CN115109155A - Preparation method of anti-human PLA2R antibody standard substance - Google Patents
Preparation method of anti-human PLA2R antibody standard substance Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- G01N2800/34—Genitourinary disorders
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Abstract
The invention relates to the field of bioengineering, in particular to a preparation method of an anti-human PLA2R antibody standard substance. The method for substituting the affinity (chimeric) human-mouse antibody not only has simple preparation and reliable source, but also has high stability, and particularly avoids the potential ethical problem and the biological safety problem of adopting the serum of a patient.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a preparation method of an anti-human PLA2R antibody standard substance.
Background
Membranous Nephropathy (MN) is the most common pathological type of adult Nephrotic Syndrome (NS), the second leading cause of primary glomerular disease in our country, with 70% of patients with Membranous Nephropathy presenting with persistent and repetitive proteinuria, resulting in kidney damage and even life-threatening. Membranous Nephropathy can be classified into Idiopathic Membranous Nephropathy (IMN) and Secondary Membranous Nephropathy (SMN), with about 70-80% of IMN patients and about 20-30% of SMN patients. Among them, 75-85% of patients with IMN have proteinuria of renal disease degree, and about 40-60% of patients with IMN develop terminal renal failure within 5-20 years. The traditional clinical diagnosis method of IMN is kidney puncture for kidney histopathological examination, including light mirror, fluorescence and electron microscope, which is an invasive detection method, not only needs higher technical requirements, but also has certain damage to patients.
It has been proved that the PLA2R receptor, i.e. the phospholipase A2 receptor, is the main target antigen of autoantibodies, and research shows that 75% of IMN patients have positive antibody of PLA2R receptor in serum, while the patients with secondary membranous nephropathy rarely show positive antibody, and other types of glomerular disease patients and healthy people are all negative antibody. Therefore, the development of a non-invasive, low-risk, convenient-to-operate and high-sensitivity quantitative detection method for PLA2R is urgently needed in clinic.
In the existing related kit, the detection standard substance of the anti-human PLA2R antibody adopts the tested mixed serum of patients with positive PLA2R antibody or the prepared antibody, and the method is not only complicated in preparation, limited in source and lack of stability, but also has the problems of often relating to ethics and biological safety.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide an alternative method for affinity (chimeric) human-mouse antibodies, which is not only simple to prepare, reliable in source, but also highly stable, in particular avoiding potential ethical and biosafety problems with patient sera.
To achieve the above and other related objects, the first aspect of the present invention provides an immunogenic protein for immunizing an organism to produce antibodies against PLA 2R.
In one embodiment, the immunizing antigen protein is an extracellular domain sequence of PLA2R receptor.
In one embodiment, the amino acid sequence of the immunity antigen protein is SEQ ID NO.1 or conservative variation thereof.
In a second aspect of the invention, an antibody against PLA2R is provided, which is prepared by using the above-mentioned immunoantigen protein as an antigen.
Further, the antibody against PLA2R is capable of specifically binding to the extracellular antigen-binding region of the PLA2R receptor. The extracellular antigen binding region of PLA2R contains an amino acid fragment as shown in SEQ ID NO. 1.
In one embodiment, the antibody to PLA2R is a monoclonal antibody.
Further, the monoclonal antibody is selected from anti-PLA 2R monoclonal antibody 6G2 or anti-PLA 2R monoclonal antibody 3C 12.
The anti-PLA 2R monoclonal antibody 6G2 comprises a heavy chain and a light chain, wherein the heavy chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.12, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.13, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 14; the light chain comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO.15, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.16, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 17.
The anti-PLA 2R monoclonal antibody 3C12 comprises a heavy chain and a light chain, wherein the heavy chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.18, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.19, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 20; the light chain comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown in SEQ ID No.21, the amino acid sequence of the LCDR2 is shown in SEQ ID No.22, and the amino acid sequence of the LCDR3 is shown in SEQ ID No. 23.
The heavy and light chains are linked by disulfide bonds.
Furthermore, the amino acid sequence of the heavy chain variable region of the anti-PLA 2R monoclonal antibody 6G2 is SEQ ID NO.5 or a conservative variation sequence thereof, and the amino acid sequence of the light chain variable region of the anti-PLA 2R monoclonal antibody is SEQ ID NO.7 or a conservative variation sequence thereof. The coding nucleotide sequence of the heavy chain variable region of the anti-PLA 2R monoclonal antibody 6G2 is SEQ ID NO.4 or a conservative variant sequence thereof, and the coding nucleotide sequence of the light chain variable region is SEQ ID NO.6 or a conservative variant sequence.
The amino acid sequence of the heavy chain variable region of the anti-PLA 2R monoclonal antibody 3C12 is SEQ ID NO.9 or a conservative variant sequence thereof, and the amino acid sequence of the light chain variable region of the anti-PLA 2R monoclonal antibody is SEQ ID NO.11 or a conservative variant sequence thereof. The coding nucleotide sequence of the heavy chain variable region of the PLA 2R-resistant monoclonal antibody 3C12 is SEQ ID NO.8 or a conservative variant sequence thereof, and the coding nucleotide sequence of the light chain variable region is SEQ ID NO.10 or a conservative variant sequence thereof.
Further, the monoclonal antibody is of murine origin.
Further, the monoclonal antibody is an immunoglobulin of IgG1 heavy chain and Kappa type light chain subtypes.
The monoclonal antibody aiming at PLA2R provided by the invention can be specifically bound with human PLA2R protein.
The monoclonal antibody or derivative thereof against PLA2R may be a single chain antibody, a double chain antibody, a chimeric antibody, a humanized antibody, as long as it binds to human PLA2R protein.
The third aspect of the present invention further provides a derivative of the monoclonal antibody against PLA2R, which can be the monoclonal antibody fragment, or a fusion protein comprising the monoclonal antibody or monoclonal antibody fragment, which can be Fab, Fab ', F (ab') 2, Fv, scFv, or the like. So long as it binds to human PLA2R protein.
A chimeric antibody against PLA2R comprising a Fab against a monoclonal antibody of PLA2R and an Fc of human IgG 4. The chimeric antibody is capable of binding to human PLA2R protein and human IgG 4.
That is, not only was it possible to capture the human PLA2R protein as a primary antibody by Fab of a monoclonal antibody against PLA2R, but also it was possible to bind to a secondary antibody by Fc of human IgG 4.
Further, the chimeric antibody against PLA2R includes a heavy chain including the heavy chain variable region VH of anti-PLA 2R mab, constant region 1CH1, Fc of human IgG4, and a light chain including the light chain variable region VL of anti-PLA 2R mab, kappa chain constant region CL.
Further, the heavy chain variable region VH of the anti-PLA 2R monoclonal antibody is selected from the heavy chain variable region VH of the anti-PLA 2R monoclonal antibody 6G2 or the heavy chain variable region VH of the anti-PLA 2R monoclonal antibody 3C 12. The amino acid sequence of the heavy chain variable region VH of the anti-PLA 2R monoclonal antibody 6G2 is shown in SEQ ID NO. 5. The amino acid sequence of the heavy chain variable region VH of the anti-PLA 2R monoclonal antibody 3C12 is shown in SEQ ID NO. 9. The amino acid sequence of the constant region 1CH1 is shown in SEQ ID NO. 34. The amino acid sequence of the Fc of the human IgG4 is shown in SEQ ID NO. 35. The light chain variable region VL of the anti-PLA 2R monoclonal antibody is selected from the light chain variable region VL of the anti-PLA 2R monoclonal antibody 6G2 or the light chain variable region VL of the anti-PLA 2R monoclonal antibody 3C 12. The amino acid sequence of the light chain variable region VL of the anti-PLA 2R monoclonal antibody 6G2 is shown as SEQ ID NO. 7. The amino acid sequence of the light chain variable region VL of the PLA2R monoclonal antibody 3C12 is shown in SEQ ID NO. 11. The amino acid sequence of the kappa chain constant region CL is shown as SEQ ID NO. 36.
In one embodiment, the amino acid sequence of the heavy chain of the chimeric antibody is SEQ ID No.37 or a conservative variant thereof. The amino acid sequence of the light chain is SEQ ID NO.38 or a conservative variant thereof.
In one embodiment, the amino acid sequence of the heavy chain of the chimeric antibody is SEQ ID No.39 or a conservative variation thereof. The amino acid sequence of the light chain is SEQ ID NO.40 or a conservative variant sequence thereof.
The heavy and light chains are linked by disulfide bonds.
In a fourth aspect, the invention provides an isolated DNA molecule encoding the variable region or full length amino acids of the heavy and/or light chain of the monoclonal or chimeric antibody directed against PLA 2R.
A fifth aspect of the invention provides a construct comprising the isolated DNA molecule.
Further, the construct is constructed by inserting the isolated DNA molecule into a multiple cloning site of an expression vector. The expression vector may be, in particular, an expression vector commonly used in the art, which is well known to those skilled in the art, and particularly employable is an expression vector including, but not limited to: pET series expression vector, pGEX series expression vector, pcDNA series expression vector, etc.
In the sixth aspect of the invention, an expression system of the monoclonal antibody or the chimeric antibody is provided, which is constructed by transfecting the construct into a host cell.
In a seventh aspect of the present invention, there is provided a method for producing the monoclonal antibody or the chimeric antibody, comprising the steps of: culturing said monoclonal or chimeric antibody expression system under conditions suitable for expression of said antibody, thereby expressing said monoclonal or chimeric antibody, and purifying and isolating said monoclonal or chimeric antibody.
In an eighth aspect of the invention, the use of the monoclonal antibody or the chimeric antibody in the preparation of a PLA2R detection kit is provided.
The ninth aspect of the invention provides a PLA2R detection kit, which includes the aforementioned monoclonal antibody or chimeric antibody.
In one embodiment, the kit comprises the chimeric antibody described above. The chimeric antibody can not only serve as a primary antibody to capture human PLA2R protein by Fab of a monoclonal antibody against PLA2R, but also can bind to a secondary antibody by Fc of human IgG 4.
Further, the detection kit further comprises a secondary antibody, and the secondary antibody can be combined with Fc of human IgG4 in the chimeric antibody. Can also be combined with Fc of human IgG1 and IgG 2.
Optionally, the secondary antibody is an IgG monoclonal antibody comprising a heavy chain and a light chain, the heavy chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 is shown as SEQ ID No.28, the amino acid sequence of HCDR2 is shown as SEQ ID No.29, and the amino acid sequence of HCDR3 is shown as SEQ ID No. 30; the light chain comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO.31, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.32, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 33.
The heavy and light chains are linked by disulfide bonds.
Furthermore, the amino acid sequence of the heavy chain variable region of the IgG monoclonal antibody is SEQ ID No.25 or a conservative variant sequence thereof, and the amino acid sequence of the light chain variable region of the IgG monoclonal antibody is SEQ ID No.27 or a conservative variant sequence thereof. The encoding nucleotide sequence of the heavy chain variable region of the IgG monoclonal antibody is SEQ ID NO.24 or a conservative variation sequence thereof, and the encoding nucleotide sequence of the light chain variable region is SEQ ID NO.26 or a conservative variation sequence thereof.
In one embodiment, the secondary antibody is labeled with a marker molecule, and the amount of secondary antibody bound to the primary antibody, and hence the amount of PLA2R bound to the primary antibody, is known from the marker molecule. For example, the labeling molecule may be HRP.
A membranous nephropathy detection product comprises the PLA2R detection kit.
As described above, the technical solution of the present invention has the following beneficial effects:
the method for substituting the affinity (chimeric) human-mouse antibody not only has simple preparation and reliable source, but also has high stability, and particularly avoids the potential ethical problem and the biological safety problem of adopting the serum of a patient.
Drawings
FIG. 1: mouse anti-human/monkey IgG Fc mab 3D2 binds to human IgG and monkey IgG effects.
FIG. 2 is a schematic diagram: mouse anti-human/monkey IgG Fc mab 13E9 binds to human IgG and monkey IgG effect data.
FIG. 3: mouse anti-human/monkey IgG Fc mab 3D2 bound IgG1, IgG2 and IgG4 effects.
FIG. 4: mouse anti-human/monkey IgG Fc mab 13E9 bound IgG1, IgG2 and IgG4 effects.
FIG. 5: 3C12,5a5,6G2,6G11,7D5,8F4 bind human PLA2R effects.
FIG. 6: schematic structure diagram of recombinant chimeric monoclonal antibody.
FIG. 7: chimeric 3C12 mab (ch3C12) bound to human PLA2R (hPLA 2R).
FIG. 8: chimeric 6G2 mab (ch6G2) bound human PLA2R (hPLA 2R).
FIG. 9: anti-human IgG Fc monoclonal antibody bio-3D2 detects anti-human PLA2R serum standard.
FIG. 10: the anti-hPLA 2R chimeric monoclonal antibody ch6G2 is used as a standard substance, and the human serum standard substance is detected by combining with a biotin-labeled 3D2 monoclonal antibody.
Detailed Description
The term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic spot with high specificity. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
The term "variable" as used herein means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called the Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are substantially in a p-fold configuration, connected by three CDRs forming a concatemeric loop, and in some cases may form part of a p-fold structure. The CDRs in each chain are held closely together by the FR regions and form the antigen binding site of an antibody with the CDRs of the other chain, and the constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, e.g., participation in antibody-dependent cytotoxicity of the antibody.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
EXAMPLE 1 preparation of mouse anti-human PLA2R monoclonal antibody
1) Recombinant human PLA2R immunized mice
Immunogen: the recombinant human PLA2R protein is expressed by adopting HEK293 cells, and the amino acid sequence of the protein is shown as SEQ ID NO.1, and specifically comprises the following steps:
EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSDAESGCQEGWERHGGFCYKIDTVLRSFDQASSGYYCPPALVTITNRFEQAFITSLISSVVKMKDSYFWIALQDQNDTGEYTWKPVGQKPEPVQYTHWNTHQPRYSGGCVAMRGRHPLGRWEVKHCRHFKAMSLCKQPVENQEKAEYEERWPFHPCYLDWESEPGLASCFKVFHSEKVLMKRTWREAEAFCEEFGAHLASFAHIEEENFVNELLHSKFNWTEERQFWIGFNKRNPLNAGSWEWSDRTPVVSSFLDNTYFGEDARNCAVYKANKTLLPLHCGSKREWICKIPRDVKPKIPFWYQYDVPWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVNNQSQRCGFISSITGLWGSEECSVSMPSICKRKKVWLIEKKKDTPKQHGTCPKGWLYFNYKCLLLNIPKDPSSWKNWTHAQHFCAEEGGTLVAIESEVEQAFITMNLFGQTTSVWIGLQNDDYETWLNGKPVVYSNWSPFDIINIPSHNTTEVQKHIPLCALLSSNPNFHFTGKWYFEDCGKEGYGFVCEKMQDTSGHGVNTSDMYPMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHVPPETRQSEHPELCSETSIPWIKFKSNCYSFSTVLDSMSFEAAHEFCKKEGSNLLTIKDEAENAFLLEELFAFGSSVQMVWLNAQFDGNNETIKWFDGTPTDQSNWGIRKPDTDYFKPHHCVALRIPEGLWQLSPCQEKKGFICKMEADIHTA。
the immune process comprises the following steps: on day 1, after emulsification of recombinant human PLA2R protein (SEQ ID No.1) with complete freund's complete adjuvant (CFA, Sigma), Balb/c mice (purchased from shanghai ling biotechnology limited) were injected subcutaneously at multiple points (50 ug/mouse/0.5 ml), 21, 41 days, after emulsification of recombinant human PLA2R protein (SEQ ID No.1) with incomplete freund's complete adjuvant (IFA, Sigma), Balb/c mice were injected subcutaneously (50 ug/mouse/0.5 ml), 61 days, after intraperitoneal injection of recombinant human PLA2R protein (SEQ ID No.1) (50 ug/mouse/0.2 ml), 3-4 days, spleen was taken for fusion experiments.
2) Hybridoma cell preparation and screening
3-4 days after the last immunization of the mice, the mouse spleen cells were electrofused with the mouse myeloma cells SP2/0 in an electrofusion apparatus (BTX) using a conventional hybridoma protocol (Salhi et al, biochem. J.2004). The fused cells were suspended in complete medium uniformly as 10 5 Cells/100 ul/well were divided into a total of 40 96-well plates and cultured overnight, and the next day 100 ul/well of complete medium containing 2XHAT (Sigma) was added to make the culture medium in the 96-well plate 200 ul/well (containing 1 XHAT). After 7-12 days, the supernatants were harvested and screened for human PLA2R binding activity by indirect enzyme-linked immunosorbent assay (ELISA) to yield 289 positive wells. The 289 positive well culture supernatant was diluted, 14 wells were screened by ELISA, and first and second rounds of subcloning were performed by limiting dilution to obtain 6 strains of positive clone hybridoma, which were named 3C12,5A5,6G2,6G11,7D5, and 8F4, respectively.
Example 2 preparation of mouse anti-human/monkey IgG Fc monoclonal antibody
1) Human/monkey IgG Fc immunized mice
On day 1, after emulsifying human IgG antibody protein (human serum obtained by protein G affinity chromatography, polyclonal antibody, or commercially available) with complete freunds complete adjuvant (CFA, Sigma), Balb/c mice (purchased from shanghai ling G biotechnology limited) were injected subcutaneously (50 ug/mouse/0.5 ml), 21, 41 days, after emulsifying monkey IgG antibody protein (monkey serum obtained by protein G affinity chromatography, polyclonal antibody, or commercially available) with incomplete freunds complete adjuvant (IFA, Sigma), Balb/c mice were injected subcutaneously (50 ug/mouse/0.5 ml), 61 days, after intraperitoneal injection of human IgG (25 ug/mouse/0.2 ml), and 3-4 days, mice spleen was taken for experiments.
2) Hybridoma cell preparation and screening
3-4 days after the last immunization of mice, the conventional hybridoma protocol (Salh) was usedi.e., biochem. J.2004), and the mouse spleen cells were electrofused with the mouse myeloma cells SP2/0 in an electrofusion apparatus (BTX). The fused cells were suspended in complete medium uniformly as 10 5 Cells/100 ul/well were divided into a total of 40 96-well plates and cultured overnight, and the next day 100 ul/well of complete medium containing 2XHAT (Sigma) was added to make the culture medium in the 96-well plate 200 ul/well (containing 1 XHAT). After 7-12 days, the supernatant was harvested, screened for positive binding by indirect enzyme-linked immunosorbent assay (ELISA) for IL-2-hIgG4Fc (SEQ ID No. 2), (hTNFR) IL2-hIgG1 Fc (SEQ ID No. 3) binding positive, cynomolgus monkey IgG (obtained by protein G affinity chromatography of cynomolgus monkey serum, polyclonal antibodies, or commercially available) bound to positive wells, and subcloned by limiting dilution in first and second rounds to obtain positive clonal hybridoma cell line 4, designated 1a6,3D2,5E1,13E 9. The IL-2-hIgG4Fc is expressed by adopting HEK293 cells, and the protein amino acid sequence is shown as SEQ ID NO.2, and specifically comprises the following steps:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK are provided. The IL2-hIgG1 Fc is expressed by adopting an HEK293 cell, and the protein amino acid sequence is shown as SEQ ID NO.3, and specifically comprises the following components:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
EXAMPLE 3 Mass production and characterization of murine antibodies
1) Preparation of murine monoclonal antibody
Complete culture in serumThe hybridoma cell lines obtained in example 1 and example 2 were amplified in culture medium, centrifuged to medium SFM (Life technologies,12045-076) without serum, and the cell density was 1-2 × 10 7 Per ml at 5% CO 2 After culturing at 37 ℃ for 1 week, the culture supernatant was centrifuged and purified by protein G affinity chromatography to obtain mouse anti-human PLA2R mab 6 strains (designated 3C12,5a5,6G2,6G11,7D5, and 8F4, respectively) and mouse anti-human/monkey IgG Fc mab 4 strains (designated 1a6,3D2,5E1, and 13E9, respectively).
2) Determination of anti-human/monkey IgG Fc monoclonal antibody binding to human IgG, monkey IgG by ELISA method
Biotinylated mouse anti-human/monkey IgG Fc monoclonal antibodies (1a6,3D2,5E1,13E9) were obtained by labeling 4 strains of mouse anti-human/monkey IgG Fc monoclonal antibodies (1a6,3D2,5E1,13E9) with biotin.
Human IgG and monkey IgG were coated at 0.1 ug/well (1ug/ml x 100ul) in a microplate (4 ℃ overnight), after 1% BSA was blocked (37 ℃ for 1 hour), each biotinylated mouse anti-human/monkey IgG Fc monoclonal antibody (1A6,3D2,5E1,13E9) (37 ℃ for 1 hour) was added at different concentrations, after washing the plates for 3 times with PBST, HRP-labeled SA (Pierce Co., 37 ℃ for 30 minutes) was added, after washing the plates for 5 times with PBST, TMB developing solution (BD Co.) was added, after 5-10 minutes, 2M sulfuric acid stop solution was added, and OD450 was measured.
For monoclonal antibodies 3D2 and 13E9, binding of human IgG and monkey IgG was consistent, and OD values were consistent at each point of detection (1000ng/ml to 8ng/ml), with an error of less than 3%. Mouse anti-human/monkey IgG Fc monoclonal antibody 3D2 binding human IgG and monkey IgG efficacy data are shown in table 1 and figure 1. Mouse anti-human/monkey IgG Fc mab 13E9 binding human IgG and monkey IgG efficacy data are presented in table 2 and figure 2. That is, the experiments demonstrated that these antibodies were able to bind human/monkey IgG simultaneously, i.e. to a common part of both IgG, as exemplified by mabs 3D2 and 13E 9.
TABLE 1 mouse anti-human/monkey IgG Fc monoclonal antibody 3D2 binding human IgG and monkey IgG Effect data
TABLE 2 mouse anti-human/monkey IgG Fc monoclonal antibody 13E9 binding human IgG and monkey IgG Effect data
3) Determination of anti-human/monkey IgG Fc mAbs binding to human IgG1 Fc, IgG2 Fc and IgG4Fc by ELISA
Human IgG1, human IgG2, human IgG40.1 ug/well (1ug/ml x 100ul) were coated in a microplate (4 ℃ overnight), after 1% BSA was blocked (37 ℃ for 1 hour), biotinylated mouse anti-human/monkey IgG Fc monoclonal antibodies (1A6,3D2,5E1,13E9) were added at different concentrations, respectively, after washing the plates 3 times with PBST, HRP-labeled SA (Pierce Co., Ltd.) was added, after washing the plates 5 times with PBST, 30 minutes at 37 ℃ and TMB developing solution (BD Co., Ltd.) were added, after washing the plates 5 times with PBST, 2M sulfuric acid stop solution was added after 5 to 10 minutes at room temperature, and OD450 was measured.
For monoclonal antibodies 3D2 and 13E9, binding to human IgG1, IgG2 and IgG4 were consistent, and at each point of detection (1000ng/ml to 4ng/ml), OD values were consistent, with an error of less than 3%. Mouse anti-human/monkey IgG Fc mab 3D2 binding IgG1, IgG2 and IgG4 effect data are shown in table 3 and figure 3. Mouse anti-human/monkey IgG Fc mab 13E9 binding IgG1, IgG2 and IgG4 effect data are shown in table 4 and figure 4.
TABLE 3 mouse anti-human/monkey IgG Fc mAb 3D2 binding IgG1, IgG2 and IgG4 Effect data
Monoclonal antibody: 3D2
TABLE 4 mouse anti-human/monkey IgG Fc mAb 13E9 binding IgG1, IgG2 and IgG4 Effect data
Monoclonal antibody: 13E9
| Antibody concentration (ng/ml) | Mean value (OD450) | IgG1 error (%) | IgG2 error (%) | IgG4 error (%) |
| 1000.00 | 1.9449 | -2.3 | 0.8 | 1.5 |
| 333.33 | 1.5833 | -0.8 | -0.1 | 1.0 |
| 111.11 | 0.9768 | -1.7 | 1.5 | 0.2 |
| 37.04 | 0.4399 | -2.2 | 0.4 | 1.8 |
| 12.35 | 0.1996 | -1.2 | -0.7 | 1.9 |
| 4.12 | 0.0555 | 5.8 | -7.6 | 1.8 |
| 1.37 | 0.0652 | -5.5 | -19.9 | 25.4 |
4) ELISA method for determining that anti-human PLA2R monoclonal antibody binds to human PLA2R
Biotinylated mouse anti-human PLA2R monoclonal antibodies (3C12,5A5,6G2,6G11,7D5,8F4) were obtained by labeling 6 mouse anti-human PLA2R monoclonal antibodies (3C12,5A5,6G2,6G11,7D5,8F4) with biotin.
Recombinant human PLA2R protein (SEQ ID NO.1)0.1 ug/well (1ug/ml x 100ul) was coated in a microplate (4 ℃ overnight), after 1% BSA was blocked (37 ℃ for 1 hour), biotinylated mouse anti-human PLA2R monoclonal antibodies (3C12,5A5,6G2,6G11,7D5,8F4) of different concentrations were added, after washing the plate for 3 times with PBST, goat anti-mouse IgG labeled with HRP (Merk Co.) was added, at 37 ℃ for 30 minutes, after washing the plate for 5 times with PBST, TMB developing solution (BD Co.) was added, after 5 to 10 minutes at room temperature, 2M sulfuric acid stop solution was added, and OD450 was measured.
The results are shown in fig. 5, and each monoclonal antibody (3C12,5a5,6G2,6G11,7D5,8F4) can bind well to human PLA 2R.
5) Monoclonal antibody gene and amino acid sequence
Extracting RNA from hybridoma by TRIZol method, and SMARTRace technologyReverse transcription of RNA into cDNA, and use
Performing PCR amplification by using a FastPfu DNA Polymerase kit to obtain the full length of a heavy chain and a light chain, connecting a target fragment to a vector by using ligase, transforming a connection product into an escherichia coli competent cell, then selecting a monoclonal for sequencing, comparing sequencing results by using an IMGT/V-QUEST database, and further analyzing.
Nucleotide and amino acid sequence of mouse anti-PLA 2R monoclonal antibody
Monoclonal antibody 6G2 variable region and CDRs nucleotide and amino acid sequence
The nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody 6G2 is shown in SEQ ID NO. 4. The method comprises the following specific steps:
GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACCTTCAGTACCTATGTGATCCACTGGGTGAAGAAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTCATCCTTACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGACGACTCTGCGGTCTATTACTGTGCAAGATTACGACGGTATGCTATGGCCTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCC
the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody 6G2 is shown in SEQ ID NO. 5. The method specifically comprises the following steps: EVQLQQSGPELVKPGASVKMSCKASGYTFSTYVIHWVKKKPGQGLEWIGYIHPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSDDSAVYYCARLRRYAMAYWGQGTSVTVSS are provided.
The amino acid sequence of complementarity determining region 1(HCDR1) of monoclonal antibody 6G2 heavy chain is shown in SEQ ID NO. 12. The method specifically comprises the following steps: GYTFSTYV.
The amino acid sequence of complementarity determining region 2(HCDR2) of monoclonal antibody 6G2 heavy chain is shown in SEQ ID NO. 13. The method specifically comprises the following steps: IHPYNDGT.
The amino acid sequence of the complementarity determining region 3(HCDR3) of monoclonal antibody 6G2 heavy chain is shown in SEQ ID NO. 14. The method comprises the following specific steps: ARLRRYAMAY are provided.
The nucleotide sequence of the light chain variable region (VL) of monoclonal antibody 6G2 is shown in SEQ ID NO. 6. The method specifically comprises the following steps:
GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAACAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。
the amino acid sequence of the light chain variable region (VL) of monoclonal antibody 6G2 is shown in SEQ ID NO. 7. The method comprises the following specific steps:
DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPYTFGGGTKLEIK。
the amino acid sequence of complementarity determining region 1(LCDR1) of monoclonal antibody 6G2 light chain is shown in SEQ ID NO. 15. The method specifically comprises the following steps: ENIYSN.
The amino acid sequence of complementarity determining region 2(LCDR2) of monoclonal antibody 6G2 light chain is shown in SEQ ID NO. 16. The method specifically comprises the following steps: AATNLA.
The amino acid sequence of the complementarity determining region 3(LCDR3) of the monoclonal antibody 6G2 light chain is shown in SEQ ID NO. 17. The method specifically comprises the following steps: QHFWGTPYT is added.
The nucleotide sequence of the heavy chain variable region (VH) of mAb 3C12 is shown in SEQ ID NO. 8. The method comprises the following specific steps:
CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCAGGGTTCTCATTAACCAGCTATGGTGTAAGCTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATTTGGGGTGACGGGAGGAAAAATTATCATTCAGCTCTCATATCCAGACTGAGCATCAGCAAGGATAACTCCAAGAGCCAAGTTTTCTTAAAACTGAACAGTCTGCAAACTGATGACACAGCCACGTACTACTGTGCCAAAACCTCCAAGGTTTATGGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。
the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody 3C12 is shown in SEQ ID NO. 9. The method specifically comprises the following steps:
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPPGKGLEWLGVIWGDGRKNYHSALISRLSISKDNSKSQVFLKLNSLQTDDTATYYCAKTSKVYGYAMDYWGQGTSVTVSS。
the amino acid sequence of the complementarity determining region 1(HCDR1) of the monoclonal antibody 3C12 heavy chain is shown in SEQ ID NO. 18. The method specifically comprises the following steps: GFSLTSYG.
The amino acid sequence of complementarity determining region 2(HCDR2) of monoclonal antibody 3C12 heavy chain is shown in SEQ ID NO. 19. The method specifically comprises the following steps: IWGDGRK.
The amino acid sequence of the complementarity determining region 3(HCDR3) of the monoclonal antibody 3C12 heavy chain is shown in SEQ ID NO. 20. The method specifically comprises the following steps: AKTSKVYGYAMDY is added.
The nucleotide sequence of the variable region of the light chain (VL) of monoclonal antibody 3C12 is shown in SEQ ID NO. 10. The method specifically comprises the following steps:
GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。
the amino acid sequence of the light chain variable region (VL) of monoclonal antibody 3C12 is shown in SEQ ID NO. 11. The method specifically comprises the following steps:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK。
the amino acid sequence of complementarity determining region 1(LCDR1) of monoclonal antibody 3C12 light chain is shown in SEQ ID NO. 21. The method specifically comprises the following steps:
QSIVHSNGNTY。
the amino acid sequence of complementarity determining region 2(LCDR2) of monoclonal antibody 3C12 light chain is shown in SEQ ID NO. 22. The method comprises the following specific steps: KVSNRF.
The amino acid sequence of complementarity determining region 3(LCDR3) of monoclonal antibody 3C12 light chain is shown in SEQ ID NO. 23. The method specifically comprises the following steps: FQGSHVPYT.
Mouse anti-human/monkey IgG Fc monoclonal antibody nucleotide and amino acid sequence
Sequencing results of anti-human/monkey IgG Fc monoclonal antibodies (1A6,3D2,5E1 and 13E9) show that the monoclonal antibodies 3D2 and 13E9 are completely consistent on the level of nucleotide sequences and amino acid sequences and are the same monoclonal antibodies.
Monoclonal antibody 3D2 variable region and CDRs nucleotide and amino acid sequence
The nucleotide sequence of the heavy chain variable region (VH) of the monoclonal antibody 3D2 is shown in SEQ ID NO. 24. The method specifically comprises the following steps:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACATTGGGGGTTACTACGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。
the amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody 3D2 is shown in SEQ ID NO. 25. The method comprises the following specific steps:
EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHWGLLRAMDYWGQGTSVTVSS。
the amino acid sequence of the complementarity determining region 1(HCDR1) of the monoclonal antibody 3D2 heavy chain is shown in SEQ ID NO. 28. The method specifically comprises the following steps:
GFTFSSYG。
the amino acid sequence of the complementarity determining region 2(HCDR2) of the monoclonal antibody 3D2 heavy chain is shown in SEQ ID NO. 29. The method specifically comprises the following steps:
ISSGGSYT。
the amino acid sequence of the complementarity determining region 3(HCDR3) of the monoclonal antibody 3D2 heavy chain is shown in SEQ ID NO. 30. The method specifically comprises the following steps:
ARHWGLLRAMDY。
the nucleotide sequence of the variable region of the light chain (VL) of mAb 3D2 is shown in SEQ ID NO. 26. The method specifically comprises the following steps:
GACATTTTGTTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAACCATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。
the amino acid sequence of the variable region of the light chain (VL) of mAb 3D2 is shown in SEQ ID NO. 27. The method specifically comprises the following steps:
DILLTQSPAILSVSPGERVSFSCRASQSIGTTIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPWTFGGGTKLEIK。
the amino acid sequence of complementarity determining region 1(LCDR1) of monoclonal antibody 3D2 light chain is shown in SEQ ID NO. 31. The method specifically comprises the following steps: QSIGTT.
The amino acid sequence of complementarity determining region 2(LCDR2) of monoclonal antibody 3D2 light chain is shown in SEQ ID NO. 32. The method comprises the following specific steps: YASESI.
The amino acid sequence of complementarity determining region 3(LCDR3) of monoclonal antibody 3D2 light chain is shown in SEQ ID NO. 33. The method comprises the following specific steps: QQSNSWPWT is added.
Example 4 recombinant chimeric monoclonal antibody preparation and characterization
(1) Structural description of recombinant chimeric monoclonal antibodies
The recombinant chimeric monoclonal antibody of the present embodiment refers to an antibody formed by linking the variable region (VH) and constant region 1(CH1) of the heavy chain of a mouse anti-PLA 2R monoclonal antibody (e.g., 3C12 or 6G2) to the Fc of human IgG4 to form a mouse-human hybrid heavy chain, and then linking the mouse-human hybrid heavy chain to the light chain of a mouse anti-PLA 2R monoclonal antibody (e.g., 3C12 or 6G 2). Thus, the chimeric antibody retains the ability to bind to the antigen PLA2R, while the Fc portion of the included human antibody is bound by the aforementioned mouse anti-human Fc mab 3D 2. The schematic structure of the recombinant chimeric monoclonal antibody is shown in fig. 6, and comprises a heavy chain and a light chain, wherein the heavy chain comprises VH of a mouse anti-PLA 2R monoclonal antibody, CH1 of a mouse anti-PLA 2R monoclonal antibody, and Fc (namely CH2CH3) of human IgG4, and the light chain comprises VL of a mouse anti-PLA 2R monoclonal antibody and a constant region (CL) of a mouse kappa chain.
(2) Preparation and effect of recombinant chimeric 3C12 monoclonal antibody (ch3C12)
The amino acid sequence of the heavy chain variable region (VH) of the mouse anti-PLA 2R monoclonal antibody 3C12 is shown in SEQ ID NO. 9. The amino acid sequence of the constant region 1(CH1) of the mouse anti-PLA 2R monoclonal antibody 3C12 is shown in SEQ ID NO.34, and specifically comprises the following steps: AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKI are provided. The amino acid sequence of Fc of human IgG4 is shown in SEQ ID NO.35, and specifically comprises:
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK are provided. The amino acid sequence of the light chain variable region (VL) of the mouse anti-PLA 2R monoclonal antibody 3C12 is shown in SEQ ID NO. 11. The amino acid sequence of the constant region (CL) of the mouse kappa chain is shown in SEQ ID NO. 36. The method comprises the following specific steps:
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
the preparation method of the recombinant chimeric 3C12 monoclonal antibody (ch3C12) of the embodiment comprises the following steps:
the encoding nucleotide sequence of the VH of the heavy chain variable region of the mouse anti-PLA 2R monoclonal antibody 3C12 (namely, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.9), the constant region 1CH1 encoding nucleotide sequence (namely, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.34) and the Fc encoding nucleotide sequence of the human IgG4 (namely, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.35) are spliced to obtain the heavy chain encoding nucleotide sequence. Splicing the encoding nucleotide sequence of the light chain variable region VL of the mouse anti-PLA 2R monoclonal antibody 3C12 (namely the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.11) and the constant region CL encoding nucleotide sequence of the mouse kappa chain (namely the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.36) to obtain the light chain encoding nucleotide sequence. Constructing the obtained heavy chain coding nucleotide sequence and light chain coding nucleotide sequence to pcDNA3.4 expression vector, transfecting HEK-293F cell, purifying by Protein A to obtain chimeric antibody, determining the molecular weight of the expressed antibody to be about 150kD and the antibody purity to be more than 95 percent by SDS-PAGE electrophoresis and SEC-HPLC, quantifying, subpackaging, and freezing at-80 ℃ for later use.
The resulting chimeric 3C12 mab (ch3C12) was identified to include a heavy chain and a light chain. The amino acid sequence of the heavy chain of the chimeric 3C12 monoclonal antibody (ch3C12) is shown as SEQ ID NO. 37. The method specifically comprises the following steps:
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPPGKGLEWLGVIWGDGRKNYHSALISRLSISKDNSKSQVFLKLNSLQTDDTATYYCAKTSKVYGYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK are provided. Namely, the polypeptide sequentially comprises a heavy chain variable region VH amino acid sequence (SEQ ID NO.9), a constant region 1CH1 amino acid sequence (SEQ ID NO.34) and a human IgG4F amino acid sequence (SEQ ID NO.35) of a mouse anti-PLA 2R monoclonal antibody 3C 12.
The amino acid sequence of the light chain of the chimeric 3C12 monoclonal antibody (ch3C12) is shown as SEQ ID NO. 38. The method comprises the following specific steps:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC are provided. Namely, the amino acid sequence (SEQ ID NO.11) of the light chain variable region VL of the mouse anti-PLA 2R monoclonal antibody 3C12 is included in sequence. The amino acid sequence of the constant region CL of the mouse kappa chain (SEQ ID NO. 36).
The chimeric 3C12 mab (ch3C12) obtained in this example bound human PLA2R (hPLA2R) for effect:
recombinant human PLA2R protein (SEQ ID NO.1)0.1 ug/well (1ug/ml x 100ul) was coated in a microplate (4 ℃ overnight), after 1% BSA was blocked (37 ℃ for 1 hour), mouse monoclonal antibodies [ chimeric 3C12 monoclonal antibody (ch3C12) ] of different concentrations were added, respectively, at 37 ℃ for 1 hour, after PBST was washed for 3 times, goat anti-human IgG labeled with HRP (Merk Co., Ltd.) was added, at 37 ℃ for 30 minutes, after PBST was washed for 5 times, TMB developing solution (BD Co., Ltd.) was added, after 5-10 minutes at room temperature, 2M sulfuric acid stop solution was added, and OD450 was determined.
The results are shown in FIG. 7, indicating that: the chimeric 3C12 mab (ch3C12) maintained binding activity to PLA2R antigen, while being recognized by an anti-human IgG secondary antibody because it contained human IgG4 Fc.
The chimeric 3C12 monoclonal antibody (ch3C12) obtained in this example differs from 3C12 in that the Fab of 3C12 is murine, and the Fc portion is derived from human IgG 4. Fab of ch3C12 bound to human PLA2R, and Fc of ch3C12 was recognized by anti-human IgG secondary antibody.
(3) Preparation and effect of recombinant chimeric 6G2 monoclonal antibody (ch6G2)
The amino acid sequence of the heavy chain variable region (VH) of the mouse anti-PLA 2R monoclonal antibody 6G2 is shown in SEQ ID NO. 5. The amino acid sequence of the constant region 1(CH1) of the mouse anti-PLA 2R monoclonal antibody 3C12 is shown in SEQ ID NO. 34. The amino acid sequence of Fc of human IgG4 is shown in SEQ ID NO. 35. The amino acid sequence of the light chain variable region (VL) of the mouse anti-PLA 2R monoclonal antibody 6G2 is shown in SEQ ID NO. 7. The amino acid sequence of the constant region (CL) of the mouse kappa chain is shown in SEQ ID NO. 36.
The preparation method of the recombinant chimeric 6G2 monoclonal antibody (ch6G2) of the embodiment comprises the following steps:
the encoding nucleotide sequence of the VH of the heavy chain variable region of the mouse anti-PLA 2R monoclonal antibody 6G2 (namely, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.5), the constant region 1CH1 encoding nucleotide sequence (the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.34) and the Fc encoding nucleotide sequence of the human IgG4 (namely, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.35) are spliced to obtain the heavy chain encoding nucleotide sequence. The encoding nucleotide sequence of the light chain variable region VL (namely the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.7) of the mouse anti-PLA 2R monoclonal antibody 6G2 and the constant region CL encoding nucleotide sequence (namely the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.36) of the mouse kappa chain are spliced to obtain the light chain encoding nucleotide sequence. Constructing the obtained heavy chain coding nucleotide sequence and light chain coding nucleotide sequence to pcDNA3.4 expression vector, transfecting HEK-293F cell, purifying by Protein A to obtain chimeric antibody, determining the molecular weight of the expressed antibody to be about 150kD and the antibody purity to be more than 95 percent by SDS-PAGE electrophoresis and SEC-HPLC, quantifying, subpackaging, and freezing at-80 ℃ for later use.
The resulting chimeric 6G2 mab (ch6G2) was identified to include a heavy chain and a light chain. The amino acid sequence of the heavy chain of the chimeric 6G2 monoclonal antibody (ch6G2) is shown as SEQ ID NO. 39. The method specifically comprises the following steps:
EVQLQQSGPELVKPGASVKMSCKASGYTFSTYVIHWVKKKPGQGLEWIGYIHPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSDDSAVYYCARLRRYAMAYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK are provided. Namely, the polypeptide sequentially comprises a heavy chain variable region VH amino acid sequence (SEQ ID NO.5), a constant region 1CH1 amino acid sequence (SEQ ID NO.34) and a human IgG4F amino acid sequence (SEQ ID NO.35) of the mouse anti-PLA 2R monoclonal antibody 6G 2.
The amino acid sequence of the light chain of the chimeric 6G2 monoclonal antibody (ch6G2) is shown as SEQ ID NO. 40. The method specifically comprises the following steps: DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC are provided. That is, the amino acid sequence of the light chain variable region VL of the mouse anti-PLA 2R monoclonal antibody ch6G2 (SEQ ID NO.7) is included in turn. The amino acid sequence of the constant region CL of the mouse kappa chain (SEQ ID NO. 36).
The chimeric 6G2 mab (6G2) obtained in this example binds human PLA2R (hPLA2R) with the following effects:
recombinant human PLA2R protein (SEQ ID NO.1)0.1 ug/well (1ug/ml x 100ul) was coated in a microplate (4 ℃ overnight), 1% BSA was blocked (37 ℃ for 1 hour), and then mouse monoclonal antibody [ 6G2 monoclonal antibody (6G2) ] with different concentrations was added, 1 hour at 37 ℃, goat anti-human IgG labeled with HRP (Merk corporation) was added after PBST washing the plate for 3 times, 30 minutes at 37 ℃, TMB developing solution (BD corporation) was added after PBST washing the plate for 5 times, 2M sulfuric acid stop solution was added after 5-10 minutes at room temperature, and OD450 was determined.
The results are shown in FIG. 8, indicating that: ch6G2 maintained binding activity to PLA2R antigen, and at the same time, was recognized by a secondary anti-human IgG antibody because it contained human IgG4 Fc.
The chimeric 6G2 monoclonal antibody (ch6G2) obtained in this example was different from 6G2 in that Fab of ch6G2 was murine, and the Fc portion was derived from human IgG 4. Fab of ch6G2 bound to human PLA2R, and Fc of ch6G2 was recognized by anti-human IgG secondary antibody.
Example 5 detection of human serum standards by anti-hPLA 2R chimeric monoclonal antibodies as standards
1. anti-hPLA 2R human serum standard, combined with biotin-labeled anti-human/monkey IgG Fc monoclonal antibody 3D2 to detect human chimeric monoclonal antibodies ch3C12 and ch6G2
The specific detection method comprises the following steps:
(1) biotin-labeled mab 3D2(bio-3D 2): biotin-labeled anti-human/monkey IgG Fc monoclonal antibody 3D2 (same as example 3) was prepared using NHS-activated biotin (product of Sigma) according to the instructions. NHS-activated biotin powder was dissolved in DMSO so that the concentration was 20mg/ml, and 50ul (1mg) of the aforementioned NHS-activated biotin solution was slowly added to 5ml (2mg/ml) of 3D2 monoclonal antibody solution, and after mixing, overnight at 4 ℃, TBS buffer (20mM Tris, 0.15N NaCl, pH 7.2) was removed for thorough dialysis. Obtaining the biotin-labeled monoclonal antibody 3D 2.
(2) Recombinant human PLA2R protein (SEQ ID NO.1)1ug/ml CBS coating, 4 ℃, overnight; PBST plate washing 3 times, no protein blocking liquid (Sigma company) 200 ul/hole, 37 degrees C, 1 hours; PBST wash plate 3 times, dilute to PLA2R chimeric monoclonal antibody ch3C12 (same as example 4) to the concentration of 20, 10, 5ng/ml respectively; the chimeric mAb ch6G2 to PLA2R (same as example 4) was diluted to concentrations of 40, 20, 10ng/ml, 100 ul/well added at 37 ℃ for 1 hour; diluting PLA2R antibody standard (1500RU/ml, Germany Europe company) to 500RU/ml, diluting 3 times, diluting for 7 gradients, adding 100 ul/well, 37 deg.C, 1 hr; PBST washing plate 3 times, dilution biotin mark Fc monoclonal antibody 3D2 [ this example 1 part of the step (1) preparation ] to 0.1ug/ml, 100 ul/hole add, 37 degrees C, 1 hours; PBST washing plate 3 times, adding SA-HRP (Pierce company, USA), 37 degrees, 0.5 hours; the plates were washed 6 times, TMB100 ul/well, developed for 10 min, 4M HCl50 ul/well stopped, OD450 was measured with a microplate reader (BioTek, USA), a standard curve was plotted against the standard measurements using the ELISA Calc regression/fitting calculation program V1.0 and the ch3C12/ch6G2 values were calculated.
1) Anti-human IgG Fc monoclonal antibody bio-3D2 detection anti-human PLA2R serum standard substance
As shown in FIG. 9, the standard test value is fit to the four-parameter regression method and the correlation coefficient R of the curve is fitted 2 The value is greater than 0.99.
As shown in Table 5, within the range of 6.2-166.7RU/ml, the errors of the serum standard substance detection are less than 1%, and the standard with the error of less than 15% is met.
TABLE 5
| RU/ml | OD450 | Back calculation RU/ml | Error (%) |
| 500.0 | 3.7370 | 664.3823 | 32.88 |
| 166.7 | 3.0320 | 166.7 | 0.02 |
| 55.6 | 1.9222 | 55.6 | 0.08 |
| 18.5 | 0.5088 | 18.5 | -0.10 |
| 6.2 | 0.1676 | 6.2 | 0.44 |
| 2.1 | 0.1195 | 4.31 | 109.32 |
| 0 | 0.0782 |
2) Detection of ch3C12 and ch6G2 by combining human serum standard of anti-human PLA2R with anti-human IgG Fc monoclonal antibody bio-3D2
TABLE 6
| ch3C12 | |||
| ng/ml | OD450 | Calculated value (RU/ml) | RU/ng |
| 20 | 1.7028 | 40.6191 | 1.0155 |
| 10 | 0.7389 | 20.3363 | 1.0168 |
| 5 | 0.3013 | 11.5981 | 1.1598 |
As shown in Table 6,3 concentrations of ch3C12, namely 20ng/ml, 10ng/ml and 5ng/ml, were tested, which contained PLA2R at concentrations of 48.46/24.23/12.29RU/ml, respectively, and the specific activity corresponding to ch3C12 was 2.42/2.42/2.45RU/ng, respectively, which was very consistent, indicating that ch3C12 contained 2.4RU per ng.
TABLE 7
| ch6G2 | |||
| ng/ml | OD450 | Calculated value (RU/ml) | RU/ng |
| 40 | 1.4213 | 40.6191 | 1.0155 |
| 20 | 0.5797 | 20.3363 | 1.0168 |
| 10 | 0.3102 | 11.5981 | 1.1598 |
As shown in Table 7, 3 concentrations of ch6G2, namely 40ng/ml, 20ng/ml and 10ng/ml, were tested, which contained PLA2R at concentrations of 40.62/20.34/11.6RU/ml, respectively, and the specific activities corresponding to ch6G2 at 1.02/1.02/1.16RU/ng, respectively, which were very consistent, and it was confirmed that ch6G2 contained 1.0RU per ng.
2. anti-hPLA 2R chimeric monoclonal antibody ch6G2 as standard substance, combined with biotin-labeled 3D2 monoclonal antibody to detect human serum standard substance
Recombinant human PLA2R protein (SEQ ID NO.1)1ug/ml CBS coating, 4 ℃, overnight; PBST plate washing 3 times, no protein blocking liquid (Sigma company) 200 ul/hole, 37 degrees C, 1 hours; PBST wash plate 3 times, dilute to PLA2R chimeric monoclonal antibody ch6G2 (same as example 4) to concentration 160, 80, 40, 20, 10, 5, 2.5, 0ng/ml respectively; diluting PLA2R antibody standard (1500RU/ml, Germany Europe company) to concentrations of 160, 80, 20RU/ml, respectively, adding 100 ul/well, 37 deg.C, 1 hr; PBST washing plate 3 times, dilution biotin mark Fc monoclonal antibody 3D2 [ this example 1 part of the step (1) preparation ] to 0.1ug/ml, 100 ul/hole add, 37 degrees C, 1 hours; PBST washing plate 3 times, adding SA-HRP (Pierce company, USA), 37 degrees, 0.5 hours; the plates were washed 6 times, TMB100 ul/well, developed for 10 min, 4M HCl50 ul/well stopped, OD450 was measured with a microplate reader (BioTek, USA), a standard curve was plotted against the standard measurements using the ELISA Calc regression/fitting calculation program V1.0 and the ch3C12/ch6G2 values were calculated.
TABLE 8
| ch6G2 | |||
| ng/ml | OD450 | Back calculation concentration (ng/ml) | Error (%) |
| 160 | 3.7209 | 161.0279 | 0.6425 |
| 80 | 2.6528 | 78.7522 | -1.5597 |
| 40 | 1.6603 | 40.3432 | 0.8580 |
| 20 | 0.9191 | 20.9068 | 4.5341 |
| 10 | 0.4198 | 10.0400 | 0.4004 |
| 5 | 0.1834 | 5.1467 | 2.9334 |
| 2.5 | 0.0952 | 3.2847 | 31.3865 |
As shown in Table 8, 3 concentrations of human serum against human PLA2R, 160ng/ml, 80ng/ml and 20ng/ml, containing 159.17/78.82/19.11ng/ml for ch6G2 antibody and 1.01/1.01/1.05RU/ng for ch6G2, respectively, were tested. Within the range of 5-160ng/ml, the errors of the detection of the ch6G2 standard are less than 5 percent.
As shown in FIG. 10, the standard test value is fit to a four-parameter regression method to fit a curve correlation coefficient R 2 The value is greater than 0.99.
TABLE 9
| Standard article | |||
| RU/ml | OD450 | Calculated value (ng/ml) | RU/ng |
| 160 | 3.7052 | 159.1682 | 1.0052 |
| 80 | 2.6542 | 78.8227 | 1.0149 |
| 20 | 0.8404 | 19.1089 | 1.0466 |
As shown in table 9, the results show that: within the range of ch6G 25-160 ng/ml, namely 5-160RU/ml, the error of the concentration detection of each standard substance calculated by the standard curve is less than 5 percent. The high concentration (160RU/ml), the medium concentration (80RU/ml) and the low concentration (20RU/ml) of the serum standard were detected by taking ch6G2 as a standard, and the activity of the serum standard at the three concentrations was calculated to be 159.2/78.8/19.1RU/ml respectively, and the errors from the labeled activity were-0.5%, -1.5% and-4.5%, both of which are < + > -5%, respectively, indicating that the binding of ch6G2 to biotinylated 3D2 can accurately determine the serum-derived PLA2R antibody standard.
3. Stability of ch6G2 and ch3C12 as detection standards
ch6G2 (same as example 4) was diluted to 16ug/ml with the above reagents No.1, No.2, No.3, No.4, No.5, and split-packaged into 0.5ml tubes (1.5ml centrifuge tubes), sealed and stored at 4 deg.C and-80 deg.C, and stored at 37 deg.C in a wet box. After one week (7 days) and 4 weeks (28 days), the samples stored at-80 ℃ were used as standards, the samples stored at 4 ℃ and 37 ℃ were measured according to the above-mentioned detection method, the concentrations of the samples corresponding to-80 ℃ were calculated, and the stability of the ch6G2 sample at 4 ℃ and 37 ℃ was judged by the error value.
ch3C12 (same as example 4) was diluted to 5ug/ml with the above 1#, 2#, 3#, 4#, 5# reagents, and subpackaged into 0.5 ml/tube (1.5ml centrifuge tube), sealed and stored at 4 deg.C and-80 deg.C, and stored at 37 deg.C in a wet box. After one week (7 days) and 4 weeks (28 days), the samples stored at-80 ℃ were used as standards, the samples stored at 4 ℃ and 37 ℃ were measured according to the above-mentioned detection method, the concentrations of the samples corresponding to-80 ℃ were calculated, and the stability of the ch3C12 sample at 4 ℃ and 37 ℃ was judged by the error value.
Standard (Euron corporation) stability of ch6G2 sample in reagent # 1 was measured as described above, and RU values were calculated for samples at-80 deg.C, 4 deg.C, and 37 deg.C, and the RU value of each sample was calculated and compared with the value measured for the sample at-80 deg.C to determine the stability of ch6G2 in reagent # 1.
Stability results for ch3C 12:
stability of ch3C12 in each reagent at 4 deg.C
As shown in table 10, ch3C12 contained > 15% measured points with errors between the measured and labeled values at 4 concentrations (50/10/2/0.4ng/ml) in 5 buffers after one week at 4 ℃ in all 5 common protein stabilizer buffers, 1#4/4, 2#2/4, 3#2/4, 4#3/4, and 5#2/4, while the error of the calculated concentration of the standard curve point was < 5%, which met the standard, indicating that ch3C12 was unstable at 4 ℃ in 5 buffers.
Watch 10
One week (7 days)
As shown in table 11, the errors of the detection value and the identification value of ch3C12 at 4 concentrations (50/10/2/0.4ng/ml) in all 5 common protein stabilizer buffers after one week at 37 ℃ each contain > 15% of measurement points, wherein 1#1/4, 2#3/4, 3#3/4, 4#3/4 and 5#4/4 are measured, and the error of the calculated concentration of the standard curve point is < 5%, which meets the standard and indicates that ch3C12 is unstable at 37 ℃ in 5 buffers.
TABLE 11
One week (7 days)
Stability results for ch6G 2:
as shown in Table 12, the difference between the detection value and the identification value of ch6G2 in all 5 common protein stabilizer buffers is less than 15% in 4 concentrations (50/10/2/0.4ng/ml) after one week at 4 ℃, and the difference in the back-calculated concentration of the standard curve point is less than 5%, which meets the standard and shows that ch6G2 is stable in 5 buffers at 4 ℃ for 1 week.
1 week (7 days)
TABLE 12
As shown in Table 13, the difference between the detection value and the identification value of 3 concentrations (80/40/20ng/ml) in all 1# protein stabilizer buffers after 4 weeks at 4 ℃ is less than 5%, and the difference between the calculated concentration of the standard curve point and the identification value is less than 15%, which meets the standard and indicates that ch6G2 is stable in 1# buffer at 4 ℃ for 4 weeks.
Watch 13
4 weeks (28 days)
The stability of ch6G2 at 37 ℃ in each reagent, as shown in table 14, ch6G2 detected at 4 concentrations (50/10/2/0.4ng/ml) with a difference from the labeled value of > 15% at 1#0/4, 2#0/4, 3#1/4 (100ng/ml),4#2/4 (100ng/ml, 20ng/ml),5#1/4 (100ng/ml) in all 5 common protein stabilizer buffers after one week at 37 ℃, and the error of the calculated concentration of the standard curve point is < 5%, which meets the standard and indicates that ch6G2 is stable at 37 ℃ for 1 week in 1# and 2# buffers.
TABLE 14
1 week (7 days)
As shown in Table 15, the activity of the ch6G2 samples stored at-80 ℃,4 ℃ and 37 ℃ for 1 week was determined using human serum-derived standards, and the activity of the samples stored at-80 ℃,4 ℃ and 37 ℃ was 0.99/0.99/1.00RU/ng, respectively, at the detection point of 160/80/40/20/10ng/ml, which is very consistent with that of the samples stored at-80 ℃,4 ℃ and 37 ℃, further indicating that ch6G2 is stable in the buffer # 1.
Watch 15
As shown in Table 16, after 4 weeks at 37 ℃ in the No.1 protein stabilizer buffer solution, the errors of 3 concentration (80/40/20ng/ml) detection values and the identification values are all less than 5%, and the errors of the calculated concentration of the standard curve point are less than 15%, which meets the standard and indicates that the ch6G2 is stable at 37 ℃ in the No.1 protein stabilizer buffer solution for 4 weeks.
TABLE 16
4 weeks (28 days)
As shown in Table 17, the activity of the ch6G2 samples stored at-80 ℃,4 ℃ and 37 ℃ for 4 weeks was measured using human serum-derived standards at a detection point of 80/20ng/ml for samples stored at-80 ℃,4 ℃ and 37 ℃ of 1.01/1.03,1.01/1.04 and 0.99/1.04RU/ng, respectively, which are very consistent with each other, further indicating that ch6G2 is stable in the 1# buffer.
TABLE 17
Stability results of biotinylated 3D2 mab as detection antibody:
as shown in table 18, it was shown that: biotinylated 3D2 was stable in buffer # 3 at 37 ℃ for 2 weeks.
Watch 18
2 weeks
| RU/ml | bio3D2-80 |
1#37 |
2#37 |
3#37 |
4#37 |
5#37℃ |
| 160 | -5.32 | 5.81 | 66.86 | 1.34 | -3.01 | 2.63 |
| 80 | 3.09 | -5.05 | -17.72 | -4.38 | 6.95 | -8.59 |
| 40 | -1.95 | 5.02 | 6.22 | 5.02 | -5.14 | 11.85 |
| 20 | 4.28 | -6.57 | 0.28 | 4.97 | 0.00 | 1.52 |
| 10 | 5.22 | -4.23 | -4.76 | -18.88 | 3.58 | -19.50 |
| r^2 | 0.99949 | 0.99804 | 0.99695 | 0.99888 | 0.99675 | 0.9936 |
As shown in table 19, it was shown that: biotinylated 3D2 was stable in buffer # 3 at 37 ℃ for 4 weeks.
Watch 19
4 weeks
4. The anti-hPLA 2R chimeric monoclonal antibody ch6G2 is used as a standard substance, and combined with a biotin-labeled anti-human IgG Fc monoclonal antibody ch3D2, a human serum standard substance and patient serum are detected
Recombinant human PLA2R (same as example 1) was diluted to 1ug/ml with PBS, 100 ul/well was added to the plate, and overnight at 4 ℃; adding 200ul of protein-free blocking solution (THERMO corporation) per well, and heating at 37 deg.C for 1 hr; PBST plate washing 3 times; diluting the protein-free confining liquid by 3 times by PBS, and uniformly mixing to obtain a diluent; diluting the ch6G2 to 160ng/ml, 80ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml and adding 100ul of diluent into an ELISA plate; diluting the standard substance (1500RU/ml) in the kit to 160RU/ml, 80RU/ml, 40RU/ml, 20RU/ml, 10RU/ml, 5RU/ml and 100 ul/hole by using the diluent, and adding the enzyme label plate; diluting the serum of a patient by 101 times by using a diluent, and adding an enzyme label plate into each 100 ul/hole; diluting 100 ul/well, adding enzyme label plate as reference; incubating for 1 hour at 37 ℃; PBST plate washing 3 times; diluting biotin-labeled anti-human IgG Fc monoclonal antibody 3D2(bio-3D2) to 100ng/ml with the diluent, and incubating for 1 hour at 37 ℃; PBST plate washing 3 times; diluting HRP-labeled SA (SA-HRP, THERMO company) 8000 times (according to the instruction) by using the diluent, and adding an ELISA plate at 100 ul/hole; incubating for 30 minutes at 37 ℃; PBST washing plate for 5 times, adding 100 ul/hole TMB color developing solution (BD company), developing for 5-10 minutes at room temperature; adding 50 ul/well stop solution (4M HCl, made by manufacturer); OD450 was measured with a microplate reader (BioTEK Co.); the RU values for the PLA2R autoantibodies were calculated for each serum sample using the ELISA Calc regression/fitting calculation program V1.0.
Watch 20
As shown in table 20, the two standard methods detect the content of PLA2R antibody in the sera of 58 idiopathic membranous nephropathy patients, and the result shows that the two standard methods detect 45 positive cases with the same detection sensitivity of 77.6% and detect the sera of 77 other nephropathy patients with 1 weak positive case and none of 100 healthy people with positive cases according to the test instruction of the omon kit. The consistency of the detection results of all related diseases and non-related diseases detected by the two methods and healthy people is 100 percent.
As shown in table 21, the difference between the detection values of the two detection methods for the same detection example was compared, and when the detection value was <100RU, the two methods showed good consistency, and the error was within 5%, and the difference between the detection values of the two methods increased with the increase of the detection value, and particularly when the detection value was >160RU/ml, the error reached 100% or more, which was caused because both detection methods could not be accurately quantified due to the HOOK effect when the detection value was >500 RU.
TABLE 21
| Detection value Range (RU) | Number of examples | Detection difference range of the two (%) |
| <20 | 13 | -3.1—4.3 |
| 20-40 | 3 | -4.6--3.8 |
| 40-80 | 9 | -2.8--4.2 |
| 80-160 | 16 | -4.8—5.1 |
| >160 | 17 | -25.3—16.8 |
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (25)
1. An immunogenic protein for immunizing an organism to produce antibodies against PLA 2R.
2. The immunizing antigen protein according to claim 1, wherein said immunizing antigen protein is the extracellular region sequence of PLA2R receptor, and the amino acid sequence of said immunizing antigen protein is SEQ ID NO.1 or conservative variation thereof.
3. An antibody against PLA2R, prepared by using the immunoantigen protein of claim 1 or 2 as an antigen.
4. An antibody against PLA2R, wherein the antibody is capable of specifically binding to the extracellular antigen-binding region of PLA2R receptor, and the extracellular antigen-binding region of PLA2R comprises an amino acid fragment as set forth in SEQ ID No. 1.
5. The antibody of claim 4, wherein said antibody to PLA2R is a monoclonal antibody.
6. The antibody of claim 5, further comprising any one or more of the following features: (1) the monoclonal antibody is selected from anti-PLA 2R monoclonal antibody 6G2 or anti-PLA 2R monoclonal antibody 3C 12; (2) the anti-PLA 2R monoclonal antibody 6G2 comprises a heavy chain and a light chain, wherein the heavy chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.12, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.13, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 14; the light chain comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO.15, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.16, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 17; (3) the anti-PLA 2R monoclonal antibody 3C12 comprises a heavy chain and a light chain, wherein the heavy chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.18, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.19, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 20; the light chain comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO.21, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.22, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 23; (4) the heavy and light chains are linked by disulfide bonds.
7. The antibody of claim 6, further comprising any one or more of the following features: (1) the amino acid sequence of the heavy chain variable region of the anti-PLA 2R monoclonal antibody 6G2 is SEQ ID NO.5 or a conservative variant sequence thereof, and the amino acid sequence of the light chain variable region of the anti-PLA 2R monoclonal antibody 6G2 is SEQ ID NO.7 or a conservative variant sequence thereof; (2) the amino acid sequence of the heavy chain variable region of the anti-PLA 2R monoclonal antibody 3C12 is SEQ ID NO.9 or a conservative variant sequence thereof, and the amino acid sequence of the light chain variable region of the anti-PLA 2R monoclonal antibody is SEQ ID NO.11 or a conservative variant sequence thereof.
8. The antibody of claim 5, further comprising any one or more of the following features: (1) the monoclonal antibody is murine; (2) the monoclonal antibodies are immunoglobulins of the IgG1 heavy chain and Kappa type light chain subtypes.
9. A chimeric antibody against PLA2R, comprising the Fab of any one of claims 5-8 directed to a monoclonal antibody of PLA2R and the Fc of human IgG 4.
10. The chimeric antibody according to claim 9, characterized in that it comprises a heavy chain comprising the heavy chain variable region VH, constant region 1CH1, Fc of human IgG4 of a monoclonal antibody directed against PLA2R and a light chain comprising the light chain variable region VL, kappa chain constant region CL of a monoclonal antibody directed against PLA 2R.
11. The chimeric antibody of claim 10, further comprising any one or more of the following characteristics: (1) the monoclonal antibody aiming at the PLA2R is selected from anti-PLA 2R monoclonal antibody 6G2 or anti-PLA 2R monoclonal antibody 3C 12; (2) the amino acid sequence of the constant region 1CH1 is shown in SEQ ID NO. 34; (3) the amino acid sequence of the Fc of the human IgG4 is shown in SEQ ID NO. 35; (4) the amino acid sequence of the kappa chain constant region CL is shown as SEQ ID NO. 36.
12. The chimeric antibody according to claim 10, wherein the chimeric antibody is selected from any one of the following: (1) the amino acid sequence of the heavy chain of the chimeric antibody is SEQ ID NO.37 or a conservative variant sequence thereof; the amino acid sequence of the light chain is SEQ ID NO.38 or a conservative variant sequence thereof; (2) the amino acid sequence of the heavy chain of the chimeric antibody is SEQ ID NO.39 or a conservative variant sequence thereof; the amino acid sequence of the light chain is SEQ ID NO.40 or a conservative variant sequence thereof.
13. An isolated DNA molecule encoding the variable region or full length amino acids of the heavy and/or light chain of a monoclonal antibody against PLA2R according to any one of claims 5-8 or a chimeric antibody against PLA2R according to any one of claims 10-12.
14. A construct comprising the isolated DNA molecule of claim 13.
15. An expression system for monoclonal or chimeric antibodies constructed by transfecting the construct of claim 14 into a host cell.
16. A method for preparing a monoclonal antibody or a chimeric antibody, comprising the steps of: culturing the monoclonal or chimeric antibody expression system of claim 15 under conditions suitable for expression of said antibody, thereby expressing said monoclonal or chimeric antibody, and purifying and isolating said monoclonal or chimeric antibody.
17. Use of a monoclonal antibody against PLA2R according to any one of claims 5-8 or a chimeric antibody against PLA2R according to any one of claims 10-12 in the preparation of a PLA2R test kit.
18. A PLA2R test kit comprising the monoclonal antibody against PLA2R of any one of claims 5-8 or the chimeric antibody against PLA2R of any one of claims 10-12.
19. The test kit of claim 18, further comprising a secondary antibody capable of binding to the Fc of human IgG4 in the chimeric antibody.
20. The test kit of claim 19, wherein the secondary antibody is selected from a species IgG monoclonal antibody.
21. The test kit of claim 20, wherein the IgG monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID No.28, the amino acid sequence of HCDR2 is set forth in SEQ ID No.29, and the amino acid sequence of HCDR3 is set forth in SEQ ID No. 30; the light chain comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO.31, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.32, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 33.
22. The detection kit of claim 20, wherein the IgG monoclonal antibody has the amino acid sequence of SEQ ID No.25 or a conservative variant thereof in its heavy chain variable region and the amino acid sequence of SEQ ID No.27 or a conservative variant thereof in its light chain variable region.
23. The test kit of claim 20, wherein the secondary antibody carries a labeling molecule.
24. Use of the test kit according to any one of claims 18 to 23 for the manufacture of a product for the detection of membranous nephropathy.
25. A membranous nephropathy detection product comprising the detection kit of any one of claims 18 to 23.
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