CN116535510B - Anti-human PLA2R antibody, kit and application thereof - Google Patents

Anti-human PLA2R antibody, kit and application thereof Download PDF

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CN116535510B
CN116535510B CN202310786674.8A CN202310786674A CN116535510B CN 116535510 B CN116535510 B CN 116535510B CN 202310786674 A CN202310786674 A CN 202310786674A CN 116535510 B CN116535510 B CN 116535510B
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pla2r
antibody
antigen
binding fragment
cdr
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CN116535510A (en
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李贵森
吴姝焜
李怡
熊琳
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Sichuan Peoples Hospital of Sichuan Academy of Medical Sciences
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Abstract

The invention discloses an antibody for resisting human PLA2R, a kit and application thereof, and relates to the technical field of bioengineering. An antibody or antigen-binding fragment thereof directed against human PLA2R includes a heavy chain complementarity determining region and a light chain complementarity determining region; the heavy chain complementarity determining regions include CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NO.3-5, respectively; the light chain complementarity determining regions include the CDR-L1, CDR-L2 and CDR-L3 shown in SEQ ID NOS.6-8, respectively. Antibodies against human PLA2R can target three domains, cysR, fnII, CTLD1, of PLA2R and have a higher affinity for PLA2R proteins. The anti-human PLA2R antibody or antigen-binding fragment thereof is capable of specifically recognizing a PLA2R protein; can be applied to the detection fields of IHC, ELISA and the like.

Description

Anti-human PLA2R antibody, kit and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to an antibody for resisting human PLA2R, a kit and application thereof.
Background
Membranous nephropathy (membranous nephropathy, MN), i.e. membranous glomerulonephritis, specifically nephrotic syndrome resulting from excessive deposition of immune complexes in glomerular capillaries, is one of the most common pathological types in adult nephrotic syndrome, with a global incidence of about 12/100 ten thousand (Mc Grogan A, franssen CF, de Vries CS. Nephrol Dial Transplay.2011; 26 (2): 414-430). In recent years, the incidence of MN has increased at a rate of 13% per year (Lwezaula BF, ameh OI, ekrikpo UE, et al BMC Nephrol 2021; 22 (1): 15), and has become the second leading cause of chronic glomerulonephritis. Clinically MN is mainly manifested by massive proteinuria, hypoproteinemia, edema and hyperlipidemia, diagnosis depends on kidney biopsy histopathological examination, and the pathological diagnosis characteristics include thickening of glomerular capillary walls, deposition of IgG and complement C3 along the capillary walls under immunofluorescence conditions, and formation of glomerular epithelial cell subelectron dense matters under electron microscopy.
Depending on the etiology, MN can be classified into idiopathic membranous nephropathy (idiopathic membranous nephropathy, IMN) and secondary membranous nephropathy (secondary membranous nephropathy, SMN). Among them, IMN is a major type of MN, and its cause is not clear, but is also called primary membranous nephropathy (primary membranous nephropathy, PMN), with a ratio of about 70% to 80%. At present, the pathogenesis of PMNs is not well defined, but most students consider that PMNs are an autoimmune glomerular disease mediated by antibodies, target antigens located on podocytes are recognized by the autoantibodies and combined with the target antigens to form immune complexes to deposit under podocytes of basement membrane, the complement system is activated to cause damage and shedding of podocytes, the permeability of the basement membrane is increased, and a large amount of proteinuria occurs.
The first report in 2009 that the phospholipase A2 receptor (PLA 2R) is one of the major antigens responsible for PMN production was that anti-phospholipase A2 receptor antibodies (phospholipase-A2 receptor antibody, PLA 2R-Ab) could be detected in 70% of PMN patient blood and kidney tissue eluate (Beck LH Jr, bonegio RG, lambeau G, et al N Engl J Med 2009; 361 (1): 11-21), which has attracted considerable attention from the medical community to PLA2R. PLA2R-Ab can be specifically combined with target antigen (namely PLA 2R) on podocyte in Kidney tissue, can be expressed in 70% -80% of serum of PMN patients (Bobart SA, de Vriese AS, pawar AS, et al Kidney int.2019; 95 (2): 429-438), and has extremely low PLA2R-Ab antibody positive expression rate in serum of healthy people, SMN and other glomerular disease patients, so that the specificity of diagnosing MN by PLA2R-Ab is higher. A recent Meta analysis shows (J.Ind. Lu Rong, zhang Jiaqin, et al. J.clinical laboratory, 2017, 35 (7): 545-549) that serum PLA2R-Ab has a total sensitivity of 69% and a total specificity of 97% for diagnosing PMNs, further demonstrates that serum PLA2R-Ab has better sensitivity and very high specificity for PMNs, and can be used as a specific biomarker for diagnosing PMNs.
PLA2R is composed of multiple domains (FIG. 1), and studies have shown that the domains responsible for PLA2R-Ab production consist essentially of three domains, cysR, FNII, CTLD (Liyo Kao, vinson Lam, et al J Am Soc Nephrol.2015 Feb; 26 (2): 291-301). Therefore, monoclonal antibodies targeting these three domains are of great significance for cell and animal model establishment, mechanism research, diagnosis and drug development of PLA2R type membranous nephropathy. However, no commercially available mouse monoclonal antibodies can target these three domains, and the present invention aims to solve this problem.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an antibody for resisting human PLA2R, a kit and application thereof, which are used for solving the technical problems.
The invention is realized in the following way:
in a first aspect, the invention provides an antibody or antigen-binding fragment thereof directed against human PLA2R, comprising a heavy chain complementarity determining region and a light chain complementarity determining region;
the heavy chain complementarity determining regions include CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NO.3-5, respectively;
the light chain complementarity determining regions include the CDR-L1, CDR-L2 and CDR-L3 shown in SEQ ID NOS.6-8, respectively.
The inventors screened for an anti-human PLA2R antibody or antigen binding fragment thereof that could target three domains of PLA2R CysR, fnII, CTLD, and that has a higher affinity for PLA2R proteins or fragments comprising part of the PLA2R domain. Can specifically recognize PLA2R protein. Can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like. The antibody or antigen binding fragment thereof for resisting the human PLA2R provides a strong and favorable technical means for accurately detecting the PLA2R protein and diagnosing membranous nephropathy, and has good application prospect.
In a preferred embodiment of the invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 1; the heavy chain variable region has the structure: FR1-H-CDR-H1-FR2-H-CDR-H2-FR3-H2-CDR-H3-FR4-H4. The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 2. The structure of the light chain variable region is:
FR1-L-CDR-L1-FR2-L-CDR-L2-FR3-L2-CDR-L3-FR4-L4。
in a preferred embodiment of the invention for use, the antigen binding fragment is selected from the group consisting of Fab ', fab, F (ab') 2 Any of scFv and Fv.
In an alternative embodiment, the constant region of the antibody is selected from the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD;
in an alternative embodiment, the constant region of the antibody is of human, mouse, rat, bovine, equine, ovine, rabbit or canine origin.
The antigen binding fragments of the above antibodies typically have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the teachings herein that antigen binding fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The antigen binding fragments described above are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Antigen binding fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In a second aspect, the invention also provides the use of an antibody or antigen-binding fragment thereof directed against human PLA2R in the preparation of a PLA2R assay product.
In PLA2R detection product applications, detection of PLA2R is achieved by targeting a PLA2R protein fragment containing the three domains CysR, fnII, CTLD with an antibody or antigen binding fragment thereof against human PLA2R. In addition, antibodies against human PLA2R or antigen binding fragments thereof also target PLA2R protein fragments that contain CysR, fnII, CTLD, CTLD2 domains.
In an alternative embodiment, the PLA2R detection product is selected from a reagent, a kit or a chip.
The reagent comprises an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof is labeled with a detectable label. A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
Fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-photoprotein (FAM), tetrachlorophotoprotein (TET), and the like, or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof, for example, including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazoxanthin (chlorophyll), and the like, for example.
In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, nanoparticle-based labels include, but are not limited to, nanoparticles, colloids; nanoparticles include, but are not limited to: organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In a third aspect, the invention also provides the use of an antibody or antigen-binding fragment thereof directed against human PLA2R in the manufacture of a product for the detection or diagnosis of membranous nephropathy, the diagnostic marker of which is PLA2R.
In an alternative embodiment, the membranous nephropathy is primary membranous nephropathy or secondary membranous nephropathy.
Membranous kidney disease detection or diagnosis products include, but are not limited to, reagents, kits, chips.
In a fourth aspect, the invention also provides a detection reagent or kit comprising an antibody or antigen-binding fragment thereof directed against human PLA2R.
In a fifth aspect, the invention also provides immunoconjugates, bispecific molecules, chimeric and antigen receptors or pharmaceutical compositions comprising an antibody or antigen binding fragment thereof directed against human PLA2R.
The immunoconjugate is for example selected from antibody-drug conjugates, and the corresponding antibody-drug conjugate is prepared by reacting an antibody with a drug having a specific linker. It is within the scope of the invention for a person skilled in the art to engineer antibodies such that a certain site on the antibody serves as a drug conjugation site.
The bispecific molecule is for example selected from bispecific antibodies.
The chimeric antigen receptor also includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.
In a sixth aspect, the invention also provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above against human PLA2R.
Considering the degeneracy of codons, the sequence of the genes encoding the above antibodies may be modified in the coding region thereof without changing the amino acid sequence to obtain genes encoding the same antibodies; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.
In a seventh aspect, the invention also provides a vector comprising a nucleic acid molecule as described above.
The vector is an expression vector or cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can be used to introduce the DNA fragment of interest directly or indirectly (e.g., packaged into a virus) into a host cell by transformation, transfection or transduction for expression of the gene of interest.
One type of vector is a plasmid, i.e., a circular double stranded DNA molecule, into which a DNA fragment of interest can be ligated into a plasmid loop.
In an eighth aspect, the invention also provides a recombinant cell comprising the vector described above.
The recombinant cell is selected from mammalian cells;
the mammalian cells are selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, vero, BHK cells, CV1 cells, heLa cells, MDCK cells, hep-2 cells, and Per6 cells. Among them, 293 series cells, per6 cells and CHO cells are common mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.
In a ninth aspect, the invention also provides the use of an antibody or antigen-binding fragment thereof directed against human PLA2R in the detection of human PLA2R levels or the detection of pharmaceutical activity in a cellular model expressing PLA2R, for non-diagnostic and therapeutic purposes. The drug activity detection comprises the steps of constructing cells which stably express human PLA2R or express membrane proteins comprising three domains CysR, fnII, CTLD1, inducing antibody-induced cell killing effect (including CDC, ADCC, ADCP) by anti-human PLA2R antibodies to establish a cell damage model, and detecting the inhibition of the killing effect by drugs, thereby being used as a quantitative activity detection and drug quality control method. The anti-human PLA2R antibodies provided by the invention can be used in two aspects, namely, determining that cells stably expressing human PLA2R or expressing membrane proteins comprising three domains CysR, fnII, CTLD1 are successfully established and detecting the expression level. And secondly, triggering antibody-induced cell killing to establish a damage model.
The invention has the following beneficial effects:
the invention screens for an anti-human PLA2R antibody or antigen binding fragment thereof that can target three domains of PLA2R CysR, fnII, CTLD1 and that has a higher affinity for the PLA2R protein or fragments comprising a portion of the PLA2R domain. The anti-human PLA2R antibody or antigen-binding fragment thereof is capable of specifically recognizing a PLA2R protein; can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like. The antibody or antigen binding fragment thereof for resisting the human PLA2R provides a strong and favorable technical means for accurately detecting the PLA2R protein and diagnosing membranous nephropathy, and has good application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic structural diagram of PLA2R;
FIG. 2 is a schematic view showing the structure of an expression frame in embodiment 1 of the present invention;
FIG. 3 is a diagram showing the analysis of CC1H SDS-PAGE in example 3 of the present invention; lane 1 (lane labeled M in the figure), molecular weight reference; lane 2, purified CC1H non-reduced; lane 3, purified CC1H reduction;
FIG. 4 is a SDS-PAGE identification of the antibodies of example 4 of the present invention; lane 1, maker; lane 2, antibody non-reduction; lane 3, maker; lane 4: antibody reduction;
FIG. 5 is a graph showing the fitting of murine anti-human monoclonal antibody in example 5 of the present invention;
FIG. 6 is a graph of protein standard curve (y= 0.6849x+0.6325, R=0.9925) measured by the Bradford method in example 6 of the present invention;
FIG. 7 is a diagram showing the purification effect of SDS-PAGE for detecting CC1H-Biotin in example 6 of the present invention, lane 1: CC1H-Biotin in DTT-containing reduction loading buffer, lane 2: CC1H-Biotin of non-reducing loading buffer, marker: a molecular weight reference;
FIG. 8 is a graph showing actual measurement or fitting of anti-human mice at different concentrations in example 6 of the present invention;
FIG. 9 is a plasmid map of the present invention for expressing membrane protein mCC1 in example 7;
FIG. 10 is a plasmid map of the present invention for expressing membrane protein mCC2 in example 7;
FIG. 11 is an electrophoresis chart of CC1H, mCC, mCC2 and plasmid HEK293 in example 7 of the present invention; lane 1, maker; lane 2, CC1H; lane 3, mCC2; lane 4: mCC1; lane 5: plasmid HEK293.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
Construction of PLA2R major antigen region recombinant protein (CC 1H) expression vector.
The plasmid vector adopts pcDNA3.4 transient expression vector, the vector comprises a native full-length CMV promoter and WPRE elements downstream of cloning sites, the vector based on the CMV promoter generally has good expression level for a CHO cell transient expression system, the WPRE elements are positioned downstream of multiple cloning sites, the transcription and the expression of genes can be effectively improved, and the vector has ampicillin resistance genes in escherichia coli. Then, the vector gene pcDNA3.4 was amplified by PCR technique, and the gene sequences linked to the target gene (gene of interest) and signal peptide (signal peptide) were ligated. The PCR template is obtained through gene synthesis, pcDNA3.4 carrier fragments and synthetic genes are connected through a homologous recombination kit (the gene fragments obtained after connecting target genes and signal peptide sequences are called synthetic genes for short), DH5 alpha competence is transformed to obtain monoclone, sequencing is carried out after monoclone amplification, and plasmid extraction is carried out after sequencing confirmation, so as to obtain the synthetic gene recombination expression plasmid. Wherein the synthetic gene was cloned between the CMV promoter and WPRE genes of plasmid pcDNA3.4, see in particular FIG. 2.
Wherein the amino acid sequence of the signal peptide is shown as SEQ ID NO. 11. The nucleotide sequence of the signal peptide is shown as SEQ ID NO. 12.
The structure of PLA2R is schematically shown in FIG. 1, wherein the target gene is the gene sequence corresponding to the amino acid sequence from 21 to 367 (Glu) of the PLA2R full-length protein sequence (UniProt sequence accession number: Q13018). The constructed transient expression plasmid was named pcDNA3.4-CC1H.
The amino acid sequence of the synthetic gene is shown as SEQ ID NO. 13. The nucleotide sequence of the synthetic gene is shown as SEQ ID NO. 14.
Example 2
The expression vector constructed in example 1 was used for transient expression of CC1H.
About 24 hours before transfection, freeStyle ™ CHO-S cube cells (cell density 5-6X10) 5 /mL, from Gibco corporation, freeStyle ™ MAX CHO expression System, cat: k900020 At 37℃with 5% CO 2 And culturing at 120-135 rpm/min.
On the day of transfection, cells were diluted to a cell density of 1X 10 6 Per mL, 10 mL cells (cell viability above 95%) were added to each flask.
Gently mix FreeStyle ™ MAX Reagent several times, note that vortex shaking is not possible.
pcDNA3.4-CC1H 12.5. Mu.g was taken, optiPRO ™ SFM was added to a total volume of 0.2 mL and gently mixed.
12.5. Mu.L of FreeStyle ™ MAX Reagent was taken, opti-Pro ™ SFM was added to a total volume of 0.2 mL and gently mixed. After mixing well, add to plasmid mix, mix gently to obtain 0.4 mL of DNA-FreeStyle ™ MAX mixture and incubate at room temperature for 10 minutes to allow complex formation.
The 0.4 mL DNA-FreeStyle ™ MAX complex described above was slowly added to the cells while the flask was slowly rotated.
At 37℃with 5% CO 2 The cells were cultured at 120-135 rpm/min without the need to replace or supplement the medium, and the cell culture supernatant was collected after 7 days.
Example 3
And purifying and identifying the CC1H.
Sample: cell culture supernatant 25 mL. The sample was centrifuged at 7000 rpm at 4℃for 10 min and passed through a 0.22 μm needle filter. And loading on a Ni sepharose 6FF column at 2 mL/min. Washing with 20mM phosphate buffer, 500mM NaCl,20mM imidazole, pH 6.8. 5 times the column volume of 0-100% 20mM phosphate buffer, 1M NaCl,500mM imidazole, pH 6.8. The fraction was collected 2 mL/tube.
The elution peak 30 kD was ultrafiltered 3 times in an ultrafiltration centrifuge tube and the solution was replaced with 50mM phosphate buffer, 150mM NaCl, pH7.4. Quantification was performed using the Bradford method. The expression level of CC1H was found to be 37mg/L. The protein sizes of the CC1H recombinant proteins are shown in Table 1.
Table 1: molecular weight of CC1H recombinant protein
SDS-PAGE analysis is carried out on the CC1H, and the analysis result is shown in figure 3, so that the CC1H protein has correct molecular weight and no impurity band, and the protein is purer.
Example 4
Mice were immunized with the purified CC1H of example 3 to obtain monoclonal antibodies and sequences thereof.
Immunized mice, hybridoma screening and sequencing were performed by Beijing Yiqiao Shenzhou technologies, inc.
5 mice were immunized with antigen CC1H. Antigen CC1H was coated and serum titers were measured by ELISA with serum dilutions of 8000 and 16000. And (5) fusing after the serum titer ELISA is qualified.
According to the serum titer result, selecting the optimal 1 mouse to carry out hybridoma fusion screening and limiting dilution, taking the immunized mouse spleen cells, and carrying out the following steps of 1: 1. mixing the mixture with SP2/0 myeloma cells in proportion, and fusing by using an electrofusion method to obtain hybridoma cells. Whole spleen is plated, immunogen is used as screening antigen, cell supernatant is measured by ELISA method, positive hole combined with the immunogen is selected as positive main clone, subcloning is carried out by limiting dilution until hybridoma cell strain which stably secretes monoclonal antibody is obtained, positive hybridoma cell is obtained, cell freezing and antibody production are carried out.
Positive clones obtained by screening were cultured on appropriate scale, and the supernatant protein a was purified to obtain 0.1-0.5mg of antibodies, which were identified by UV, SDS-PAGE (fig. 4). The molecular weights of the murine mab proteins are shown with reference to table 2.
Sequencing positive hybridoma cells to obtain the mouse monoclonal antibody sequence.
Table 2: molecular weight of murine monoclonal antibody protein
The amino acid sequence of murine monoclonal antibody is as follows:
the heavy chain is shown as SEQ ID NO.1, and the light chain is shown as SEQ ID NO. 2.
The heavy chain complementarity determining regions include CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NO.3-5, respectively;
the light chain complementarity determining regions include the CDR-L1, CDR-L2 and CDR-L3 shown in SEQ ID NOS.6-8, respectively.
Sequencing a monoclonal antibody gene to obtain the DNA sequence of the mouse monoclonal antibody as follows: the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 9, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 10.
Example 5
This example performed a murine anti-human PLA2R mab ELISA experiment.
Binding of the antibodies to human PLA2R CC1H was detected by ELISA. The reagents in this example were all from solebao unless specifically indicated.
10 mug/mL of CC1H is plated, gradient diluted mouse anti-human PLA2R monoclonal antibody (monoclonal antibody prepared in example 4) is added, goat anti-mouse HRP secondary antibody is added, and color development detection is carried out. The preparation method comprises the following steps:
[1] 100. Mu.L/well of a coated liquid seal plate containing 10. Mu.g/mL CC1H, overnight at 4 ℃;
[2] discarding liquid, washing a plate by using PBST, washing 4 wheels by using a plate washer, spin-drying by using a plate slinger, and beating the plate;
[3] adding 100 mu L/hole sealing liquid, and incubating at 37 ℃ for 60 min;
[4] discarding the solution, adding 100 mu L/hole mouse anti-human PLA2R monoclonal antibody (PBST dilution), sealing the plate, and incubating for 30 min at room temperature;
[5] repeating the step [2] of washing the plate;
[6] mu.L of detection antibody (EASYBIO, BE0102,1:2000 diluted with PBST) was added to each well, the plates were closed and incubated for 30 min at room temperature;
[7] repeating the step [2] of washing the plate;
[8] after adding 100. Mu.L of TMB substrate chromogenic solution to each well and incubating for 5 min at 25℃in the dark, 100. Mu.L of stop solution was added to each well and OD450 was measured.
A murine anti-human mab fit curve was drawn by the measurements (fig. 5).
ELISA experimental results show that the purified antibody prepared in example 4 is positive for ELISA binding with immunogen (CC 1H).
Example 6
The kinetics and thermodynamics of antibody interaction with CC1H were analyzed in this example.
The binding constant (Ka), dissociation constant (Kd) and affinity constant (KD) of the interaction of murine anti-human PLA2R mab with Biotin (Biotin) labeled CC1H were detected using a Sartorius (Sartorius) Octet 2 molecular interaction instrument based on the biological membrane interference technique (Bio-Layer Interferometry, BLI).
The specific experimental steps are as follows:
(1) Biotin labelling of CC1H
The purified CC1H protein was labeled with Biotin (Biotin) using the EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, 21335) kit:
the kit is equilibrated to room temperature;
10 mM biotin reagent solution was prepared. Adding 90 [ mu ] L of sterile double distilled water into each 0.5mg biotin reagent for full dissolution;
adding 20 times mol number of biotin reagent solution of protein to the CC1H protein solution;
the system was incubated on ice for 2 hours, during which time it was inverted several times to allow for thorough mixing.
(2) Purification of CC1H-Biotin
Purification of Biotin-labeled CC1H-Biotin using a G-25 prefilled desalting column (Bogurone, EG 001):
20mM PBS buffer, pH 7.2, was prepared according to Table 3;
table 3 PBS buffer formulation table
Balance:
the PBS buffer was withdrawn with a 10 mL syringe, each time a "drop-to-drop" column and syringe, to prevent air bubbles;
cutting off the outlet end;
removing ethanol in the column by 25 mL PBS buffer solution at 5 mL/min (120 drops/min), and discarding column effluent;
loading:
1.5 mL (less than 1.5 mL supplemented with PBS buffer) was loaded at 5 mL/min using a3 mL syringe, and the column effluent was discarded;
continuing to inject 3 mL of PBS buffer; together about 2 mL.
Column cleaning and preservation:
25 mL of PBS buffer was passed through the column using a 10 mL syringe, followed by 25 mL water, and the column effluent was discarded;
25 mL of 20% ethanol was passed through a 10 mL syringe and stored at 4 ℃;
sample storage:
and (5) performing suction filtration sterilization on the collected sample by using a 0.2 mu m pore size filter, and subpackaging and preserving.
(3) Measurement of CC1H-Biotin protein concentration by Bradfod method (Biyun, P0060):
protein standard (5 mg/ml BSA) was completely thawed and mixed, diluted with PBS buffer, and 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5 mg/ml protein standard was formulated and thoroughly mixed;
mu.L of protein standard with different concentration is added into standard wells of a 96-well immune plate (Thermo Scientific, 468667);
taking 5 μl of sample into the sample wells of a 96-well immunization plate (less than 5 μl supplemented with PBS buffer);
adding 250 mu LG250 staining solution into each well;
measuring A595 by using an enzyme-labeled instrument;
the protein concentration in the samples was calculated from a standard curve, as in fig. 6, where y=0.6849x+0.6325, r=0.9925.
The concentration of CC1H-Biotin protein was calculated to be 2.36 mg/mL.
(4) Sample purity, concentration were identified by reducing/non-reducing SDS-PAGE:
sample purity, concentration were identified by reducing/non-reducing SDS-PAGE. The purification effect of the CC1H-Biotin is shown in FIG. 7, wherein lanes from left to right are the CC1H-Biotin containing the DTT reduction loading buffer and the CC1H-Biotin containing the non-reduction loading buffer in sequence.
The molecular weight of CC1H-Biotin is about 41 kDa, and the purity is more than 98%.
(5) Biotin incorporation levels were detected using the Pierce ™ Biotin Quantitation Kit (HABA assay) (Thermo Scientific ™, 28005) kit:
balancing an ABA/Avidin premix to room temperature;
adding 100 mu L of ultrapure water into the HABA/Avidin premix tube, and uniformly blowing by using a gun head;
160 μl of PBS buffer was added to wells of 96-well immunoplates (Thermo Scientific, 468667);
adding 20 mu L of HABA/Avidin premix solution into the hole containing the PBS buffer solution, shaking and uniformly mixing, and measuring the absorbance of A500;
adding 20 mu L of biotinylated sample into the hole containing HABA/Avidin, shaking and mixing uniformly, and measuring the absorbance of the A500, and keeping constant for at least 15 seconds;
biotin incorporation levels were calculated from beer's law. The results are shown in Table 4. The Biotin incorporation level of CC1H-Biotin was calculated to be 1.80 mol Biotin/mol protein.
TABLE 4 Biotin incorporation levels
(6) Interaction determination of CC1H-Biotin and mouse anti-human PLA2R monoclonal antibody
SA sensors (Sartorius, octet square Streptavidin (SA) Biosensor, 18-5019) were selected using a Sartorius (Sartorius) Octet square N1 molecular interaction instrument:
immersing the sensor in PBSTB buffer (PBS buffer with 0.02% Tween-20 and 0.1% BSA) for pre-wetting for >10 min, and loading onto a molecular interaction instrument;
the sensor was immersed in different buffers sequentially and tested using a 5-step method:
baseline 1 (300 μl of PBSTB buffer, 1 min) - > cured (CC 1H-biotin,20 μg/mL 300 μl,2 min) - > baseline 2 (PBSTB buffer, 3 min) - > bound (murine anti-human PLA2R mab 300 μl,2 min) - > dissociated (PBSTB buffer 300 μl,5 min) with a gradient of PBSTB buffer);
different concentrations of murine anti-human measured or fitted curves were plotted (fig. 8).
The binding constant (Ka), dissociation constant (Kd) and affinity constant (KD) were calculated and the results are shown in Table 5. The result shows that CC1H-Biotin has higher affinity with the mouse anti-human PLA2R monoclonal antibody.
TABLE 5 Ka, kd, KD values for CC1H-Biotin and murine anti-human PLA2R monoclonal antibody
KD (M) ka (1/Ms) kd (1/s) R^2
1.89E-07 4.11E+04 7.77E-03 0.9859
Example 7
In this example, membrane proteins containing three domains CysR, fnII, CTLD1 were detected by western blot.
(1) Construction of plasmid stable expression of membrane proteins comprising the CysR, fnII, CTLD1 domain.
Respectively constructing two membrane proteins, wherein a mCC1 protein coding region comprises a human PLA2R CysR, fnII, CTLD structural domain and a transmembrane region, namely an amino acid sequence of 1 to 367 (Glu) of a PLA2R full-length protein sequence (UniProt sequence accession number: Q13018) and an amino acid sequence of 1386 to 1463 transmembrane regions of the PLA2R full-length protein sequence; the mCC protein coding region comprises the human PLA2R CysR, fnII, CTLD, CTLD2 domains and transmembrane regions, i.e., amino acid sequences comprising amino acid sequences 1 to 510 (Asp) of the PLA2R full-length protein sequence (UniProt sequence accession number: Q13018) and the 1386 to 1463 transmembrane region of the PLA2R full-length protein sequence.
The plasmid map for expressing membrane protein mCC1 is shown in FIG. 9 and the plasmid map for expressing membrane protein mCC2 is shown in FIG. 10.
pNEO as plasmid vector
mCC1 and mCC signal peptides (SEQ ID NO. 15): MLLSPSLLLLLLLGAPRGCA; the coding sequence of the signal peptide is as follows: atgctgctgtcgccgtcgctgctgctgctgctgctgctgggggcgccgcggggctgcgcc (SEQ ID NO. 20);
mCC1 amino acid sequence (SEQ ID NO. 16): 48.5kD;
mCC1 (SEQ ID NO. 18);
mCC2 sequence (SEQ ID NO. 17): 65.0kD; mCC2 (SEQ ID NO. 19).
(2) Cell lines stably expressing mCC or mCC2 were screened.
HEK293 cells were transfected with pNeo-mCC1 or pNeo-mCC and screened with G418 to be essentially free of floating cells and >95% cell viability.
(3) The mouse anti-human PLA2R monoclonal antibodies were detected by western blot detection mCC1 or mCC.
HEK293 cells stably transfected with pNeo-mCC1 or pNeo-mCC2, and untransfected HEK293 cells were lysed with RIPA lysate (Bio, C500005-0050), SDS-PAGE was performed and transferred to PVDF membrane. Conventional western blot. Antibodies were added as in table 6.
TABLE 6 antibody addition Table
Western blot (FIG. 11) shows that lanes CC1H, mCC, mCC all appear striped at the expected molecular weight positions, and that the negative control untransfected plasmid HEK293 cell lanes are striped, showing that murine anti-human PLA2R monoclonal antibodies can be used for Western blot detection of proteins comprising the CysR, fnII, CTLD1 domain.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. An antibody against human PLA2R, or an antigen-binding fragment thereof, comprising a heavy chain complementarity determining region and a light chain complementarity determining region;
the heavy chain complementarity determining region comprises CDR-H1, CDR-H2 and CDR-H3 with amino acid sequences shown in SEQ ID NO.3-5 respectively;
the light chain complementarity determining regions include CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences shown in SEQ ID NO.6-8, respectively.
2. The anti-human PLA2R antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID No. 1; the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 2.
3. The anti-human PLA2R antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said antigen-binding fragment is selected from the group consisting of Fab ', fab, F (ab') 2 Any one of scFv and Fv;
the constant region of the antibody is selected from the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD;
the constant regions of the antibodies are derived from human, mouse, rat, bovine, equine, ovine, rabbit or canine species.
4. Use of an antibody or antigen-binding fragment thereof against human PLA2R according to any one of claims 1-3 for the preparation of a PLA2R detection product;
the PLA2R detection product is selected from a reagent, a kit or a chip;
the reagent comprises the antibody or antigen binding fragment thereof, and the antibody or antigen binding fragment thereof is labeled with a detectable label.
5. Use of an antibody or antigen-binding fragment thereof against human PLA2R according to any one of claims 1-3 for the preparation of a product for the detection or diagnosis of membranous nephropathy, wherein the diagnostic marker of membranous nephropathy is PLA2R;
the membranous nephropathy is primary membranous nephropathy or secondary membranous nephropathy.
6. A detection reagent or kit comprising an antibody or antigen-binding fragment thereof against human PLA2R according to any one of claims 1-3.
7. An immunoconjugate, bispecific molecule, chimeric and antigen receptor or pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of claims 1-3 against human PLA2R.
8. A vector comprising a nucleic acid molecule encoding the anti-human PLA2R antibody or antigen binding fragment thereof of any one of claims 1-3.
9. A recombinant cell comprising the vector of claim 8.
10. Use of an antibody or antigen-binding fragment thereof against human PLA2R according to any one of claims 1-3 for the detection of human PLA2R levels or the detection of pharmaceutical activity in a cellular model expressing PLA2R, wherein said use is for non-diagnostic and therapeutic purposes.
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