2 subtype (IgG 2), and the application also relates to the application of the anti-human immunoglobulin G2 subtype (IgG 2) specific rabbit monoclonal antibody in an immunodetection tool, including but not limited to the chemiluminescent, fluorescent and chromogenic detection for primary antibodies, and is suitable for various applications, such as cell imaging, flow cytometry detection, western immunoblotting and immunohistochemistry, and also provides a basis for the preparation of the next engineering antibody.
Drawings
FIG. 1 is an electrophoretogram of full-length amplification products of heavy and light chains of a rabbit monoclonal antibody mAb48, M being a DNA molecular weight Marker.
FIG. 2 is a Western blot analysis result of specific recognition of natural hIgG2 antibody by rabbit monoclonal antibody mAb48, and the samples in lanes 1-4 are hIgG1, hIgG2, hIgG3, and hIgG4, respectively.
FIG. 3 is a graph showing ELISA detection results of specific recognition of hIgG2 antibody by rabbit monoclonal antibody mAb 48.
FIG. 4 is a graph showing the Western blot detection results of specific recognition of natural hIgG2 antibody by horseradish peroxidase-labeled rabbit monoclonal antibody mAb 48.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally performed under conventional conditions, those described in the laboratory manual, or those suggested by the manufacturer.
EXAMPLE 1 preparation of human IgG2 rabbit monoclonal antibodies
1) Preparation of immunogens
An abcam hIgG2 antibody was purchased as an immunogen.
2) Immunization of animals
The purchased hIgG2 antibody is emulsified by complete Freund's adjuvant, about 2kg of New Zealand white rabbits are immunized by subcutaneous injection, the immunization dose is 500 mug/animal, the second immunization is carried out after two weeks interval, the incomplete Freund's adjuvant is used for emulsification, and the immunization dose is 250 mug/animal. And taking tail blood after two immunization, performing gradient dilution by ELISA method to determine serum titer, judging whether to collect PBMCs or continue immunization according to the result by taking OD450 when ELISA titer 128000 is larger than 1.0 as a standard, and selecting rabbits with highest antibody titers for collecting the PBMCs.
3) PBMCs separation, specific B cell separation, cloning recombination
Fixing the rabbit on the operating table in a supine manner, removing hair from the heart part, sterilizing skin with alcohol, selecting the most obvious part of heart beat, puncturing with a 50ml syringe, flushing blood into the syringe after the needle is punctured into the heart, rapidly pulling out the needle after the required blood volume is obtained, transferring whole blood in the syringe into a sterile 50ml tube, uniformly mixing with equal amount of PBS, slowly adding dropwise above lymphocyte separation liquid, centrifuging at room temperature of 400 Xg for 30 min, separating the liquid level into four layers from top to bottom, namely a yellow plasma layer, a white film layer, a mononuclear cell layer, a separation liquid layer and a red blood cell layer, carefully sucking the mononuclear cell layer, and washing with PBS to remove platelets and lymphocyte separation liquid to obtain the rabbit PBMCs.
Antigen-specific B cells were further sorted from rabbit PBMCs for culture, and the supernatant of the cultured B cells was screened for positive clones using antigen-coated ELISA plates. The full-length sequence of the light chain and heavy chain of the naturally paired rabbit monoclonal antibody is amplified from cDNA of the corresponding positive clone, a rabbit monoclonal antibody expression vector is constructed by a cloning recombination method, and the sequence is determined by sequencing. The results of the amplified full length PCR products are shown in FIG. 1.
4) Preparation and purification of monoclonal antibodies
In order to obtain the rabbit monoclonal antibody for recognizing the human hIgG2 protein, the heavy chain and light chain genes of the rabbit monoclonal antibody are loaded on an expression vector, HEK293 cells are transfected with plasmids, and the recombinant rabbit monoclonal antibody for recognizing the human hIgG2 protein is obtained from the culture supernatant after 120-144 hours of transfection. Collecting cell suspension, centrifuging to obtain supernatant, and performing antibody purification by affinity chromatography. The purified monoclonal antibody concentration was measured by BCA method, and then sub-packaged and lyophilized to give the purified antibody as human IgG2 rabbit monoclonal antibody mAb48.
EXAMPLE 2 specific identification of anti-hIgG 2 rabbit monoclonal antibody mAb48
1) Western blot identification of rabbit monoclonal antibody mAb48
Western Blot (WB) detection was used. 1 sample was selected for each subtype of hIgG1, hIgG2, hIgG3, and hIgG4, and 100ng of each protein was subjected to SDS-PAGE, and after transfer, WB detection was performed.
The results show that rabbit monoclonal antibody mAb48 can specifically recognize hIgG2, but does not recognize hIgG1, hIgG3 and hIgG4, and the results are shown in FIG. 2.
2) Identification of rabbit monoclonal antibody mAb48 ELISA
The ELISA plate is coated by hIgG1, hIgG2, hIgG3 and hIgG4 antibodies at 4 ℃ overnight, the ELISA plate is taken out the next day, PBST is washed once, 1% BSA solution is blocked for 2 hours at 37 ℃, PBST is washed 3 times, 48 mu l of rabbit monoclonal antibody MAB is added to each hole, the concentration is respectively 10, 3, 1, 0.3 and 0ng/ml, the incubation is carried out for 1 hour at 37 ℃, the ELISA plate is taken out after the incubation is finished, PBST is washed 3 times, HRP-labeled goat anti-rabbit secondary antibodies are respectively added as detection antibodies, the incubation is carried out for 1 hour at 37 ℃, the ELISA plate is taken out after the incubation is finished, the PBST is washed 5 times, TMB substrate is added, and color development is carried out for 10 minutes at 37 ℃. After removal, stop solution was added and OD450 readings were measured on an microplate reader.
The results show that rabbit monoclonal antibody mAb48 can specifically recognize hIgG2, but does not recognize hIgG1, hIgG3 and hIgG4, and the results are shown in FIG. 3.
Example 3 affinity identification of anti-hIgG 2 rabbit monoclonal antibody mAb48
Affinity constants (Ka) were determined by non-competitive ELISA.
Coating, namely diluting antigen with carbonate buffer solution to the concentration of 1, 0.5, 0.1 and 0.05 mug/mL, adding the antigen to a 96-well ELISA plate according to 100 mug/well, coating respectively, and incubating for 24 hours at 4 ℃;
blocking, namely, washing the plate 4 times by using PBST, adding BSA solution according to 200 mu L/hole, and incubating for 2 hours at 37 ℃;
adding the monoclonal antibody, namely washing the plate 4 times by using PBST, diluting the rabbit monoclonal antibody by a starting multiple ratio of 100 mu g/mL by using carbonate buffer solution, adding 100 mu L of the rabbit monoclonal antibody into each hole, and incubating for 2 hours at 37 ℃;
Adding enzyme-labeled secondary antibody, namely washing the plate with PBST for 4 times, adding 100 mu L of goat anti-rabbit Ig secondary antibody which is diluted 1:10000 times and is marked by HRP enzyme into each hole, and standing for 30min at 37 ℃;
color development and termination, namely, washing the plate for 4 times by using PBST, adding 100 mu L of a substrate color development solution into each hole, carrying out light-shielding reaction for 15min at 37 ℃, and adding 50 mu L of 1.0mol/L H 2SO4 termination solution into each hole to terminate the reaction;
Detection the absorbance at a wavelength of 450nm (A450 nm) was measured.
The logarithmic scale of the antibody concentration is taken as the abscissa, the OD value is taken as the ordinate, an S-shaped curve is drawn, and the affinity constant Ka=9× 8 L/mol of the monoclonal antibody mAb3 of the T3 sandwich method is calculated.
Example 4 analysis of variable region genes and amino acid sequences of rabbit monoclonal antibody mAb48 recombinant plasmids of mAb48 antibodies were used as DNA templates, light chain variable region and heavy chain variable region sequencing primers were designed from the 5' end vector sequences of the light chain and heavy chain on the templates, and sequencing was performed using a sequencer ABI 3730. The nucleotide sequence of the variable region of the light chain and the heavy chain of the mAb48 of the rabbit monoclonal antibody is obtained through sequencing.
The light chain and heavy chain nucleotide sequences are respectively subjected to sequencing result data analysis by using IMGT/V-QUEST analysis software on http:// www.imgt.org through the Internet to obtain the light chain amino acid sequence of the rabbit monoclonal antibody mAb48 shown as SEQ ID NO.7, and the heavy chain amino acid sequence is shown as SEQ ID NO. 8.
The total length of the light chain variable region was 104 amino acids, the number of FR 4 domain amino acids was 26, 17, 36 and 11, respectively, the number of LCDR3 domain amino acids was 6, 3 and 5, and the regions of LCDR1, LCDR2 and LCDR3 were 27aa-32aa,50aa-52aa and 89aa-93aa, respectively, with amino acid sequences EDIGYG (SEQ ID NO. 1), GAS (SEQ ID NO. 2) and QQGFS (SEQ ID NO. 3), respectively.
The total length of the heavy chain variable region was 112 amino acids, the number of FR 4 domain amino acids was 25, 17, 38 and 10, the number of HCDR3 domain amino acids was 8, 7 and 6, and HCDR1, HCDR2 and HCDR3 were 26aa-33aa,51aa-57aa and 96aa-101aa, respectively, and the amino acid sequences were GFSLSNYE (SEQ ID NO. 4), ISSSGST (SEQ ID NO. 5), ARNNNN (SEQ ID NO. 6), respectively
EXAMPLE 5 identification of HRP-labeled Rabbit monoclonal antibody mAb48 as secondary antibody
(1) HRP-labeled Rabbit monoclonal antibody mAb48
1. The rabbit monoclonal antibody mAb48 was dissolved in sodium bicarbonate solution at PH 9.6;
2. Dissolving HRP with a certain mass in deionized water, adding sodium periodate for reaction for 30min, adding ethylene glycol for continuous reaction for 30min, and dialyzing overnight;
3. Weighing a certain amount of sodium borohydride, dissolving the sodium borohydride in deionized water, adding the sodium borohydride into the crosslinked antibody-HRP solution, reacting for 2 hours, and dialyzing overnight;
4. The HRP-labeled antibody obtained was stored at-20℃with the same amount of glycerol.
(2) HRP-labeled rabbit monoclonal antibody mAb48 Western blot identification
Western Blot (WB) detection was used. 100ng of hIgG2 antibody was subjected to SDS-PAGE, and after transfer, the HRP-labeled rabbit monoclonal antibody mAb48 was incubated for WB detection.
The results show that the HRP-labeled rabbit monoclonal antibody mAb48 can well recognize the hIgG2 Fc region, and the molecular weight is about 55KD, and the results are shown in FIG. 4.
(3) ELISA identification of HRP-labeled rabbit monoclonal antibody mAb48
Sheep anti-mouse IgG coats the ELISA plate at 4 ℃ overnight, the ELISA plate is taken out the next day, PBST is washed once, 1% BSA solution is blocked at 37 ℃ for 2 hours, PBST is washed 3 times, hIgG2 antibody is added into each hole, the concentration is 0.2ug/ml,37 ℃ is incubated for 1 hour, the ELISA plate is taken out after incubation is completed, PBST is washed 3 times, HRP-labeled mAb48 is added as a detection antibody, 1ug/ml is diluted by a factor of 7 gradients, 37 ℃ is incubated for 1 hour, the ELISA plate is taken out after incubation is completed, PBST is washed 5 times, TMB substrate is added, and color development is carried out at 37 ℃ for 10 minutes. After removal, stop solution was added and the OD450 reading was determined on an ELISA reader, the results of which are shown in Table 1.
TABLE 1 ELISA detection results of HRP-labeled rabbit anti-hIgG 2 mAb48 specifically recognizing hIgG2 antibody
The results show that HRP-labeled rabbit anti-hIgG 2 mAb48 can be used as a secondary antibody to well detect hIgG2 antibody signals, and is superior to a commercial goat anti-mouse IgG polyclonal antibody (used as a secondary antibody to simultaneously detect hIgG1, hIgG2, hIgG3 and hIgG 4).
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (8)
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