CN112390890B - Enzyme-linked immunoassay method for quantitatively detecting content of anti-human interleukin 17 monoclonal antibody in serum - Google Patents
Enzyme-linked immunoassay method for quantitatively detecting content of anti-human interleukin 17 monoclonal antibody in serum Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- C07K2317/00—Immunoglobulins specific features
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Abstract
The present application relates to an antibody; its heavy and light chains; a respective variable domain and a respective complementary decision domain; and an enzyme-linked immunoassay method established using the antibody as a secondary antibody in a BA-ELISA assay.
Description
The application is a divisional application of an invention patent application with the application date of 11/06/2020 and the application number of 202011233020.5, and the invention name of the method is 'an enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human interleukin 17 monoclonal antibody in serum'.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an enzyme-linked immunoassay method for quantitatively detecting the content of an anti-human interleukin 17 monoclonal antibody in serum.
Background
Interleukin 17(IL-17), also known as CTLA-8 or IL-17A, is a proinflammatory cytokine which stimulates non-immune cells such as fibroblasts, keratinocytes, epithelial and endothelial cells, synovial cells, etc., to secrete a variety of cytokines such as IL-6, IL-8, PGE2, MCP-1, CXCL-1 and G-CSF, and also has biological effects of inducing ICAM-1 surface expression, T cell proliferation, etc. IL-17A is produced primarily by a class of activated CD4+ T cells called Th17 and functions by binding to a complex of IL-17RA and IL-17RC (Toy et al, 2006, J.Immunol.177 (11); 36-39). The anti-human interleukin 17 monoclonal antibody is an IgG1 kappa type humanized monoclonal antibody which is specifically combined with human interleukin 17A (IL-17A); the parent antibody is obtained by screening a new Zealand rabbit B cell immunized by human IL-17A, and the parent rabbit antibody is subjected to humanized transformation to obtain the human interleukin 17-resistant monoclonal antibody.
The human interleukin 17-resistant monoclonal antibody to be detected in the present application is QX002N of jiangsu bio-medicine limited, and is described in chinese patent document CN 108640991B. In particular, over-expressed IL-17A can cause a number of inflammatory diseases: for example, IL-17A acts on macrophages and DC cells, induces high expression of IL-1, IL-6, TNF and CRP, causes inflammatory reaction, and participates in the pathological process of psoriasis and transplant rejection; IL-17A acts on endothelial cells, induces high expression of IL-6, MMP and blood coagulation factors, leads to blood vessel activation, and participates in the pathological processes of reperfusion injury, thrombus and atherosclerosis; IL-17A acts on fibroblasts, induces high expression of IL-6, chemokines, growth factors and MMP, causes matrix destruction, and participates in pathological processes of multiple sclerosis and Crohn's disease; IL-17A acts on osteoblasts and chondrocytes, induces RANKL, MMP, osteoclastogenesis, causes bone erosion, cartilage damage, and is involved in pathological processes of rheumatoid Arthritis and periodontal disease (N Engl J Med.2009Aug 27; 361(9):888-98.Nat Rev Drug Discov.2012Oct; 11(10):763-76.Semin Arthritis Rheum.2013Oct; 43(2):158-70.Trends Mol Med.2016Mar; 22(3): 230-41.). IL-17A neutralizing antibodies can inhibit the high expression of IL-17A in patients with autoimmune diseases and reduce the production of IL-6, an important inflammatory factor (Chabaud M, Durand JM, Buchs N, et al. Many animal model experiments of autoimmune diseases have demonstrated that neutralization of IL-17A with antibodies can effectively inhibit the pathological development of inflammation (Lubberts E, Koenders MI, Oppers-Walgreen B, et al. Currently, IL-17A related antibody drugs have been approved for the market, Secukinumab (IL-17A targeting antibody, SEC) for the treatment of plaque psoriasis, ankylosing spondylitis and psoriatic arthritis, Ixekizumab (IL-17A targeting antibody, IXE) for the treatment of plaque psoriasis and psoriatic arthritis, respectively. Jiangsu Quanxin biomedicine GmbH developed an anti-IL-17 monoclonal antibody with the product code of QX002N for use as a medicament.
For drugs, pharmacokinetics is the process of applying the principle of dynamics and mathematical processing methods to quantitatively describe the dynamic change rule of drugs in vivo and study the absorption, distribution, metabolism and excretion of drugs entering the human body through various routes. Thus, pharmacokinetic studies will facilitate the evaluation of the safety and efficacy of the drug. Common analytical methods for biological samples include chromatography and immunoassay. The chromatographic method has low sensitivity, complicated test process and unstable recovery rate. The immunoassay is an analysis method based on specific antigen-antibody reaction, is the most commonly used method in the current clinical and non-clinical biological sample detection methods, because the anti-human interleukin 17 monoclonal antibody is protein macromolecules, the immunoassay method is suitable for determination by using an enzyme-linked immunoassay method, and the biotinylated rabbit anti-human interleukin 17 monoclonal antibody is used as a second antibody to be combined with a detection antibody, so that the interference of irrelevant proteins in serum is reduced.
BA-ELISA refers to a detection system established by combining the high amplification effect between biotin (B) and avidin (A) on the basis of the conventional ELISA principle. Avidin is a basic glycoprotein extracted from ovalbumin and has very high affinity for biotin (binding constant as high as 1015M-1). Biotin is easily bonded to proteins (such as antibodies) through covalent bonds, so that avidin molecules bonded with enzyme react with biotin molecules bonded with specific antibodies, a multi-stage amplification effect is achieved, and color is developed due to the catalytic action of the enzyme when the enzyme meets corresponding substrates, and the purpose of detecting unknown antigen (or antibody) molecules is achieved. Has the advantages of high sensitivity, high specificity, high stability and the like.
Disclosure of Invention
Based on the reasons, the development of a method for quantitatively determining the content of the anti-human interleukin 17 monoclonal antibody in serum based on a biotin-avidin enzyme-linked immunoassay method with strong specificity and high sensitivity is urgently needed.
1. A monoclonal antibody comprising a heavy chain and a light chain, said heavy and light chains each comprising 3 complementarity determining region CDR regions having amino acid sequences:
heavy chain complementarity determining region HCDR1 region: SEQ ID NO. 1: SYGMR;
heavy chain complementarity determining region HCDR2 region: SEQ ID NO. 2: VVSSNGNIYYANWAKG, respectively;
heavy chain complementarity determining region HCDR3 region: SEQ ID NO. 3: DLRAGWSVGPFNL, respectively;
light chain complementarity determining region LCDR1 region: SEQ ID NO. 4: QASQSIASALA, respectively;
light chain complementarity determining region LCDR2 region: SEQ ID No. 5: DASDLAS;
light chain complementarity determining region LCDR3 region: SEQ ID NO. 6: QSYYHSGSSFYGYNG is added.
2. The monoclonal antibody of claim 1, said antibody comprising a heavy chain and a light chain, wherein said heavy chain and light chain each comprise a variable domain CVR region having the amino acid sequence:
heavy chain variable domain HCVR region: SEQ ID NO. 7:
QSVKESGGRLVTPGGSLTLTCTVSGIDLSSYGMRWVRQAPGKGLEWIGVVSSNGNIYYANWAKGRFTISKTSTTVDLKITSLTTEDTATYFCARDLRAGWSVGPFNLWGPGTLVTVSS；
light chain variable domain LCVR region: SEQ ID NO. 8:
DIVMTQTPSPVSAAVGGTVTIKCQASQSIASALAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTITDLECADVATYYCQSYYHSGSSFYGYNGFGGGTEVVVK。
3. the monoclonal antibody of claim 2, said antibody comprising a heavy chain and a light chain having the amino acid sequences:
heavy chain: SEQ ID NO. 9:
QSVKESGGRLVTPGGSLTLTCTVSGIDLSSYGMRWVRQAPGKGLEWIGVVSSNGNIYYANWAKGRFTISKTSTTVDLKITSLTTEDTATYFCARDLRAGWSVGPFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGGGGSGLNDIFEAQKIEWHE；
light chain: SEQ ID NO. 10:
DIVMTQTPSPVSAAVGGTVTIKCQASQSIASALAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTITDLECADVATYYCQSYYHSGSSFYGYNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
4. the monoclonal antibody of item 3, wherein said antibody is labeled with biotin.
4-1. use of the monoclonal antibody of item 4 in the preparation of a kit for detecting a human IL-17A monoclonal antibody.
4-2 the use according to the item 4-1, wherein the anti-human interleukin 17 monoclonal antibody as an object of detection is QX 002N.
4-3. the use according to item 4-1, wherein the kit comprises an antigen and a second antibody, the second antibody being the monoclonal antibody of item 4.
4-4. the use according to item 4-2, wherein the antigen is (human) interleukin 17 and it is immobilized on a solid support.
4-5 the use according to items 4-4, wherein the solid support is an enzyme-linked plate.
4-6. the use according to items 4-3, characterized in that the kit further comprises:
horseradish peroxidase-labeled streptavidin;
washing liquor, substrate developing liquor and stopping liquor;
the standard substance of the anti-human interleukin 17 monoclonal antibody with known content and corresponding diluent are used for preparing a standard curve.
4-7. the use according to item 4-1, characterized in that said kit is a biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) kit.
5. A kit for detecting an anti-human interleukin 17 monoclonal antibody, wherein the kit comprises: an antigen and a second antibody;
wherein the antigen is interleukin 17 and is immobilized on an enzyme-linked plate; while
The second antibody is a monoclonal antibody according to claim 4.
6. The kit according to item 5, wherein the anti-human interleukin 17 monoclonal antibody to be detected is QX 002N; of said QX002N
LCDR1 has the amino acid sequence of SEQ ID NO:11(QASQNIGGSLA),
LCDR2 has the amino acid sequence of SEQ ID NO:12(GASSLAS),
LCDR3 has the amino acid sequence of SEQ ID NO 13(QSYNTISTYGLA),
HCDR1 has the amino acid sequence of SEQ ID NO:14(LFYMS),
HCDR2 has the amino acid sequence of SEQ ID NO. 15(TIHEVASSYYASWAKG),
HCDR3 has the amino acid sequence of SEQ ID NO 16 (ETYSSRYPYPNI).
7. The kit of claim 5 or 6, further comprising:
horseradish peroxidase-labeled streptavidin; washing liquor, substrate developing liquor and stopping liquor;
the standard substance of the anti-human interleukin 17 monoclonal antibody with known content and corresponding diluent are used for preparing a standard curve.
8. The kit according to item 7, wherein,
the washing solution is a mixed solution (V/V:100/0.05) of a PBS solution and Tween 20, the color development solution is a TMB color development solution, and the stop solution is a mixed solution (V/V:4/1) of purified water and phosphoric acid.
9. The kit of claim 8, wherein the kit is a biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) kit.
10. A method for assaying an anti-human interleukin 17 monoclonal antibody according to the BA-ELISA method using the kit according to item 9.
11. The method for assaying the content of a monoclonal antibody according to item 10, which comprises the steps of:
-coating the enzyme-linked plate with interleukin 17 as antigen; washing the plate with a washing solution;
-diluting a standard anti-human interleukin 17 monoclonal antibody with a known concentration into a diluted column with the diluent and reacting with the coating antigen; incubation; washing the plate with a washing solution;
-adding the monoclonal antibody according to item 4 as a second antibody; washing the plate with a washing solution;
-adding the chromogenic antibodies sequentially; a substrate developing solution; stopping liquid;
-measuring the absorbance of the reaction solution obtained above at a specific wavelength to produce a standard curve;
and:
-coating the enzyme-linked plate with interleukin 17 as antigen; washing the plate with a washing solution;
-reacting a sample of a monoclonal antibody against human interleukin 17 to be tested with the envelope antigen; incubation; washing the plate with a washing solution;
-adding the monoclonal antibody according to item 4 as a second antibody; washing the plate with a washing solution;
-adding the chromogenic antibodies sequentially; a substrate developing solution; a stop solution;
measuring the absorbance of the reaction solution obtained above at a specific wavelength, and calculating the concentration of the anti-human interleukin 17 monoclonal antibody sample to be tested from the standard curve obtained in the above step.
12. The method for assaying monoclonal antibody content according to item 11, wherein the monoclonal antibody sample is incubated under the conditions for reacting with an antigen: the mixture was placed on a shaker at room temperature at about 110rpm for about 2 hours.
13. The method for measuring the content of a monoclonal antibody according to item 11, wherein the washing reagent, the developing solution and the stopping solution used are, respectively: a mixed solution (V/V:100/0.05) of PBS solution and Tween 20, a TMB color development solution, a mixed solution (V/V:4/1) of purified water and phosphoric acid; and the specific wavelength is 450 nm.
14. The method for measuring the content of a monoclonal antibody according to item 11, wherein the conditions for development after addition of the chromogenic antibody and the substrate are as follows: placed on a shaker at room temperature at about 110rpm protected from light.
15. The method for measuring the content of a monoclonal antibody according to any one of claims 10 to 14, wherein the anti-human interleukin 17 monoclonal antibody to be detected is QX 002N; of said QX002N
LCDR1 has the amino acid sequence of SEQ ID NO:11(QASQNIGGSLA),
LCDR2 has the amino acid sequence of SEQ ID NO:12(GASSLAS),
LCDR3 has the amino acid sequence of SEQ ID NO 13(QSYNTISTYGLA),
HCDR1 has the amino acid sequence of SEQ ID NO:14(LFYMS),
HCDR2 has the amino acid sequence of SEQ ID NO:15(TIHEVASSYYASWAKG),
HCDR3 has the amino acid sequence of SEQ ID NO 16 (ETYSSRYPYPNI).
Technical effects of the invention
By adopting the technical scheme of the application, the following beneficial effects are obtained: firstly, establishing an enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human interleukin 17 monoclonal antibody in serum by taking a biotinylated rabbit anti-human interleukin 17 monoclonal antibody as a second antibody; secondly, the effective quantitative range of the method established by the application is very wide and is 100-4000 ng/ml, the lower limit of the quantitative limit of the anti-human interleukin 17 monoclonal antibody is 100ng/ml, and the sensitivity is high; thirdly, the method established by the invention has good repeatability, and the coefficient of variation of repeated experiments among plates is 1.7-10.6 percent and is less than 20 percent; fourthly, the method established by the invention has good specificity and can tolerate 50 mug/ml of intravenous injection human immunoglobulin and 8ng/ml of human interleukin 17; fifthly, the invention can be used for detecting the content of the anti-human interleukin 17 monoclonal antibody in serum and clinically evaluating the pharmacokinetic characteristics of the anti-human interleukin 17 monoclonal antibody so as to evaluate the safety and the effectiveness of the anti-human interleukin 17 monoclonal antibody.
Drawings
FIG. 1 is a schematic diagram of an enzyme-linked immunoassay method of the present invention;
FIG. 2 is a graph of a four parameter fit of a method standard curve.
Detailed description of the preferred embodiments
It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
The present application relates in a first aspect to an antibody.
In a specific embodiment, a monoclonal antibody is provided, the monoclonal antibody comprising a heavy chain and a light chain, the heavy chain and the light chain comprising 3 complementarity determining region CDR regions having amino acid sequences of:
heavy chain complementarity determining region HCDR1 region: 1, SEQ ID NO. 1: SYGMR;
heavy chain complementarity determining region HCDR2 region: SEQ ID NO. 2: VVSSNGNIYYANWAKG, respectively;
heavy chain complementarity determining region HCDR3 region: SEQ ID No. 3: DLRAGWSVGPFNL, respectively;
light chain complementarity determining region LCDR1 region: SEQ ID NO. 4: QASQSIASALA, respectively;
light chain complementarity determining region LCDR2 region: SEQ ID No. 5: DASDLAS;
light chain complementarity determining region LCDR3 region: SEQ ID NO. 6: QSYYHSGSSFYGYNG are provided.
In the context of the present specification, "monoclonal antibody" means an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies constituting said population are identical and/or bind the same epitope, each monoclonal antibody of a monoclonal antibody preparation being directed against a single determinant on an antigen. Thus, the term "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies being described herein. Monoclonal antibodies structurally include a heavy chain and a light chain.
In the context of this specification, a CDR is an abbreviation for a complementary determining region, also referred to as "hypervariable region". LCDR is an abbreviation for light chain hypervariable region and HCDR is an abbreviation for heavy chain hypervariable region. In general, both the heavy and light chains of an antibody have 3 CDRs which together constitute the antigen-binding site of an Ig. At this site, the antibody can be sterically complementary to the epitope. Hereinafter, the monoclonal antibody claimed in the present application is used in the assay technique as a "secondary antibody" or "anti-antibody" to specifically bind to an anti-human interleukin 17 monoclonal antibody, i.e., relying on the specific binding ability of the CDR regions defined herein to a specific antigenic determinant on the anti-human interleukin 17 monoclonal antibody.
In yet another embodiment, a monoclonal antibody is provided comprising a heavy chain and a light chain, wherein the heavy chain and the light chain each comprise a variable domain CVR region having the amino acid sequences:
heavy chain variable domain HCVR region: SEQ ID NO. 7:
QSVKESGGRLVTPGGSLTLTCTVSGIDLSSYGMRWVRQAPGKGLEWIGVVSSNGNIYYANWAKGRFTISKTSTTVDLKITSLTTEDTATYFCARDLRAGWSVGPFNLWGPGTLVTVSS；
light chain variable domain LCVR region: SEQ ID NO. 8:
DIVMTQTPSPVSAAVGGTVTIKCQASQSIASALAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTITDLECADVATYYCQSYYHSGSSFYGYNGFGGGTEVVVK。
in the context of this specification, CVR is the english abbreviation for "variable domain", which definition is relative to "constant domain". The variable domains of both the heavy and light chains include the three "complementarity determining regions" described above.
In yet another embodiment, a monoclonal antibody is provided, the monoclonal antibody comprising a heavy chain and a light chain having the amino acid sequences:
heavy chain: SEQ ID NO. 9:
QSVKESGGRLVTPGGSLTLTCTVSGIDLSSYGMRWVRQAPGKGLEWIGVVSSNGNIYYANWAKGRFTISKTSTTVDLKITSLTTEDTATYFCARDLRAGWSVGPFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGGGGSGLNDIFEAQKIEWHE；
light chain: SEQ ID NO. 10:
DIVMTQTPSPVSAAVGGTVTIKCQASQSIASALAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTITDLECADVATYYCQSYYHSGSSFYGYNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。
in yet another embodiment, a monoclonal antibody is provided, the monoclonal antibody bearing a biotin label.
In the context of the present specification, when reference is made to the BA-ELISA technique, BA refers to the Biotin-Avidin System (Biotin-Avidin-System, BAS). It is a novel biological reaction amplification system developed in the end of the 70 s. With the advent of various biotin derivatives, BAS is rapidly becoming widely used in various fields of medicine. Numerous studies in recent years have demonstrated that the biotin-avidin system can bind almost exclusively to the various markers that have been successfully studied. The strong binding with high affinity between biotin and avidin and the multi-stage amplification effect make BAS immune labeling and related tracing analysis more sensitive. It has become a new technology widely used for qualitative and quantitative detection and positioning observation research of trace antigens and antibodies. The BA-ELISA method has further detailed classification, and the technology type of the application is that avidin and enzyme-labeled biotin form an avidin-biotin-peroxidase complex together, and when the avidin and the enzyme-labeled biotin contact with biotinylated anti-antibody, an antigen-antibody reaction system and an ABC labeling system are connected into a whole, which is also called as an ABC method. The method can amplify the signal of a trace amount of antigen by thousands of times so as to facilitate detection. The technical scheme of the application is to select the 'biotinylated anti-antibody' as the best and carry out technical improvement to obtain the ABC method with excellent performance.
The present application relates in a second aspect to a kit.
In a specific embodiment, a kit for detecting an anti-human interleukin 17 monoclonal antibody is provided, wherein the kit comprises: an antigen and a second antibody;
wherein the antigen is interleukin 17 and is immobilized on an enzyme-linked plate; while
The second antibody is the aforementioned biotin-added monoclonal antibody.
In yet another embodiment, a kit is provided, wherein the anti-human interleukin 17 monoclonal antibody to be detected is QX 002N; of said QX002N
LCDR1 has the amino acid sequence of SEQ ID NO:11(QASQNIGGSLA),
LCDR2 has the amino acid sequence of SEQ ID NO:12(GASSLAS),
LCDR3 has the amino acid sequence of SEQ ID NO 13(QSYNTISTYGLA),
HCDR1 has the amino acid sequence of SEQ ID NO:14(LFYMS),
HCDR2 has the amino acid sequence of SEQ ID NO:15(TIHEVASSYYASWAKG),
HCDR3 has the amino acid sequence of SEQ ID NO 16 (ETYSSRYPYPNI).
In the context of the present specification, Human Interleukin 17A (Human Interleukin-17A, hIL-17A) denotes a protein of Human origin. IL-17A is a proinflammatory cytokine produced by Th17 cells, stimulates cytokine production by cells by binding to their receptors, and is involved in and promotes a variety of diseases mediated by aberrant immune responses. The monoclonal antibodies of the present application are neutralizing antibodies that specifically recognize IL-17A, and are capable of specifically binding to IL-17A.
In the context of the present specification, "anti-human IL-17A monoclonal antibody" means a monoclonal antibody that: it is capable of binding human interleukin 17A with sufficient affinity such that the monoclonal antibody can be used for identifying/detecting human interleukin 17A, and also as an antibody drug, for example, as a drug for treating autoimmune diseases. Therefore, the BA-ELISA method established hereinafter with the kit of the present application can be used as an analytical means for pharmacokinetic studies of such antibody drugs. In another specific embodiment, a kit is provided, wherein the kit further comprises:
horseradish peroxidase-labeled streptavidin;
washing liquor, substrate developing liquor and stopping liquor;
the standard substance of the anti-human interleukin 17 monoclonal antibody with known content and corresponding diluent are used for preparing a standard curve.
In the context of the present specification, a "standard curve" refers to a linear relationship between the content of a sample to be tested and a measurable quantity of an analytical method (e.g. absorbance value) with the aid of which a quantitative analysis of the sample to be tested is achieved.
In still another embodiment, there is provided a kit, wherein the washing reagent is a mixture of a PBS solution and Tween 20 (V/V:100/0.05), the color-developing solution is a TMB color-developing solution, and the stop solution is a mixture of purified water and phosphoric acid (V/V: 4/1).
In yet another embodiment, a kit is provided, wherein the kit is a biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) kit.
The present application relates in a third aspect to a method for assaying monoclonal antibodies.
In one particular embodiment, there is provided: a method for measuring the content of the anti-human interleukin 17 monoclonal antibody by using the kit according to the BA-ELISA method.
In yet another embodiment, there is provided: the method for measuring the content of the anti-human interleukin 17 monoclonal antibody comprises the following steps:
-coating the enzyme-linked plate with interleukin 17 as antigen; washing the plate with a washing solution;
-diluting a standard anti-human interleukin 17 monoclonal antibody with a known concentration into a diluted column with the diluent and reacting with the coating antigen; incubation; washing the plate with a washing solution;
-adding the aforementioned biotin-labeled monoclonal antibody as a second antibody; washing the plate with a washing solution;
-adding the chromogenic antibodies sequentially; a substrate developing solution; a stop solution;
-measuring the absorbance of the reaction solution obtained above at a specific wavelength to produce a standard curve;
and:
-coating the enzyme-linked plate with interleukin 17 as antigen; washing the plate with a washing solution;
-reacting a sample of the anti-human interleukin 17 monoclonal antibody to be tested with the coating antigen; incubation; washing the plate with a washing solution;
-adding the aforementioned biotin-labeled monoclonal antibody as a second antibody; washing the plate with a washing solution;
-adding the chromogenic antibodies sequentially; a substrate developing solution; a stop solution;
measuring the absorbance of the reaction solution obtained above at a specific wavelength, and calculating the concentration of the anti-human interleukin 17 monoclonal antibody sample to be tested from the standard curve obtained in the above step.
In yet another embodiment, a method for determining the amount of a monoclonal antibody is provided, wherein the monoclonal antibody sample is incubated under the following conditions when reacting with an antigen: the mixture was placed on a shaker at room temperature at about 110rpm for about 2 hours.
In still another embodiment, there is provided a method for measuring the content of a monoclonal antibody, wherein the washing reagent, the developing solution and the stopping solution used are a mixture of a PBS solution and Tween 20 (V/V:100/0.05), a TMB developing solution, a mixed solution of purified water and phosphoric acid (V/V: 4/1); and the specific wavelength is 450 nm.
In another embodiment, a method for determining the content of monoclonal antibodies is provided, wherein the chromogenic conditions after adding chromogenic antibodies and substrates are as follows: placed on a shaker at room temperature at about 110rpm protected from light.
In one embodiment, a monoclonal antibody assay method is provided, wherein the monoclonal antibody against human interleukin 17 detected is QX 002N; of said QX002N
LCDR1 has the amino acid sequence of SEQ ID NO:11(QASQNIGGSLA),
LCDR2 has the amino acid sequence of SEQ ID NO:12(GASSLAS),
LCDR3 has the amino acid sequence of SEQ ID NO 13(QSYNTISTYGLA),
HCDR1 has the amino acid sequence of SEQ ID NO:14(LFYMS),
HCDR2 has the amino acid sequence of SEQ ID NO:15(TIHEVASSYYASWAKG),
HCDR3 has the amino acid sequence of SEQ ID NO 16 (ETYSSRYPYPNI).
The BA-ELISA method established according to the present application can be seen in FIG. 1.
< example >
Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
EXAMPLE 1 preparation and purification of Biotin-labeled Secondary antibodies
Using monoclonal antibodies (F (ab))2) The monoclonal antibody can be used as an antigen to carry out rabbit immunization, and a non-neutralizing rabbit monoclonal antibody which can be combined with the monoclonal antibody, can not block the combination of the anti-human interleukin 17 monoclonal antibody and the target human interleukin 17 thereof, and can not have cross reaction with other human IgG of the same type is screened out by a B cell cloning technology.
After the anti-antibody light/heavy chain gene fragment and the sequence information are obtained, firstly, a 3' end primer containing an Avi-Tag gene sequence is designed and synthesized; then, amplifying by using a PCR method to obtain a heavy chain gene of which the 3' end contains an Avi-Tag sequence; inserting the heavy chain gene containing the Avi-Tag into an expression plasmid by a one-step cloning method to construct a heavy chain expression plasmid containing the Avi-Tag; then, the heavy chain expression plasmid containing the Avi-Tag and the light chain expression plasmid are co-transfected into an ExpicHO-S cell, samples are collected after 8 days of expression, and the antibody of the heavy chain C end fused with the Avi-Tag is obtained by utilizing protein A affinity chromatography purification. As the BirA enzyme can effectively and specifically mark biotin on the Avi-Tag, the BirA enzyme is utilized to mark the biotin on the Avi-Tag, thereby obtaining the biotin site-specific marked antibody.
Example 2 characterization of the monoclonal antibody produced in example 1
1. Identification of binding Activity
Using anti-human interleukin 17 monoclonal antibody and human IgG as antigen coating enzyme linked plate; reacting serially diluted monoclonal antibody of example 1 with a coating antigen; adding the chromogenic antibodies in sequence; a substrate developing solution; a stop solution; binding activity experiments were performed. The result shows that the monoclonal antibody has better binding activity with the human interleukin 17 monoclonal antibody and does not have cross reaction with isotype IgG.
2. Identification of neutralizing Activity
Human interleukin 17 was used as an antigen coated enzyme conjugate plate; adding a quantitative anti-human interleukin 17 monoclonal antibody and a serial diluted incubation of the monoclonal antibody in the example 1 to react with the coating antigen; adding the chromogenic antibodies in sequence; a substrate developing solution; a stop solution; neutralization activity experiments were performed. The results show that the monoclonal antibody does not block the combination of the anti-human interleukin 17 monoclonal antibody and the target human interleukin 17, and therefore does not have neutralizing activity.
3. Biotin marker identification
Using an anti-human interleukin 17 monoclonal antibody as an antigen coated enzyme linked plate; reacting serially diluted monoclonal antibody of example 1 with a coating antigen; adding the chromogenic antibodies in sequence; a substrate color developing solution; a stop solution; activity experiments were performed. The result shows that the monoclonal antibody has better binding activity with the human interleukin 17 monoclonal antibody, high OD value response and good biotin labeling effect.
Example 3 construction of Elisa kit Using the "Secondary antibody" prepared in example 1
The antigen is interleukin 17 and is immobilized on an enzyme-linked plate; the anti-human interleukin 17 monoclonal antibody can be combined with the coating antigen; "Secondary antibody" prepared in example 1 was used as the second antibody; horse radish peroxidase-labeled streptavidin was used as the chromogenic antibody.
The experimental process comprises the following steps:
coating: human interleukin 17 was diluted to 1. mu.g/ml with 1 XPBS, 100. mu.l/well, coated overnight at 2-8 ℃;
and (3) sealing: sealing the solution at room temperature for 2h by using 200 mu l/hole sealing solution;
sample adding: uniformly diluting the standard substance and the quality control substance by using a diluent for 20 times before sample addition; 100 μ l/well, incubate for 2h at room temperature;
adding a secondary antibody: diluting the secondary antibody prepared in the example 1 by using the diluent to 10ng/ml and 100 mu l/hole, and incubating for 90min at room temperature in a dark place;
adding a chromogenic antibody: diluting the streptavidin marked by the horseradish peroxidase to 12.5ng/ml by using a diluent, and incubating for 1h at room temperature in a dark place;
color development: adding TMB color development solution into 100 μ l/hole, and keeping out of the sun at room temperature for 25 min;
termination and reading: adding stop solution into 100 mul/hole; OD reading at 450nm (650 nm as reference)
Reagent information:
1 × PBS: 23.48g of PBS powder was dissolved in 2000ml of ultrapure water;
cleaning solution: 500. mu.l Tween 20 per 1000ml1 XPBS;
diluent/blocking solution: weighing 2.5g BSA, dissolving with 1 × PBS 500ml, adding 250 μ l Tween 20 and 250 μ l Proclin 300, and mixing well;
preparing a TMB color development liquid: taking the solution A and the solution B with equal volume, cooling to room temperature, and mixing uniformly to use, keeping out of the sun, and preparing for use;
stopping liquid: 100ml of H3PO4Adding into 400ml of ultrapure water;
human pooled serum: mixing 20 individual blank sera with equal volume;
5% human mixed serum dilution: to each 1ml of the dilution, 50. mu.l of human pooled serum was added.
Example 4 establishment of enzyme-linked immunoassay method for quantitatively detecting content of anti-human interleukin 17 monoclonal antibody in serum
1. Establishment of standard curve of enzyme-linked immunoassay method for quantitatively detecting content of anti-human interleukin 17 monoclonal antibody in serum
The anti-human interleukin 17 monoclonal antibody is diluted to 39.1, 78.2, 156.3, 312.5, 625, 1250, 2500, 4000 and 5000ng/ml by using human mixed serum, and enzyme linked immunoassay experiments are carried out under the determined optimal experimental conditions. The concentration of the monoclonal antibody for detecting the human interleukin 17 is taken as an abscissa, and an OD450 value is taken as an ordinate to draw a four-parameter curve, as shown in figure 2, the concentration of the monoclonal antibody for detecting the human interleukin 17 is found to be in a range of 78.2-4000 ng/ml, and the curve has a better recovery rate.
The results are shown in Table 1 below, which shows that the standard curve concentration of the enzyme-linked immunoassay method established by the present invention is 39.1 (anchor point), 78.2(LLOQ), 156.3, 312.5, 625, 1250, 2500, 4000(ULOQ), 5000ng/ml (anchor point). The quantitative range of the method is 100-4000 ng/ml, 39.1 and 5000ng/ml are used as anchor points for improving the fitting of the standard curve (the recovery rate is not required). The standard curve established can be seen in fig. 2.
Table 1: method for establishing recovery rate statistics of each concentration point by standard curve
Note: mark as anchor point, recovery rate is not required
2. Methodology research of enzyme-linked immunoassay method for quantitatively detecting content of anti-human interleukin 17 monoclonal antibody in serum
2.1 quantitative Range
Different experimenters evaluate the standard curves of 5 independent batches by using the established enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human interleukin 17 monoclonal antibody in the serum on different days.
Results are shown in table 2, except for the anchor point, at LLOQ and ULOQ concentration levels: the accuracy RE% between batches is within the range of-4.9% -1.7%, and the precision CV% is within the range of 1.7-10.6%. 4000ng/mL and 100ng/mL can be used as the upper and lower quantitation limits of the method.
Table 2: method quantitative Range study
2.2 accuracy and precision
The experimenter uses the established enzyme-linked immunoassay method for quantitatively detecting the content of the human interleukin 17 monoclonal antibody in the serum to evaluate 5 independent batches of quality control samples (comprising LLOQ, LQC, MQC, HQC and ULOQ) for at least 2 days.
The results are shown in table 3 at LLOQ and ULOQ concentration levels: the accuracy RE% between batches is in the range of 1.1% -5.9%, and the precision CV% is in the range of 8.0% -8.5%. At QC concentration level: the accuracy RE% between batches is in the range of-8.1% -0.2%, and the precision CV% is in the range of 6.6% -7.9%.
Table 3: study of method accuracy and precision
2.3 Selectivity
The established enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human interleukin 17 monoclonal antibody in serum is used for inspecting by adding the anti-human interleukin 17 monoclonal antibody with the LLOQ concentration level to at least 10 blank matrixes with individual sources.
As shown in Table 4, the response values of blank samples from 12 individual sources are all lower than that of LLOQ, and the accuracy RE% of quality control samples from 11 individual sources in the batch is in the range of 8.1% -22.6%.
Table 4: study of Process Selectivity
2.4 dilution Linear and hook Effect
Using an established enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human interleukin 17 monoclonal antibody in serum to respectively prepare 1000000, 100000, 25000 and 2500ng/mL concentration samples, diluting the verification sample higher than the upper limit of the quantification by 1000 times, 100 times and 10 times to respectively obtain diluted linear verification samples with the concentrations of 1000, 1000 and 2500ng/mL, and detecting the diluted linear verification samples and the hook effect sample, wherein each concentration level of the verification sample comprises 3 samples, and each sample is a multi-well.
The results are shown in table 5/6, and the hook effect above ULOQ samples were not accurately recalculated in concentration and did not exhibit hook effect within the standard curve. After dilution linear verification samples are diluted by 1000 times, 100 times and 10 times, the accuracy of the final concentration of the back-calculation concentration is within the range of 4.2-8.9%, and the precision is less than or equal to 12%.
Table 5: study of hook Effect
Table 6: dilution line study
2.5 specificity
By using the established enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human interleukin 17 monoclonal antibody in serum, the concentration of an analyte is analyzed under the condition that 50000 and 5000ng/ml of an interferent 1 (human immunoglobulin for intravenous injection) and 8 and 1.6ng/ml of an interferent 2 (human interleukin 17) are respectively added into a test sample with the concentration of 0 and 200 ng/ml. Each concentration level included 3 validation samples and was a duplicate well.
The results are shown in Table 7/8, which shows good process specificity and tolerates 50000ng/ml of Interferon 1 (human immunoglobulin for intravenous injection) and 8ng/ml of Interferon 2 (human interleukin 17).
Table 7: study of method specificity
Table 8: method specificity study 2
Example 5 measurement of the content of anti-human Interleukin-17 monoclonal antibody in serum by the method established in example 4 using Elisa kit constructed in example 3
Using diluent to carry out MRD dilution on the monoclonal antibody sample of the anti-human interleukin 17 to be detected by 20 times, and then using 5% serum diluent to carry out dilution by proper times so as to be in a quantitative range of standard curve; the content of the anti-human interleukin 17 monoclonal antibody in the serum was measured by the method established in example 4 using the Elisa kit constructed in example 3.
To sum up, the enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human interleukin 17 monoclonal antibody in the serum provided by the invention is to form a solid phase carrier by a human interleukin 17 coated enzyme label plate, combine the solid phase carrier with the anti-human interleukin 17 monoclonal antibody in a biological sample to be detected, and form a solid phase antigen-antibody-enzyme labeled antibody compound by a biotinylated second antibody and a detection antibody. The biotinylated second antibody (secondary antibody) obtained by screening has strong specificity, so the method established by the invention has strong specificity, high sensitivity, good precision and accuracy.
Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments and application fields, and the above-described embodiments are illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> Jiangsu Quanxin biomedicine GmbH
<120> enzyme linked immunosorbent assay method for quantitatively detecting content of anti-human interleukin 17 monoclonal antibody in serum
<130>PD01054D1
<141> 2020-10-21
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Claims (11)
1. A monoclonal antibody composed of a heavy chain and a light chain, wherein the amino acid sequences of CDR regions of complementarity determining regions in the heavy chain and the light chain are:
heavy chain complementarity determining region HCDR1 region: SEQ ID NO. 1: SYGMR;
heavy chain complementarity determining region HCDR2 region: SEQ ID No. 2: VVSSNGNIYYANWAKG, respectively;
heavy chain complementarity determining region HCDR3 region: SEQ ID NO. 3: DLRAGWSVGPFNL;
light chain complementarity determining region LCDR1 region: SEQ ID No. 4: QASQSIASALA, respectively;
light chain complementarity determining region LCDR2 region: SEQ ID No. 5: DASDLAS;
light chain complementarity determining region LCDR3 region: SEQ ID No. 6: QSYYHSGSSFYGYNG are provided.
2. The monoclonal antibody of claim 1, said antibody consisting of a heavy chain and a light chain, wherein the amino acid sequence of the variable domain CVR region in said heavy and light chains is:
heavy chain variable domain HCVR region: SEQ ID NO. 7: QSVKESGGRLVTPGGSLTLTCTVSGIDLSSYGMRWVRQAPGKGLEWIGVVSSNGNIYYANWAKGRFTISKTSTTVDLKITSLTTEDTATYFCARDLRAGWSVGPFNLWGPGTLVTVSS, respectively;
light chain variable domain LCVR region: SEQ ID No. 8: DIVMTQTPSPVSAAVGGTVTIKCQASQSIASALAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTITDLECADVATYYCQSYYHSGSSFYGYNGFGGGTEVVVK are provided.
3. The monoclonal antibody of claim 2, said antibody being composed of a heavy chain and a light chain having the amino acid sequences:
heavy chain: SEQ ID NO. 9: QSVKESGGRLVTPGGSLTLTCTVSGIDLSSYGMRWVRQAPGKGLEWIGVVSSNGNIYYANWAKGRFTISKTSTTVDLKITSLTTEDTATYFCARDLRAGWSVGPFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGGGGSGLNDIFEAQKIEWHE;
light chain: SEQ ID No. 10: DIVMTQTPSPVSAAVGGTVTIKCQASQSIASALAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTITDLECADVATYYCQSYYHSGSSFYGYNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC is added.
4. The monoclonal antibody of claim 3, wherein said antibody is labeled with biotin.
5. Use of the monoclonal antibody according to claim 4 in the preparation of a kit for the detection of an anti-human interleukin 17 monoclonal antibody.
6. The use according to claim 5, wherein the anti-human interleukin 17 monoclonal antibody to be detected is QX 002N.
7. The use according to claim 5, wherein the kit comprises an antigen and a second antibody, the second antibody being the monoclonal antibody of claim 4.
8. Use according to claim 7, wherein the antigen is human interleukin 17 and is immobilized on a solid support.
9. Use according to claim 8, wherein the solid support is an enzyme-linked plate.
10. The use according to claim 7, wherein the kit further comprises:
horseradish peroxidase-labeled streptavidin;
washing liquor, substrate developing liquor and stopping liquor;
the standard substance of the anti-human interleukin 17 monoclonal antibody with known content and corresponding diluent are used for preparing a standard curve.
11. Use according to claim 5, characterized in that the kit is a biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) kit.
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| CN105315371B (en) * | 2015-03-05 | 2018-05-29 | 北京百特美博生物科技有限公司 | Anti-human IL-17 monoclonal antibodies |
| CN107488227A (en) * | 2016-06-12 | 2017-12-19 | 三生国健药业（上海）股份有限公司 | Anti-human IL-17 A monoclonal antibodies, its preparation method and application |
| CN108359011B (en) * | 2017-07-21 | 2019-06-25 | 华博生物医药技术（上海）有限公司 | Target the antibody, preparation method and application of interleukin-17 A |
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| CN110551215A (en) * | 2018-05-30 | 2019-12-10 | 中山康方生物医药有限公司 | anti-interleukin-17A antibodies, pharmaceutical compositions thereof, and uses thereof |
Non-Patent Citations (1)
| Title |
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| Secukinumab, an Interleukin-17A inhibitor, in ankylosing spondylitis;BAETEN D.et al.;《N Engl J Med.》;20151224;第373卷(第26期);2534-48 * |
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